U.S. patent application number 12/748168 was filed with the patent office on 2010-10-07 for methods and apparatus for single molecule sequencing using energy transfer detection.
This patent application is currently assigned to LIFE TECHNOLOGIES CORPORATION. Invention is credited to JOSEPH BEECHEM, Cheng-Yao Chen, Vi-En Choong, Guobin Luo, THEO NIKIFOROV, Xinzhan Peng, Michael Previte.
Application Number | 20100255487 12/748168 |
Document ID | / |
Family ID | 42781937 |
Filed Date | 2010-10-07 |
United States Patent
Application |
20100255487 |
Kind Code |
A1 |
BEECHEM; JOSEPH ; et
al. |
October 7, 2010 |
METHODS AND APPARATUS FOR SINGLE MOLECULE SEQUENCING USING ENERGY
TRANSFER DETECTION
Abstract
Provided herein are systems and methods for nucleotide
incorporation reactions. The systems comprise polymerases having
altered nucleotide incorporation kinetics and are linked to an
energy transfer donor moiety, and nucleotide molecules linked with
at least one energy transfer acceptor moiety. The donor and
acceptor moieties undergo energy transfer when the polymerase and
nucleotide are proximal to each other during nucleotide binding
and/or nucleotide incorporation. As the donor and acceptor moieties
undergo energy transfer, they generate an energy transfer signal
which can be associated with nucleotide binding or incorporation.
Detecting a time sequence of the generated signals, or the change
in the signals, can be used to determine the order of the
incorporated nucleotides, and can therefore be used to deduce the
sequence of the target molecule.
Inventors: |
BEECHEM; JOSEPH; (EUGENE,
OR) ; NIKIFOROV; THEO; (CARLSBAD, CA) ;
Choong; Vi-En; (Carlsbad, CA) ; Peng; Xinzhan;
(Carlsbad, CA) ; Luo; Guobin; (Oceanside, CA)
; Chen; Cheng-Yao; (Carlsbad, CA) ; Previte;
Michael; (Carlsbad, CA) |
Correspondence
Address: |
LIFE TECHNOLOGIES CORPORATION;C/O INTELLEVATE
P.O. BOX 52050
MINNEAPOLIS
MN
55402
US
|
Assignee: |
LIFE TECHNOLOGIES
CORPORATION
CARLSBAD
CA
|
Family ID: |
42781937 |
Appl. No.: |
12/748168 |
Filed: |
March 26, 2010 |
Related U.S. Patent Documents
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Application
Number |
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Patent Number |
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61307356 |
Feb 23, 2010 |
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61299917 |
Jan 29, 2010 |
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61299919 |
Jan 29, 2010 |
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61293616 |
Jan 8, 2010 |
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61293618 |
Jan 8, 2010 |
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61289388 |
Dec 22, 2009 |
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61263974 |
Nov 24, 2009 |
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61245457 |
Sep 24, 2009 |
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61242771 |
Sep 15, 2009 |
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61184770 |
Jun 5, 2009 |
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61164324 |
Mar 27, 2009 |
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Current U.S.
Class: |
435/6.12 ;
435/193 |
Current CPC
Class: |
G01N 21/6428 20130101;
C12N 9/1241 20130101; C07H 19/20 20130101; G01N 33/582 20130101;
G01N 2021/6432 20130101; C12N 9/1252 20130101; C12Y 207/07
20130101; C12Q 1/6869 20130101; C12N 9/96 20130101; C12Q 1/6818
20130101; C12Y 207/07007 20130101 |
Class at
Publication: |
435/6 ;
435/193 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12N 9/10 20060101 C12N009/10 |
Claims
1. A method for generating an energy transfer signal comprising the
steps of: contacting (i) a polymerase having altered nucleotide
incorporation kinetics and linked to an energy transfer donor
moiety with (ii) a nucleic acid molecule and with (iii) at least
one type of a nucleotide having an energy transfer acceptor moiety,
so as to incorporate the nucleotide into the nucleic acid molecule
thereby locating the polymerase and nucleotide in close proximity
with each other to generate the energy transfer signal.
2. A method for generating an energy transfer signal comprising the
steps of: contacting (i) a polymerase having altered nucleotide
incorporation kinetics and linked to an energy transfer donor
moiety with (ii) a nucleic acid molecule and with (iii) at least
one type of a hexaphosphate nucleotide having an energy transfer
acceptor moiety, so as to incorporate the hexaphosphate nucleotide
into the nucleic acid molecule thereby locating the polymerase and
nucleotide in close proximity with each other to generate the
energy transfer signal.
3. A method for generating an energy transfer signal comprising the
steps of: contacting (i) a polymerase having altered nucleotide
incorporation kinetics and linked to an energy transfer donor
moiety with (ii) a target nucleic acid molecule which is
base-paired with a polymerization initiation site having a terminal
3' OH group and with (iii) at least one type of a nucleotide having
an energy transfer acceptor moiety, so as to incorporate the
nucleotide onto the terminal 3' OH group thereby locating the
polymerase and nucleotide in close proximity with each other to
generate the energy transfer signal.
4. A method for generating an energy transfer signal comprising the
steps of: contacting (i) a polymerase having altered nucleotide
incorporation kinetics and linked to an energy transfer donor
moiety with (ii) a target nucleic acid molecule which is
base-paired with a polymerization initiation site having a terminal
3' OH group and with (iii) at least one type of a hexaphosphate
nucleotide having an energy transfer acceptor moiety, so as to
incorporate the hexaphosphate nucleotide onto the terminal 3' OH
group thereby locating the polymerase and nucleotide in close
proximity with each other to generate the energy transfer
signal.
5. The method of claim 1, further comprising the steps of: a)
exciting the energy transfer donor moiety with an excitation
source; and b) detecting the energy transfer signal from the energy
transfer donor moiety and the energy transfer acceptor moiety that
are in close proximity to each other.
6. The method of claim 1, further comprising the steps of: a)
exciting the energy transfer donor moiety with an excitation
source; b) detecting the energy transfer signal from the energy
transfer donor moiety and the energy transfer acceptor moiety which
are in close proximity to each other; and c) identifying the energy
transfer signal from the energy transfer accepter moiety.
7. The method of claim 1, wherein the polymerase is a DNA-dependent
polymerase, RNA-dependent polymerase, or reverse transcriptase.
8. The method of claim 1, wherein the altered nucleotide
incorporation kinetics includes altered polymerase binding to the
target molecule, altered polymerase binding to the nucleotide,
altered polymerase catalyzing nucleotide incorporation, altered the
polymerase cleaving the phosphate group or substituted phosphate
group, and/or altered polymerase releasing the cleavage
product.
9. The method of claim 1, wherein the polymerase is a B103
polymerase according to SEQ ID NO:1, 2 or 3.
10. The method of claim 1, wherein the energy transfer donor moiety
is a nanoparticle or a fluorescent dye.
11. The method of claim 10, wherein the nanoparticle is an
inorganic fluorescent nanoparticle.
12. The method of claim 10, wherein the nanoparticle is 1-20 nm in
its largest dimension.
13. The method of claim 10, wherein the nanoparticle is a
non-blinking nanoparticle.
14. The method of claim 1, wherein the target nucleic acid molecule
is a DNA or RNA molecule.
15. The method of claim 1, wherein the target nucleic acid molecule
is immobilized to a surface.
16. The method of claim 1, wherein the at least one type of
nucleotide comprises 3-10 phosphate groups.
17. The method of claim 1, wherein a terminal phosphate group of
the at least one type of nucleotide is linked to the acceptor
moiety.
18. The method of claim 1, wherein the at least one type of
nucleotide is adenosine, guanosine, cytosine, thymidine or
uridine.
19. The method of claim 1, wherein the polymerase is contacted with
at least two types of nucleotide.
20. The method of claim 19, wherein the at least two types of
nucleotides are each linked to a different type of energy transfer
acceptor moiety.
21. The method of claim 20, wherein the energy transfer acceptor
moiety is a fluorescent dye.
22. The method of claim 6, wherein the excitation source is
electromagnetic energy.
23. The method of claim 6, wherein the excitation source is
light.
24. The method of claim 6, wherein the energy transfer signal is a
FRET signal.
25. The method of claim 6, wherein the energy transfer signal is
optically detectable.
Description
[0001] This application claims the filing date benefit of U.S.
Provisional Application Nos.: 61/164,324, filed on Mar. 27, 2009;
61/184,770, filed on Jun. 5, 2009; 61/242,771, filed on Sep. 15,
2009; 61/245,457, filed on Sep. 24, 2009; 61/263,974, filed on Nov.
24, 2009; 61/289,388; filed on Dec. 22, 2009; 61/293,618, filed on
Jan. 8, 2010; 61/293,616, filed on Jan. 8, 2010; 61/299,919, filed
on Jan. 29, 2010; 61/299,917, filed on Jan. 29, 2010; 61/307,356,
filed on Feb. 23, 2010. The contents of each foregoing patent
applications are incorporated by reference in their entirety.
[0002] Throughout this application various publications are
referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the art to
which this invention pertains.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which
has been submitted via EFS-Web and is hereby incorporated by
reference in its entirety. Said ASCII copy, created on Jun. 14,
2010, is named LT00019.txt and is 84,083 bytes in size.
FIELD
[0004] The disclosed embodiments are related generally to single
molecule sequencing. More specifically, the disclosed embodiments
relate to an energy transfer system which permits detection and
monitoring of nucleotide polymerization.
BACKGROUND
[0005] Obtaining nucleic acid sequence information is an important
starting point for medical and academic research endeavors. The
sequence information facilitates medical studies of active disease,
genetic disease predispositions, and assists in rational design of
drugs targeting specific diseases. Sequence information is also the
basis for genomic and evolutionary studies, and many genetic
engineering applications. Reliable sequence information is critical
for paternity tests, criminal investigations, and forensic
studies.
[0006] Nucleic acid sequence information is typically obtained
using chain termination and size separation procedures, such as
those described by Sanger, et al., (1977 Proc. Nat. Acad. Sci. USA
74:5463-5467). Prior to gel separation, the nucleic acid target
molecules of interest are cloned, amplified, and isolated. Then the
sequencing reactions are conducted in four separate reaction
vessels, one for each nucleotide: A, G, C and T. These sequencing
methods are adequate for read lengths of 500-10000 nucleotides.
However, they are time-consuming and require relatively large
amounts of target molecules. Additionally, these methods can be
expensive, as they require reagents for four reaction vessels. And
the amplification steps are error-prone which can jeopardize
acquiring reliable sequence information. Furthermore, these methods
suffer from sequence-dependent artifacts including band compression
during size separation.
[0007] The technological advances in automated sequencing machines,
fluorescently-labeled nucleotides, and detector systems, have
improved the read lengths, and permit massively parallel sequencing
runs for high throughput methods. But these procedures are still
inadequate for large projects, like sequencing the human genome.
The human genome contains approximately three billion bases of DNA
sequence. Procedures that can sequence and analyze the human genome
(or the genome of any organism) in a relatively short time span and
at a reduced cost will make it feasible to deliver genomic
information as part of a healthcare program which can prevent,
diagnose, and treat disease.
[0008] The energy transfer system provided herein overcomes many
problems associated with current nucleotide incorporation
procedures. The energy transfer system requires minute amounts of
target molecule with no amplification steps, there is no need to
perform four separate nucleotide incorporation reactions, and the
reactions are not size separated or loaded on a gel. The energy
transfer system is a single molecule sequencing system which
facilitates rapid, accurate, and real-time sequencing of long
nucleic acid fragments.
SUMMARY
[0009] In one embodiment, the disclosed relates to methods for
generating an energy transfer signal, comprising: contacting (i) a
polymerase having altered nucleotide incorporation kinetics and
linked to an energy transfer donor moiety with (ii) a nucleic acid
molecule and with (iii) at least one type of a nucleotide having an
energy transfer acceptor moiety, so as to incorporate the
nucleotide into the nucleic acid molecule thereby locating the
polymerase and nucleotide in close proximity with each other to
generate the energy transfer signal.
[0010] In another embodiment, the disclosed relates to methods for
generating an energy transfer signal comprising the steps of:
contacting (i) a polymerase having altered nucleotide incorporation
kinetics and linked to an energy transfer donor moiety with (ii) a
nucleic acid molecule and with (iii) at least one type of a
hexaphosphate nucleotide having an energy transfer acceptor moiety,
so as to incorporate the nucleotide into the nucleic acid molecule
thereby locating the polymerase and nucleotide in close proximity
with each other to generate the energy transfer signal.
[0011] In another embodiment, the disclosed relates to methods for
generating an energy transfer signal comprising the steps of:
contacting (i) a polymerase having altered nucleotide incorporation
kinetics and linked to an energy transfer donor moiety with (ii) a
target nucleic acid molecule which is base-paired with a
polymerization initiation site having a terminal 3' OH group and
with (iii) at least one type of a nucleotide having an energy
transfer acceptor moiety, so as to incorporate the nucleotide onto
the terminal 3' OH group thereby locating the polymerase and
nucleotide in close proximity with each other to generate the
energy transfer signal.
[0012] In another embodiment, the disclosed relates to methods for
generating an energy transfer signal comprising the steps of:
contacting (i) a polymerase having altered nucleotide incorporation
kinetics and linked to an energy transfer donor moiety with (ii) a
target nucleic acid molecule which is base-paired with a
polymerization initiation site having a terminal 3' OH group and
with (iii) at least one type of a hexaphosphate nucleotide having
an energy transfer acceptor moiety, so as to incorporate the
nucleotide onto the terminal 3' OH group thereby locating the
polymerase and nucleotide in close proximity with each other to
generate the energy transfer signal.
DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 illustrates non-limiting examples of the general
structures of nucleotides linked with at least one energy transfer
acceptor moiety.
[0014] FIG. 2 depicts one embodiment showing an immobilized target
molecule/primer duplex to re-sequence the same target molecule, in
a direction away from the solid surface, using the reagent exchange
methods.
[0015] FIG. 3 depicts another embodiment showing an immobilized
target molecule/primer duplex to re-sequence the same target
molecule, in a direction away from solid surface, using the reagent
exchange methods.
[0016] FIG. 4 depicts another embodiment showing an immobilized,
self-primed target molecule to re-sequence the same target
molecule, in a direction away from the solid surface, using the
reagent exchange methods.
[0017] FIGS. 5A and B depict one embodiment showing an immobilized
target molecule/primer duplex to synthesize an extension product,
where the same extension product is re-sequenced in a direction
towards the solid surface, using the reagent exchange methods.
[0018] FIGS. 6A and B depict another embodiment showing an
immobilized target molecule/primer duplex to synthesize an
extension product, where the same extension product is re-sequenced
in a direction towards the solid surface, using the reagent
exchange methods.
[0019] FIG. 7 depicts one embodiment showing an immobilized
circular target nucleic acid molecule and a primer for rolling
circle replication to re-sequence the same target molecule multiple
times.
[0020] FIG. 8 depicts one embodiment showing an immobilized
double-stranded target nucleic acid molecule, which is ligated at
both ends with adaptors, for rolling circle replication to
re-sequence the same target molecule multiple times.
DETAILED DESCRIPTION
[0021] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of ordinary skill in the art to which these inventions belong. All
patents, patent applications, published applications, treatises and
other publications referred to herein, both supra and infra, are
incorporated by reference in their entirety. If a definition and/or
description is explicitly or implicitly set forth herein that is
contrary to or otherwise inconsistent with any definition set forth
in the patents, patent applications, published applications, and
other publications that are herein incorporated by reference, the
definition and/or description set forth herein prevails over the
definition that is incorporated by reference.
[0022] The practice of the present disclosure will employ, unless
otherwise indicated, conventional techniques of molecular biology,
microbiology and recombinant DNA techniques, which are within the
skill of the art. Such techniques are explained fully in the
literature. See, for example, Sambrook, J., and Russell, D. W.,
2001, Molecular Cloning: A Laboratory Manual, Third Edition;
Ausubel, F. M., et al., eds., 2002, Short Protocols In Molecular
Biology, Fifth Edition.
[0023] As used herein, the terms "comprising" (and any form or
variant of comprising, such as "comprise" and "comprises"),
"having" (and any form or variant of having, such as "have" and
"has"), "including" (and any form or variant of including, such as
"includes" and "include"), or "containing" (and any form or variant
of containing, such as "contains" and "contain"), are inclusive or
open-ended and do not exclude additional, unrecited additives,
components, integers, elements or method steps.
[0024] As used herein, the terms "a," "an," and "the" and similar
referents used herein are to be construed to cover both the
singular and the plural unless their usage in context indicates
otherwise. Accordingly, the use of the word "a" or "an" when used
in the claims or specification, including when used in conjunction
with the term "comprising", may mean "one," but it is also
consistent with the meaning of "one or more," "at least one," and
"one or more than one."
[0025] As used herein, the terms "link", "linked", "linkage" and
variants thereof comprise any type of fusion, bond, adherence or
association that is of sufficient stability to withstand use in the
particular biological application of interest. Such linkage can
comprise, for example, covalent, ionic, hydrogen, dipole-dipole,
hydrophilic, hydrophobic, or affinity bonding, bonds or
associations involving van der Waals forces, mechanical bonding,
and the like. Optionally, such linkage can occur between a
combination of different molecules, including but not limited to:
between a nanoparticle and a protein; between a protein and a
label; between a linker and a functionalized nanoparticle; between
a linker and a protein; between a nucleotide and a label; and the
like. Some examples of linkages can be found, for example, in
Hermanson, G., Bioconjugate Techniques, Second Edition (2008);
Aslam, M., Dent, A., Bioconjugation: Protein Coupling Techniques
for the Biomedical Sciences, London: Macmillan (1998); Aslam, M.,
Dent, A., Bioconjugation: Protein Coupling Techniques for the
Biomedical Sciences, London: Macmillan (1998).
[0026] As used herein, the term "linker" and its variants comprises
any composition, including any molecular complex or molecular
assembly that serves to link two or more compounds or
molecules.
[0027] As used herein, the term "polymerase" and its variants
comprise any enzyme that can catalyze the polymerization of
nucleotides (including analogs thereof) into a nucleic acid strand.
Typically but not necessarily such nucleotide polymerization can
occur in a template-dependent fashion. Such polymerases can include
without limitation naturally-occurring polymerases and any subunits
and truncations thereof, mutant polymerases, variant polymerases,
recombinant, fusion or otherwise engineered polymerases, chemically
modified polymerases, synthetic molecules or assemblies, and any
analogs, derivatives or fragments thereof that retain the ability
to catalyze such polymerization. Optionally, the polymerase can be
a mutant polymerase comprising one or more mutations involving the
replacement of one or more amino acids with other amino acids, the
insertion or deletion of one or more amino acids, or the linkage of
parts of two or more polymerases. Typically, the polymerase
comprises one or more active sites at which nucleotide binding
and/or catalysis of nucleotide polymerization can occur. Some
exemplary polymerases include without limitation DNA polymerases
(such as for example Phi-29 DNA polymerase, reverse transcriptases
and E. coli DNA polymerase) and RNA polymerases. The term
"polymerase" and its variants, as used herein, also refers to
fusion proteins comprising at least two portions linked to each
other, where the first portion comprises a peptide that can
catalyze the polymerization of nucleotides into a nucleic acid
strand and is linked to a second portion that comprises a second
polypeptide, such as, for example, a reporter enzyme or a
processivity-enhancing domain. One exemplary embodiment of such a
polymerase is Phusion.RTM. DNA polymerase (New England Biolabs),
which comprises a Pyrococcus-like polymerase fused to a
processivity-enhancing domain as described, for example, in U.S.
Pat. No. 6,627,424.
[0028] As used herein, the term "polymerase activity" and its
variants, when used in reference to a given polymerase, comprises
any in vivo or in vitro enzymatic activity characteristic of a
given polymerase that relates to catalyzing the polymerization of
nucleotides into a nucleic acid strand, e.g., primer extension
activity, and the like. Typically, but not necessarily such
nucleotide polymerization occurs in a template-dependent fashion.
In addition to such polymerase activity, the polymerase can
typically possess other enzymatic activities, for example, 3' to 5'
or 5' to 3' exonuclease activity.
[0029] As used herein, the term "nucleotide" and its variants
comprises any compound that can bind selectively to, or can be
polymerized by, a polymerase. Typically, but not necessarily,
selective binding of the nucleotide to the polymerase is followed
by polymerization of the nucleotide into a nucleic acid strand by
the polymerase; occasionally however the nucleotide may dissociate
from the polymerase without becoming incorporated into the nucleic
acid strand, an event referred to herein as a "non-productive"
event. Such nucleotides include not only naturally-occurring
nucleotides but also any analogs, regardless of their structure,
that can bind selectively to, or can be polymerized by, a
polymerase. While naturally-occurring nucleotides typically
comprise base, sugar and phosphate moieties, the nucleotides of the
present disclosure can include compounds lacking any one, some or
all of such moieties. In some embodiments, the nucleotide can
optionally include a chain of phosphorus atoms comprising three,
four, five, six, seven, eight, nine, ten or more phosphorus atoms.
In some embodiments, the phosphorus chain can be attached to any
carbon of a sugar ring, such as the 5' carbon. The phosphorus chain
can be linked to the sugar with an intervening O or S. In one
embodiment, one or more phosphorus atoms in the chain can be part
of a phosphate group having P and O. In another embodiment, the
phosphorus atoms in the chain can be linked together with
intervening O, NH, S, methylene, substituted methylene, ethylene,
substituted ethylene, CNH.sub.2, C(O), C(CH.sub.2),
CH.sub.2CH.sub.2, or C(OH)CH.sub.2R (where R can be a 4-pyridine or
1-imidazole). In one embodiment, the phosphorus atoms in the chain
can have side groups having O, BH.sub.3, or S. In the phosphorus
chain, a phosphorus atom with a side group other than 0 can be a
substituted phosphate group. Some examples of nucleotide analogs
are described in Xu, U.S. Pat. No. 7,405,281. In some embodiments,
the nucleotide comprises a label (e.g., reporter moiety) and
referred to herein as a "labeled nucleotide"; the label of the
labeled nucleotide is referred to herein as a "nucleotide label".
In some embodiments, the label can be in the form of a fluorescent
dye attached to the terminal phosphate group, i.e., the phosphate
group or substitute phosphate group most distal from the sugar.
Some examples of nucleotides that can be used in the disclosed
methods and compositions include, but are not limited to,
ribonucleotides, deoxyribonucleotides, modified ribonucleotides,
modified deoxyribonucleotides, ribonucleotide polyphosphates,
deoxyribonucleotide polyphosphates, modified ribonucleotide
polyphosphates, modified deoxyribonucleotide polyphosphates,
peptide nucleotides, metallonucleosides, phosphonate nucleosides,
and modified phosphate-sugar backbone nucleotides, analogs,
derivatives, or variants of the foregoing compounds, and the like.
In some embodiments, the nucleotide can comprise non-oxygen
moieties such as, for example, thio- or borano-moieties, in place
of the oxygen moiety bridging the alpha phosphate and the sugar of
the nucleotide, or the alpha and beta phosphates of the nucleotide,
or the beta and gamma phosphates of the nucleotide, or between any
other two phosphates of the nucleotide, or any combination
thereof.
[0030] As used herein, the term "nucleotide incorporation" and its
variants comprises polymerization of one or more nucleotides into a
nucleic acid strand.
[0031] As used herein, the term "biomolecule" and its variants
comprises any compound isolated from a living organism, as well as
analogs (including engineered and/or synthetic analogs),
derivatives, mutants or variants and/or biologically active
fragments of the same. For example, the biomolecule can be a
protein (e.g., enzyme), nucleic acid, nucleotide, carbohydrate or
lipid. In some embodiments, the biomolecule can be an engineered or
synthetic analog of a compound isolated from a living cell that is
structurally different from the compound but retains a biological
activity characteristic of that compound.
[0032] As used herein, the term "target" and its variants comprises
any compound that is capable of binding specifically to a
particular biomolecule. In one exemplary embodiment, the target of
an enzyme can be, for example, a substrate of the enzyme.
[0033] As used herein, the term "biological activity" and its
variants, when used in reference to a biomolecule (such as, for
example, an enzyme) refers to any in vivo or in vitro activity that
is characteristic of the biomolecule itself, including the
interaction of the biomolecule with one or more targets. For
example, biological activity can optionally include the selective
binding of an antibody to an antigen, the enzymatic activity of an
enzyme, and the like. Such activity can also include, without
limitation, binding, fusion, bond formation, association, approach,
catalysis or chemical reaction, optionally with another biomolecule
or with a target molecule.
[0034] As used herein, the term "biologically active fragment" and
its variants refers to any fragment, derivative or analog of a
biomolecule that possesses an in vivo or in vitro activity that is
characteristic of the biomolecule itself. For example, the
biomolecule can be an antibody that is characterized by
antigen-binding activity, or an enzyme characterized by the ability
to catalyze a particular biochemical reaction, etc. Biologically
active fragments can optionally exist in vivo, such as, for
example, fragments which arise from post transcriptional processing
or which arise from translation of alternatively spliced RNAs, or
alternatively can be created through engineering, bulk synthesis,
or other suitable manipulation. Biologically active fragments
include fragments expressed in native or endogenous cells as well
as those made in expression systems such as, for example, in
bacterial, yeast, insect or mammalian cells. Because biomolecules
often exhibit a range of physiological properties and because such
properties can be attributable to different portions of the
biomolecule, a useful biologically active fragment can be a
fragment of a biomolecule that exhibits a biological activity in
any biological assay. In some embodiments, the fragment or analog
possesses 10%, 40%, 60%, 70%, 80% or 90% or greater of the activity
of the biomolecule in any in vivo or in vitro assay of
interest.
[0035] The term "modification" or "modified" and their variants, as
used herein with reference to a protein, comprise any change in the
structural, biological and/or chemical properties of the protein,
particularly a change in the amino acid sequence of the protein. In
some embodiments, the modification can comprise one or more amino
acid mutations, including without limitation amino acid additions,
deletions and substitutions (including both conservative and
non-conservative substitutions).
[0036] The terms "resonance energy transfer" and "RET" and their
variants, as used herein, refer to a radiationless transmission of
excitation energy from a first moiety, termed a donor moiety, to a
second moiety termed an acceptor moiety. One type of RET includes
Forster Resonance Energy Transfer (FRET), in which a fluorophore
(the donor) in an excited state transfers its energy to a proximal
molecule (the acceptor) by nonradiative dipole-dipole interaction.
See, e.g., Forster, T. "Intermolecular Energy Migration and
Fluorescence", Ann. Phys., 2:55-75, 1948; Lakowicz, J. R.,
Principles of Fluorescence Spectroscopy, 2nd ed. Plenum, New York.
367-394., 1999. RET also comprises luminescence resonance energy
transfer, bioluminescence resonance energy transfer,
chemiluminescence resonance energy transfer, and similar types of
energy transfer not strictly following the Forster's theory, such
as nonoverlapping energy transfer occurring when nonoverlapping
acceptors are utilized. See, for example, Anal. Chem. 2005, 77:
1483-1487.
[0037] The term "conservative" and its variants, as used herein
with reference to any change in amino acid sequence, refers to an
amino acid mutation wherein one or more amino acids is substituted
by another amino acid having highly similar properties. For
example, one or more amino acids comprising nonpolar or aliphatic
side chains (for example, glycine, alanine, valine, leucine,
isoleucine or proline) can be substituted for each other.
Similarly, one or more amino acids comprising polar, uncharged side
chains (for example, serine, threonine, cysteine, methionine,
asparagine or glutamine) can be substituted for each other.
Similarly, one or more amino acids comprising aromatic side chains
(for example, phenylalanine, tyrosine or tryptophan) can be
substituted for each other. Similarly, one or more amino acids
comprising positively charged side chains (for example, lysine,
arginine or histidine) can be substituted for each other.
Similarly, one or more amino acids comprising negatively charged
side chains (for example, aspartic acid or glutamic acid) can be
substituted for each other. In some embodiments, the modified
polymerase is a variant that comprises one or more of these
conservative amino acid substitutions, or any combination thereof.
In some embodiments, conservative substitutions for leucine
include: alanine, isoleucine, valine, phenylalanine, tryptophan,
methionine, and cysteine. In other embodiments, conservative
substitutions for asparagine include: arginine, lysine, aspartate,
glutamate, and glutamine.
[0038] The term "primer extension activity" and its variants, as
used herein, when used in reference to a given polymerase,
comprises any in vivo or in vitro enzymatic activity characteristic
of a given polymerase that relates to catalyzing nucleotide
incorporation onto the terminal 3'OH end of an extending nucleic
acid molecule. Typically but not necessarily such nucleotide
incorporation occurs in a template-dependent fashion. The primer
extension activity is typically quantified as the total number of
nucleotides incorporated (as measured by, e.g., radiometric or
other suitable assay) by a unit amount of polymerase (in moles) per
unit time (seconds) under a particular set of reaction
conditions.
[0039] The terms "His tag" or "His-tag" and their variants as used
herein refers to a stretch of amino acids comprising multiple
histidine residues. Typically, the His tag can bind to metal ions,
for example, Zn.sup.2+, Ni.sup.2+, Co.sup.2+, or Cu.sup.2+ ions.
Optionally, the His tag comprises 2, 3, 4, 5, 6, 7, 8 or more
histidine residues. In some embodiments, the His tag is fused to
the N- or C-terminus of a protein; alternatively, it can be fused
at any suitable location within the protein.
[0040] As used herein, the term "binding pair" or "binding partner"
and its variants refers to two molecules, or portions thereof,
which have a specific binding affinity for one another and
typically will bind to each other in preference to binding to other
molecules. Typically but not necessarily some or all of the
structure of one member of a specific binding pair is complementary
to some or all of the structure possessed by the other member, with
the two members being able to bind together specifically by way of
a bond between the complementary structures, optionally by virtue
of multiple noncovalent attractions. The two members of a binding
pair are referred to herein as the "first member" and the "second
member" respectively. The following may be mentioned as
non-limiting examples of molecules that can function as a member of
a specific binding pair, without this being understood as any
restriction: thyroxin-binding globulin, steroid-binding proteins,
antibodies, antigens, haptens, enzymes, lectins, nucleic acids,
repressors, oligonucleotides, polynucleotides, protein A, protein
G, avidin, streptavidin, biotin, complement component C1q, nucleic
acid-binding proteins, receptors, carbohydrates, complementary
nucleic acid sequences, and the like. Examples of specific binding
pairs include without limitation: an avidin moiety and a biotin
moiety; an antigenic epitope and an antibody or immunogically
reactive fragment thereof; an antibody and a hapten; a digoxigen
moiety and an anti-digoxigen antibody; a fluorescein moiety and an
anti-fluorescein antibody; an operator and a repressor; a nuclease
and a nucleotide; a lectin and a polysaccharide; a steroid and a
steroid-binding protein; an active compound and an active compound
receptor; a hormone and a hormone receptor; an enzyme and a
substrate; an immunoglobulin and protein A; and an oligonucleotide
or polynucleotide and its corresponding complement.
[0041] As used herein, the term "biotin" and its variants comprises
biotin (cis-hexahydro-2-oxo-1H-thieno[3,4]imidazole-4-pentanoic
acid) and any derivatives and analogs thereof, including
biotin-like compounds. Such compounds include, for example,
biotin-.di-elect cons.-N-lysine, biocytin hydrazide, amino or
sulfhydryl derivatives of 2-iminobiotin and biotinyl-.di-elect
cons.-aminocaproic acid-N-hydroxysuccinimide ester,
sulfosuccinimideiminobiotin, biotinbromoacetylhydrazide,
p-diazobenzoyl biocytin, 3-(N-maleimidopropionyl)biocytin, and the
like. "Biotin moiety" also comprises biotin variants that can
specifically bind to an avidin moiety.
[0042] The term "biotinylated" and its variants, as used herein,
refer to any covalent or non-covalent adduct of biotin with other
moieties such as biomolecules, e.g., proteins, nucleic acids
(including DNA, RNA, DNA/RNA chimeric molecules, nucleic acid
analogs and peptide nucleic acids), proteins (including enzymes,
peptides and antibodies), carbohydrates, lipids, etc.
[0043] The terms "avidin" and "avidin moiety" and their variants,
as used herein, comprises the native egg-white glycoprotein avidin,
as well as any derivatives, analogs and other non-native forms of
avidin, that can specifically bind to biotin moieties. In some
embodiments, the avidin moiety can comprise deglycosylated forms of
avidin, bacterial streptavidins produced by selected strains of
Streptomyces, e.g., Streptomyces avidinii, to truncated
streptavidins, and to recombinant avidin and streptavidin as well
as to derivatives of native, deglycosylated and recombinant avidin
and of native, recombinant and truncated streptavidin, for example,
N-acyl avidins, e.g., N-acetyl, N-phthalyl and N-succinyl avidin,
and the commercial products ExtrAvidin.RTM., Captavidin.RTM.,
Neutravidin.RTM. and Neutralite Avidin.RTM.. All forms of
avidin-type molecules, including both native and recombinant avidin
and streptavidin as well as derivatized molecules, e.g.
nonglycosylated avidins, N-acyl avidins and truncated
streptavidins, are encompassed within the terms "avidin" and
"avidin moiety". Typically, but not necessarily, avidin exists as a
tetrameric protein, wherein each of the four tetramers is capable
of binding at least one biotin moiety.
[0044] As used herein, the term "biotin-avidin bond" and its
variants refers to a specific linkage formed between a biotin
moiety and an avidin moiety. Typically, a biotin moiety can bind
with high affinity to an avidin moiety, with a dissociation
constant K.sub.d typically in the order of 10.sup.-14 to 10.sup.-15
mol/L. Typically, such binding occurs via non-covalent
interactions.
[0045] As used herein, the term "accessory compound" and its
variants refer to any non-polymerase compound capable of attaching
to a nanoparticle through one or more attachment sites. Optionally,
the accessory compound can comprise a His tag.
[0046] As used herein, the term "modification enzyme recognition
site" refers to an amino acid recognition sequence that is
chemically modified in an enzyme-catalyzed reaction, wherein the
enzyme catalyzing the reaction exhibits specificity for the amino
acid recognition sequence. The amino acid recognition sequence may
be inserted into a protein of interest, for example by conventional
recombinant DNA techniques. Examples of modification enzyme
recognition sites include, but are not limited to a biotin ligase
modification site, for example a site comprising the amino acid
sequence GLNDIFEAQKIEWHE (SEQ ID NO: 61), for introducing a biotin
moiety; a protein kinase modification site, for example a site
comprising the amino acid sequence LRRASLG (SEQ ID NO: 19), for
introducing a phosphorothioate moiety; and a transglutaminase
modification site, for example a site comprising the amino acid
sequence PKPQQF (SEQ ID NO: 22), for introducing an amine
moiety.
[0047] The terms "reporter" and "reporter moiety" and their
variants, as used herein, refer to any moiety that generates, or
causes to be generated, a detectable signal. Any suitable reporter
moiety may be used, including luminescent, photoluminescent,
electroluminescent, bioluminescent, chemiluminescent, fluorescent,
phosphorescent, chromophore, radioisotope, electrochemical, mass
spectrometry, Raman, hapten, affinity tag, atom, or an enzyme. The
reporter moiety generates, or causes to be generated, a detectable
signal resulting from a chemical or physical change (e.g., heat,
light, electrical, pH, salt concentration, enzymatic activity, or
proximity events). A proximity event includes two reporter moieties
approaching each other, or associating with each other, or binding
each other. The appropriate procedures for detecting a signal, or
change in the signal, generated by the reporter moiety are well
known in the art. The reporter moieties can be linked to a solid
surface, polymerase, nucleotide (or analog thereof), target nucleic
acid molecule, or primer. In one embodiment, a nucleotide can be
linked to a reporter moiety. The reporter moiety can generate a
signal, or a change in a signal, upon excitation from an
appropriated energy source (e.g., electromagnetic source). Some
energy transfer reporter moieties can be optically or spectrally
detectable.
[0048] The term "label" and its variants, as used herein, comprises
any optically detectable moiety and includes any moiety that can be
detected using, for example, fluorescence, luminescence and/or
phosphoresecence spectroscopy, Raman scattering, or diffraction.
Exemplary labels according to the present disclosure include
fluorescent and luminescent moieties as well as quenchers thereof.
Some typical labels include without limitation energy transfer
moieties, nanoparticles and organic dyes.
[0049] Other objects, features and advantages of the disclosed
compositions, methods, systems and kits will become apparent from
the following detailed description. It should be understood,
however, that the detailed description and the specific examples,
while indicating specific embodiments, are given by way of
illustration only, since various changes and modifications within
the spirit and scope of the inventions provided herein will become
apparent to those skilled in the art from this detailed
description.
[0050] Provided herein are methods, compositions, systems and kits
for nucleotide polymerization using energy transfer signals, or
changes in energy transfer signals, from an energy transfer system
which permits generating, detecting, measuring, and characterizing
the energy transfer signals, which are associated with nucleotide
polymerization, and permits identification of nucleotide binding
and nucleotide incorporation events. The compositions and methods
can permit accurate base identification for single molecule
sequencing reactions.
[0051] The methods, compositions, systems and kits provided herein
can be used for sequence-by-synthesis procedures for deducing the
sequence of a target nucleic acid molecule. The methods permit
detection and monitoring of nucleotide binding and nucleotide
polymerization events. The systems comprise polymerases attached
with an energy transfer donor moiety (or acceptor moiety), and
nucleotides attached with at least one energy transfer acceptor
moiety (or donor moiety). The donor and acceptor moieties undergo
energy transfer when the polymerase and nucleotide are proximal to
each other during nucleotide binding and/or nucleotide
polymerization. As the donor and acceptor moieties undergo energy
transfer, they generate an energy transfer signal (or a change in
an energy transfer signal) which may correlate with nucleotide
binding or polymerization. Detecting a time sequence of the
generated energy transfer signals, or the change in the generated
energy transfer signals, can be used to determine the order of the
incorporated nucleotides, and can therefore be used to deduce the
sequence of the target molecule.
Nucleotide Polymerization
[0052] Provided herein are methods, compositions, systems and kits
for: nucleotide binding; nucleotide incorporation; nucleotide
polymerization; generating an energy transfer signal which is
associated with the proximity of the polymerase and nucleotide;
generating an energy transfer signal which is associated with
nucleotide incorporation; detecting an energy transfer signal which
is associated with nucleotide incorporation; and identifying the
energy transfer signal which is associated with nucleotide
incorporation.
[0053] The methods include polymerase-dependent nucleotide
polymerization in a continuous (i.e., asynchronous) manner. The
compositions, systems, methods and kits comprise polymerases
attached with at least one energy transfer donor moiety, and
nucleotides attached with at least one energy transfer acceptor
moiety. The donor and acceptor moieties undergo energy transfer
when the polymerase and nucleotide are proximal to each other
during nucleotide binding and/or nucleotide incorporation. As the
donor and acceptor moieties undergo energy transfer, they generate
an energy transfer signal (or a change in an energy transfer
signal) which may correlate with nucleotide binding to the
polymerase or with nucleotide incorporation during polymerization.
Detecting the energy transfer signal, or change in the energy
transfer signal, can be used to identify the incorporating
nucleotide. Detecting a time sequence of the generated energy
transfer signals, or the change in the generated energy transfer
signals, from successive nucleotide incorporation events can be
used to determine the order of the incorporated nucleotides, and
can therefore be used to deduce the sequence of the target
molecule. Nucleotide incorporation includes DNA polymerization and
RNA polymerization.
[0054] By way of a non-limiting example of nucleotide
polymerization, the steps or events of DNA polymerization are well
known and comprise: (1) complementary base-pairing a target DNA
molecule (e.g., a template molecule) with a DNA primer molecule
having a terminal 3' OH (the terminal 3' OH provides the
polymerization initiation site for DNA polymerase); (2) binding the
base-paired target DNA/primer duplex with a DNA-dependent
polymerase to form a complex (e.g., open complex); (3) a candidate
nucleotide binds with the DNA polymerase which interrogates the
candidate nucleotide for complementarity with the template
nucleotide on the target DNA molecule; (4) the DNA polymerase may
undergo a conformational change (e.g., to a closed complex if the
candidate nucleotide is complementary); (5) the polymerase
catalyzes nucleotide polymerization.
[0055] In one embodiment, the polymerase catalyzes nucleotide
incorporation. For example, the polymerase catalyzes bond formation
between the candidate nucleotide and the nucleotide at the terminal
end of the polymerization initiation site. The polymerase can
catalyze the terminal 3' OH of the primer exerting a nucleophilic
attack on the bond between the .alpha. and .beta. phosphates of the
candidate nucleotide to mediate a nucleotidyl transferase reaction
resulting in phosphodiester bond formation between the terminal 3'
end of the primer and the candidate nucleotide (i.e., nucleotide
incorporation in a template-dependent manner), and concomitant
cleavage to form a cleavage product. The polymerase can liberate
the cleavage product. In some embodiments, where the polymerase
incorporates a nucleotide having phosphate groups, the cleavage
product includes one or more phosphate groups. In other
embodiments, where the polymerase incorporates a nucleotide having
substituted phosphate groups, the cleavage product may include one
or more substituted phosphate groups.
[0056] The candidate nucleotide may or may not be complementary to
the template nucleotide on the target molecule. The candidate
nucleotide can dissociate from the polymerase. If the candidate
nucleotide dissociates from the polymerase, it can be liberated and
typically carries intact polyphosphate groups. When the candidate
nucleotide dissociates from the DNA polymerase, the event is known
as a "non-productive binding" event. The dissociating nucleotide
may or may not be complementary to the template nucleotide on the
target molecule.
[0057] The incorporated nucleotide may or may not be complementary
to the template nucleotide on the target. When the candidate
nucleotide binds the DNA polymerase and is incorporated, the event
is a "productive binding" event. The incorporated nucleotide may or
may not be complementary to the template nucleotide on the target
molecule.
[0058] The length of time, frequency, or duration of the binding of
the complementary candidate nucleotide to the polymerase can differ
from that of the non-complementary candidate nucleotide. This time
difference can be used to distinguish between the complementary and
non-complementary nucleotides, and/or can be used to identify the
incorporated nucleotide, and/or can be used to deduce the sequence
of the target molecule.
[0059] The energy transfer signal (or change in energy transfer
signal) generated by the energy transfer donor and/or acceptor can
be detected before, during, and/or after any nucleotide
incorporation event.
[0060] Nucleotide incorporation also includes RNA polymerization
which may not require a primer to initiate nucleotide
polymerization. Nucleotide incorporation events involving RNA
polymerization are well known in the art.
Productive and Non-Productive Binding
[0061] The methods, compositions, systems and kits disclosed herein
can be used for distinguishing between the productive and
non-productive binding events. The compositions and methods can
also provide base identity information during nucleotide
incorporation. The compositions include nucleotides and polymerases
each attached to at least one energy transfer moiety.
[0062] In a productive binding event, the nucleotide can
bind/associate with the polymerase for a time period which is
distinguishable (e.g., longer or shorter time period), compared to
a non-productive binding event. In a non-productive binding event,
the nucleotide can bind/associate with the polymerase and then
dissociate. The donor and acceptor energy transfer moieties produce
detectable energy transfer signals when they are in proximity to
each other and can be associated with productive and non-productive
binding events. Thus, the time-length difference between signals
from the productive and non-productive binding events can provide
distinction between the two types of events. Typically, the length
of time for a productive binding event is longer compared the
length of time for a non-productive event.
[0063] The detectable signals can be classified into true positive
and false positive signals. For example, the true positive signals
can arise from productive binding in which the nucleotide binds the
polymerase and is incorporated. The incorporated nucleotide can be
complementary to the template nucleotide. In another example, the
false positive signals can arise from different binding events,
including: non-specific binding, non-productive binding, and any
event which brings the energy transfer donor and acceptor into
sufficient proximity to induce a detectable signal, but the
nucleotide is not incorporated.
Nucleotide Polymerization Reactions and Methods
[0064] The methods, compositions, systems and kits disclosed herein
can be used for single molecule nucleic acid sequencing, by
generating an energy transfer signal which is associated with
nucleotide incorporation, detecting the generated energy transfer
signal, measuring the generated energy transfer signal,
characterizing the generated energy transfer signal, and
identifying the incorporated nucleotide based on the characterized
energy transfer signal.
[0065] Certain embodiments of the methods, composition, systems,
and kits offer one or more advantages over other single molecule
sequencing methods (see e.g., U.S. Pat. Nos.: Korlach 7,033,764;
7,052,847; 7,056,661; 7,056,676; 7,361,466; and Hardin 7,329,492),
although no individual embodiment necessarily displays all
advantages. The advantages of the energy transfer system and
methods include: (1) energy transfer methods, which require very
small distances (about 5-10 nm) between the polymerase and
nucleotide, to generate the energy transfer signals which are
associated with the close proximity of the polymerase and
nucleotide or are associated with nucleotide polymerization, rather
than signals which are associated with non-productive binding
events; (2) conjugates having a polymerase linked to an energy
transfer moiety (e.g., donor moiety) in which the polymerase is
enzymatically active; (3) polymerases having altered kinetics for
nucleotide binding and/or nucleotide incorporation (e.g., U.S. Ser.
Nos. 61/242,771 and 61/293,618) to improve distinction between
productive and non-productive nucleotide binding events compared to
polymerases traditionally used for nucleotide polymerization
reactions; (4) polymerases having altered kinetics for nucleotide
binding and/or nucleotide incorporation (e.g., U.S. Ser. Nos.
61/242,771 and 61/293,618) used in combination with labeled
nucleotides having six or more phosphate groups (or substituted
phosphate groups), which improve distinction between productive and
non-productive binding events compared to polymerases and
triphosphate nucleotides, which are traditionally used for
nucleotide polymerization reactions; and (5) polymerases having
improved photo-stability when exposed to electromagnetic energy
(e.g., exposed to light during the nucleotide incorporation
reactions) compared to polymerases traditionally used for
nucleotide polymerization reactions.
[0066] The methods can be practiced using suitable conditions which
mediate binding a nucleotide to the polymerase and/or which mediate
nucleotide incorporation. The suitable conditions can include: any
conjugate having a polymerase linked to at least one energy
transfer moiety (e.g., donor moiety) in which the polymerase is
enzymatically active; polymerases and/or nucleotides which improve
distinction between productive and non-productive nucleotide
binding events; and/or polymerases having improved photo-stability
when exposed to electromagnetic energy (e.g., light).
[0067] The methods provided herein are performed under any
conditions which are suitable for: forming the complex
(target/polymerase or target/initiation site/polymerase); binding
the nucleotide to the polymerase; permitting the energy transfer
and reporter moieties to generate detectable energy transfer
signals when the nucleotide binds the polymerase; incorporating the
nucleotide; permitting the energy transfer and reporter moieties to
generate an energy transfer signal upon close proximity and/or upon
nucleotide binding or nucleotide incorporation; detecting the
energy transfer signal, or change in the energy transfer signal,
from the energy transfer or reporter moieties; measuring the energy
transfer signal; and/or translocation of the polymerase to the next
position on the target molecule.
[0068] The suitable conditions include parameters for time,
temperature, pH, reagents, buffers, reagents, salts, co-factors,
nucleotides, target DNA, primer DNA, enzymes such as nucleic
acid-dependent polymerase, amounts and/or ratios of the components
in the reactions, and the like. The reagents or buffers can include
a source of monovalent cations, such as KCl, K-acetate,
NH.sub.4-acetate, K-glutamate, NH.sub.4Cl, or ammonium sulfate. The
reagents or buffers can include a source of divalent cations, such
as Mg.sup.2+ and/or Mn.sup.2+, MgCl.sub.2, or Mg-acetate. The
buffer can include Tris, Tricine, HEPES, MOPS, ACES, or MES, which
can provide a pH range of about 5.0 to about 9.5. The buffer can
include chelating agents such as EDTA and EGTA, and the like. The
suitable conditions can also include compounds which reduce
photo-damage.
Divalent Cations
[0069] The methods, compositions, systems and kits disclosed herein
can include any combination of divalent cations. The divalent
cations can include any cation which permits nucleotide binding
and/or nucleotide incorporation, including for example: manganese,
magnesium, cobalt, strontium, or barium. The divalent cations can
include any cation which promotes the formation and/or stability of
the closed complex (polymerase/target/nucleotide), including
magnesium, manganese, and chromium. The divalent cations can
include any cation which permits nucleotide binding to the
polymerase but inhibits nucleotide incorporation (e.g., calcium).
The divalent cations can include chloride or acetate forms,
including MnCl.sub.2, Mn-acetate, MgCl.sub.2, Mg-acetate, and the
like.
[0070] In practicing the nucleotide incorporation methods, some
polymerases exhibit improved nucleotide binding and/or nucleotide
incorporation kinetics when used with (i) manganese and/or
magnesium, and/or with (ii) tri-, tetra-, penta-, hexa-, or
hepta-phosphate nucleotides. In one embodiment, the disclosed
nucleotide incorporation methods can be practiced using manganese
or magnesium, or a combination of manganese and magnesium. For
example, the methods can include manganese at about 0.1-5 mM, or
about 0.2-4 mM, or about 0.3-3 mM, or about 0.4-2 mM, or about
0.5-2 mM, or about 1-2 mM.
[0071] In another example, the methods can include magnesium at
about 0.01-0.3 mM, or about 0.025-0.2 mM, or about 0.05-0.1 mM, or
about 0.075-0.1 mM, or about 0.1 mM.
[0072] In yet another example, the methods can include a
combination of manganese and magnesium at about 0.25-1 mM of
manganese and 0.025-0.2 mM of magnesium, or about 0.5-0.75 mM of
manganese and 0.05-0.075 mM of magnesium, or about 0.5 mM manganese
and 0.1 mM magnesium.
[0073] In another example, the nucleotide incorporation reaction
include B103 polymerase (SEQ ID NOS:1, 2 or 3) and labeled
hexa-phosphate nucleotides, with about 0.5-2 mM MnCl.sub.2, or with
a combination of about 0.5 mM MnCl.sub.2 and about 0.1 mM
MgCl.sub.2.
Polymerization Initiation Sites
[0074] The methods, compositions, systems and kits disclosed herein
can include a polymerization initiation site. The polymerization
initiation site can be used by the polymerase (e.g., DNA or RNA
polymerase) to initiate nucleotide polymerization. In some
embodiments, the polymerization initiation site can be a terminal
3' OH group. The 3' OH group can serve as a substrate for the
polymerase for nucleotide polymerization. The 3' OH group can serve
as a substrate for the polymerase to form a phosphodiester bond
between the terminal 3' OH group and an incorporated nucleotide.
The 3' OH group can be provided by: the terminal end of a primer
molecule; a nick or gap within a nucleic acid molecule (e.g.,
oligonucleotide) which is base-paired with the target molecule; the
terminal end of a secondary structure (e.g., the end of a
hairpin-like structure); or an origin of replication. The
polymerization initiation site can be provided by an accessory
protein (e.g., RNA polymerase or helicase/primase). The
polymerization initiation site can be provided by a terminal
protein which can be bound (covalently or non-covalently) to the
end of the target nucleic acid, including terminal protein (e.g.,
TP) found in phage (e.g., TP from phi29 phage). Thus, the
polymerization initiation site may be at a terminal end or within a
base-paired nucleic acid molecule. In other embodiments, the
polymerization initiation site used by some polymerases (e.g., RNA
polymerase) may not include a 3'0H group.
[0075] The portion of the target molecule which is base paired with
the primer or with the oligonucleotide, or the self-primed portion
of the target molecule, can form hydrogen bonding by Watson-Crick
or Hoogstein binding to form a duplex nucleic acid structure. The
primer, oligonucleotide, and self-priming sequence may be
complementary, or partially complementary, to the nucleotide
sequence of the target molecule. The complementary base pairing can
be the standard A-T or C-G base pairing, or can be other forms of
base-pairing interactions.
[0076] The polymerization initiation site can be in a position on
the target nucleic acid molecule to permit nucleotide incorporation
events in a direction away from, or towards, the solid surface.
[0077] Some polymerases exhibit a preference for binding
single-stranded nucleic acid molecules. For example, multiple
polymerases may preferentially bind the single-stranded portion of
a target nucleic acid molecule which is base-paired with a primer.
In cases where one target molecule is bound by multiple
polymerases, the efficiency of polymerization initiation can be
poor. Initiating nucleotide polymerization using a gap can improve
the number of target nucleic acid molecules which can undergo
polymerization. In one embodiment, an unexpected procedure for
improving a nucleotide polymerization can include initiating the
polymerization reaction with the terminal 3'OH within a gap, rather
than from a primer (which is base-paired with the target molecule).
In one embodiment, the polymerases which can initiate nucleotide
polymerization from a gap include strand-displacing polymerases.
For example, the strand-displacing polymerase can be a phi29-like
polymerases including: phi29, B103 (SEQ ID NO:1, 2 or 3), and GA-1.
In one embodiment, the gap can be the length of a polynucleotide
molecule which is about 2-15 nucleotides in length, or about 3-14
in length, or about 4-13 in length, or about 5-12 in length, or
about 6-11 in length, or about 7-10 in length. The gap can be
formed by annealing a target nucleic acid molecule to two primer
nucleic acid molecules. Forming a gap is well known in the art.
Primer Molecules
[0078] The methods, compositions, systems and kits disclosed herein
can include a primer molecule which can hybridize with the target
nucleic acid molecule. The sequence of the primer molecule can be
complementary or non-complementary with the sequence of the
sequence of the target molecule. The terminal 3' OH of the primer
molecule can provide the polymerization initiation site.
[0079] The primers can be modified with a chemical moiety to
protect the primer from serving as a polymerization initiation site
or as a restriction enzyme recognition site. The chemical moiety
can be a natural or synthetic amino acid linked through an amide
bond to the primer.
[0080] The primer, oligonucleotide, or self-priming portion, may be
naturally-occurring, or may be produced using enzymatic or chemical
synthesis methods. The primer, oligonucleotide, or self-priming
portion may be any suitable length including 5, 10, 15, 20, 25, 30,
40, 50, 75, or 100 nucleotides or longer in length. The primer,
oligonucleotide, or self-priming portion may be linked to an energy
transfer moiety (e.g., donor or acceptor) or to a reporter moiety
(e.g., a dye) using methods well known in the art.
[0081] The primer molecule, oligonucleotide, and self-priming
portion of the target molecule, may comprise ribonucleotides,
deoxyribonucleotides, ribonucleotides, deoxyribonucleotides,
peptide nucleotides, modified phosphate-sugar backbone nucleotides
including phosphorothioate and phosphoramidate, metallonucleosides,
phosphonate nucleosides, and any variants thereof, or combinations
thereof.
[0082] In one embodiment, the primer molecule can be a recombinant
DNA molecule. The primer can be linked at the 5' or 3' end, or
internally, with at least one binding partner, such as biotin. The
biotin can be used to immobilize the primer molecule to the surface
(via an avidin-like molecule), or for attaching the primer to a
reporter moiety. The primer can be linked to at least one energy
transfer moiety, such as a fluorescent dye or a nanoparticle, or to
a reporter moiety. The primer molecule can hybridize to the target
nucleic acid molecule. The primer molecule can be used as a capture
probe to immobilize the target molecule.
Reducing Photo-Damage
[0083] The methods, compositions, systems and kits disclosed herein
can include compounds which reduce oxygen-damage or photo-damage.
Illuminating the nucleotide binding and/or nucleotide incorporation
reactions with electromagnetic radiation can induce formation of
reactive oxygen species from the fluorophore or other components in
the reaction. The reactive oxygen species can cause photo-damage to
the fluorophores, polymerases, or any other component of the
binding or incorporation reactions. The nucleotide binding or
nucleotide incorporation reactions can include compounds which are
capable of reducing photo-damage, including:
protocatechuate-3,4-dioxygenase, protocatechuic acid;
6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic Acid (TROLOX); or
cyclooctatetraene (COT).
[0084] Other compounds for reducing photo-damage include: ascorbic
acid, astazanthin, bilirubin, biliverdin, bixin, captopril,
canthazanthin, carotene (alpha, beta, and gamma), cysteine,
beta-dimethyl cysteine, N-acetyl cysteine, diazobicyclooctane
(DABCO), dithiothreitol (DTT), ergothioneine, glucose
oxidase/catalase (GO/Cat), glutathione, glutathione peroxidase,
hydrazine (N.sub.2H.sub.4), hydroxylamine, lycopene, lutein,
polyene dialdehydes, melatonin, methionine,
mercaptopropionylglycine, 2-mercaptoethane sulfonate (MESNA),
pyridoxinel and its derivatives, mercaptoethylamine (MEA),
.beta.-mercaptoethanol (BME), n-propyl gallate, p-phenylenediamene
(PPD), hydroquinone, sodium azide (NaN.sub.3), sodium sulfite
(Na.sub.2SO.sub.3), superoxide dismutase, tocopherols,
.alpha.-tocopheryl succinate and its analogs, and zeaxanthin.
Methods for Generating an Energy Transfer Signal: Proximity
[0085] The methods, compositions, systems and kits disclosed herein
can be used for generating a signal which is associated with close
proximity of the polymerase and nucleotide.
[0086] Provided herein are methods for generating an energy
transfer signal comprising the steps of: contacting (i) a
polymerase having altered nucleotide incorporation kinetics and
linked to an energy transfer donor moiety with (ii) a nucleic acid
molecule and with (iii) at least one type of a nucleotide having an
energy transfer acceptor moiety, so as to incorporate the
nucleotide into the nucleic acid molecule thereby locating the
polymerase and nucleotide in close proximity with each other to
generate the energy transfer signal.
[0087] Provided herein are methods for generating an energy
transfer signal comprising the steps of: contacting (i) a
polymerase having altered nucleotide incorporation kinetics and
linked to an energy transfer donor moiety with (ii) a nucleic acid
molecule and with (iii) at least one type of a hexaphosphate
nucleotide having an energy transfer acceptor moiety, so as to
incorporate the hexaphosphate nucleotide into the nucleic acid
molecule thereby locating the polymerase and nucleotide in close
proximity with each other to generate the energy transfer
signal.
[0088] Provided herein are methods for generating an energy
transfer signal comprising the steps of: contacting (i) a
polymerase having altered nucleotide incorporation kinetics and
linked to an energy transfer donor moiety with (ii) a target
nucleic acid molecule which is base-paired with a polymerization
initiation site having a 3' OH group and with (iii) at least one
type of a nucleotide having an energy transfer acceptor moiety, so
as to incorporate the nucleotide onto the 3' OH group thereby
locating the polymerase and nucleotide in close proximity with each
other to generate the energy transfer signal.
[0089] Provided herein are methods for generating an energy
transfer signal comprising the steps of: contacting (i) a
polymerase having altered nucleotide incorporation kinetics and
linked to an energy transfer donor moiety with (ii) a target
nucleic acid molecule which is base-paired with a polymerization
initiation site having a 3' OH group and with (iii) at least one
type of a hexaphosphate nucleotide having an energy transfer
acceptor moiety, so as to incorporate the hexaphosphate nucleotide
onto the 3' OH group thereby locating the polymerase and nucleotide
in close proximity with each other to generate the energy transfer
signal.
[0090] Provided herein are methods for generating an energy
transfer signal comprising the steps of: contacting (i) a
polymerase having an energy transfer donor nanoparticle with (ii) a
nucleic acid molecule and with (iii) at least one type of a
nucleotide having an energy transfer acceptor moiety, so as to
incorporate the nucleotide into the nucleic acid molecule thereby
locating the polymerase and nucleotide in close proximity with each
other to generate the energy transfer signal.
[0091] Provided herein are methods for generating an energy
transfer signal comprising the steps of: contacting (i) a
polymerase having an energy transfer donor nanoparticle with (ii) a
nucleic acid molecule and with (iii) at least one type of a
hexaphosphate nucleotide having an energy transfer acceptor moiety,
so as to incorporate the hexaphosphate nucleotide into the nucleic
acid molecule thereby locating the polymerase and nucleotide in
close proximity with each other to generate the energy transfer
signal.
[0092] Provided herein are methods for generating an energy
transfer signal comprising the steps of: contacting (i) a
polymerase having an energy transfer donor nanoparticle with (ii) a
target nucleic acid molecule which is base-paired with a
polymerization initiation site having a 3' OH group and with (iii)
at least one type of a nucleotide having an energy transfer
acceptor moiety, so as to incorporate the nucleotide onto the 3' OH
group thereby locating the polymerase and nucleotide in close
proximity with each other to generate the energy transfer
signal.
[0093] Provided herein are methods for generating an energy
transfer signal comprising the steps of: contacting (i) a
polymerase having an energy transfer donor nanoparticle with (ii) a
target nucleic acid molecule which is base-paired with a
polymerization initiation site having a 3' OH group and with (iii)
at least one type of a hexaphosphate nucleotide having an energy
transfer acceptor moiety, so as to incorporate the hexaphosphate
nucleotide onto the 3' OH group thereby locating the polymerase and
nucleotide in close proximity with each other to generate the
energy transfer signal.
[0094] Provided herein are methods for generating an energy
transfer signal comprising the steps of: contacting (i) a
polymerase according to any one of SEQ ID NO:1-3 and linked to an
energy transfer donor moiety with (ii) a nucleic acid molecule and
with (iii) at least one type of a hexaphosphate nucleotide linked
to an energy transfer acceptor moiety, so as to incorporate the
hexaphosphate nucleotide into the nucleic acid molecule thereby
locating the polymerase and nucleotide in close proximity with each
other to generate the energy transfer signal.
Detecting the Energy Transfer Signal
[0095] In one embodiment, additional steps can be conducted to
detect the energy transfer signal or the change in the energy
transfer signal. The additional steps comprise: (a) exciting the
energy transfer donor moiety with an excitation source; and (b)
detecting the energy transfer signal or a change in the energy
transfer signal from the energy transfer donor moiety and the
energy transfer acceptor moiety that are in close proximity to each
other.
[0096] In one embodiment, the excitation source can be
electromagnetic energy. In another embodiment, the excitation
source can be a laser. In another embodiment, the energy transfer
signal or the change in the energy transfer signal is a FRET
signal. In yet another embodiment, the energy transfer signal or
the change in the energy transfer signal can be optically
detectable.
Identifying the Incorporated Nucleotide
[0097] In another embodiment, additional steps can be conducted to
identify the energy transfer signal, which can also identify the
incorporated nucleotide. The additional steps comprise: (a)
exciting the energy transfer donor moiety with an excitation
source; (b) detecting the energy transfer signal or a change in the
energy transfer signal from the energy transfer donor moiety and
the energy transfer acceptor moiety that are in close proximity to
each other; and (c) identifying the energy transfer signal or the
change in the energy transfer signal from the energy transfer
accepter moiety.
[0098] In one embodiment, the excitation source can be
electromagnetic energy. In another embodiment, the excitation
source can be a laser. In another embodiment, the energy transfer
signal or the change in the energy transfer signal is a FRET
signal. In yet another embodiment, the energy transfer signal or
the change in the energy transfer signal can be optically
detectable.
Embodiments of Methods for Generating an Energy Transfer Signal:
Proximity
[0099] In methods for generating an energy transfer signal, in one
embodiment, the energy transfer donor and acceptor moieties can
fluoresce in response to exposure to an excitation source, such as
electromagnetic radiation. These fluorescence responses can be an
energy transfer signal. In another embodiment, the energy transfer
acceptor moiety can fluoresce in response to energy transferred
from a proximal excited energy transfer donor moiety. These
fluorescence responses can be an energy transfer signal. The
proximal distance between the donor and acceptor moieties that
accommodates energy transfer can be dependent upon the particular
donor/acceptor pair. The proximal distance between the donor and
acceptor moieties can be about 1-20 nm, or about 1-10 nm, or about
1-5 nm, or about 5-10 nm. In another embodiment, the energy
transfer signal generated by proximity of the donor moiety to the
acceptor moiety can remain unchanged. In another embodiment, the
energy transfer signal generated by proximity of the donor moiety
to the acceptor moiety results in changes in the energy transfer
signal. In another embodiment, the changes in the energy transfer
signals from the donor or acceptor moiety can include changes in
the: intensity of the signal; duration of the signal; wavelength of
the signal; amplitude of the signal; polarization state of the
signal; duration between the signals; and/or rate of the change in
intensity, duration, wavelength or amplitude. In another
embodiment, the change in the energy transfer signal can include a
change in the ratio of the change of the energy transfer donor
signal relative to change of the energy transfer acceptor signals.
In another embodiment, the energy transfer signal from the donor
can increase or decrease. In another embodiment, the energy
transfer signal from the acceptor can increase or decrease. In
another embodiment, the energy transfer signal associated with
nucleotide incorporation includes: a decrease in the donor signal
when the donor is proximal to the acceptor; an increase in the
acceptor signal when the acceptor is proximal to the donor; an
increase in the donor signal when the distance between the donor
and acceptor increases; and/or a decrease in the acceptor signal
when the distance between the donor and acceptor increases.
[0100] In one embodiment, the detecting the energy transfer signal
can be performed using confocal laser scanning microscopy, Total
Internal Reflection (TIR), Total Internal Reflection Fluorescence
(TIRF), near-field scanning microscopy, far-field confocal
microscopy, wide-field epi-illumination, light scattering, dark
field microscopy, photoconversion, wide field fluorescence, single
and/or multi-photon excitation, spectral wavelength discrimination,
evanescent wave illumination, scanning two-photon, scanning wide
field two-photon, Nipkow spinning disc, and/or multi-foci
multi-photon.
[0101] In practicing the nucleotide binding and/or nucleotide
incorporation methods, non-desirable fluorescent signals can come
from sources including background and/or noise. In one embodiment,
the energy transfer signals can be distinguished from the
non-desirable fluorescent signals by measuring, analyzing and
characterizing attributes of all fluorescent signals generated by
the nucleotide incorporation reaction. In one embodiment,
attributes of the energy transfer signal that can permit
distinction from the non-desirable fluorescent signals can include:
duration; wavelength; amplitude; photon count; and/or the rate of
change of the duration, wavelength, amplitude; and/or photon count.
In one embodiment, the identifying the energy transfer signal,
includes measuring, analyzing and characterizing attributes of:
duration; wavelength; amplitude; photon count and/or the rate of
change of the duration, wavelength, amplitude; and/or photon count.
In one embodiment, identifying the energy transfer signal can be
used to identify the incorporated nucleotide.
[0102] In one embodiment, the nucleic acid molecule can be DNA, RNA
or DNA/RNA.
[0103] In one embodiment, the polymerase has an active site. The
nucleotide can bind the active site. In another embodiment, the
polymerase can be a DNA-dependent or RNA-dependent polymerase, or a
reverse transcriptase. In another embodiment, the polymerase having
altered nucleotide binding and/or nucleotide incorporation kinetics
can improve distinction between productive and non-productive
binding events. In another embodiment, the altered nucleotide
binding kinetics and/or altered nucleotide incorporation kinetics
can include altered kinetics for: polymerase binding to the target
molecule; polymerase binding to the nucleotide; polymerase
catalyzing nucleotide incorporation; the polymerase cleaving the
nucleotide and forming a cleavage product; and/or the polymerase
releasing the cleavage product. In another embodiment, the
polymerase can be linked to an energy transfer donor moiety to form
a conjugate. In another embodiment, the polymerase component of the
conjugate can be enzymatically active. In another embodiment, the
polymerase has altered kinetics for nucleotide binding and/or
nucleotide incorporation used in combination with labeled
nucleotides having six or more phosphate groups (or substituted
phosphate groups), which improve distinction between productive and
non-productive binding events. In another embodiment, the
polymerase can have improved photo-stability. The polymerase can be
a Phi29-like polymerase, including Phi29 or B103 polymerase. The
polymerase can be a mutant polymerase. The polymerase can be a B103
polymerase according to any one of SEQ ID NOS:1-5.
[0104] In one embodiment, the energy transfer donor moiety can be a
nanoparticle or a fluorescent dye. The nanoparticle can be about
1-20 nm in its largest dimensions. The nanoparticle can be a
core/shell nanoparticle. The nanoparticle can include a core
comprising semiconductor material(s). The core can include
materials (including binary, ternary and quaternary mixtures
thereof), from: Groups II-VI of the periodic table, including ZnS,
ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, HgTe, MgTe; Groups III-V,
including GaN, GaP, GaAs, GaSb, InN, InP, InAs, InSb, AlAs, AlP,
AlSb, AlS; and/or Group IV, including Ge, Si, Pb. The nanoparticle
can include at least one shell surrounding the core. The shell can
include semiconductor material(s). The nanoparticle can include an
inner shell and an outer shell. The shell can include materials
(including binary, ternary and quaternary mixtures thereof)
comprising: ZnO, ZnS, ZnSe, ZnTe, CdO, CdS, CdSe, CdTe, MgS, MgSe,
GaAs, GaN, GaP, GaAs, GaSb, HgO, HgS, HgSe, HgTe, InAs, InN, InP,
InSb, AlAs, AlN, AlP, or AlSb. In one embodiment, the nanoparticle
comprises a core having CdSe. In another embodiment, the
nanoparticle comprises an inner shell having CdS. In another
embodiment, the nanoparticle comprises an outer shell having ZnS.
The outermost surface of the core or shell can be coated with
tightly associated ligands which are not removed by ordinary
solvation. In some embodiments, the nanoparticle can have a layer
of ligands on its surface which can further be cross-linked to each
other. In some embodiments, the nanoparticle can have other or
additional surface coatings which can modify the properties of the
particle, for example, increasing or decreasing solubility in water
or other solvents. The nanoparticle can be water dispersible. The
nanoparticle can be a non-blinking nanoparticle. The nanoparticle
can be photo-stable. The nanoparticle may not interfere with
polymerase activity, including polymerase binding to the target
molecule, polymerase binding to the nucleotide, polymerase
catalyzing nucleotide incorporation, or the polymerase cleaving the
nucleotide and/or releasing the cleavage product.
[0105] In one embodiment, the target nucleic acid molecule can be
DNA or RNA or DNA/RNA molecule. In another embodiment, the target
nucleic acid molecule is a single nucleic acid molecule. In another
embodiment, the target nucleic acid molecule (e.g., target
molecule) is base-paired with a polymerization initiation site. In
another embodiment, the polymerization initiation site is a
terminal 3'OH of a primer molecule or of a self-primed target
molecule. In another embodiment, the polymerization initiation site
is a 3'OH within a gap or nick. In another embodiment, the target
nucleic acid molecule and/or the polymerization initiation site is
immobilized to a solid surface. In another embodiment, the target
nucleic acid molecule is a linear or circular nucleic acid
molecule.
[0106] In one embodiment, the at least one type of nucleotide can
include 3-10 phosphate groups or substituted phosphate groups, or a
combination of phosphate groups and substituted phosphate groups.
The nucleotide can include a terminal phosphate group or terminal
substituted phosphate group which can be linked to the energy
transfer acceptor moiety. The nucleotide can include the energy
transfer acceptor moiety which is linked the base, sugar, or any
phosphate group or substituted phosphate group. The nucleotide can
be adenosine, guanosine, cytosine, thymidine, uridine, or any other
type of nucleotide.
[0107] In one embodiment, the energy transfer acceptor moiety can
be a fluorescent dye. The energy transfer acceptor moiety and the
energy transfer donor moiety can be capable of energy transfer.
[0108] In one embodiment, more than one type of nucleotide can be
contacted with the polymerase. Each of the different types of
nucleotides can be linked to the same or to different types of
energy transfer acceptor moieties, or any combination of the same
or different types of acceptor moieties.
Methods for Generating an Energy Transfer Signal: Nucleotide
Incorporation
[0109] Provided herein are methods for generating an energy
transfer signal comprising the steps of: contacting (i) a
polymerase having altered nucleotide incorporation kinetics and
linked to an energy transfer donor moiety with (ii) a nucleic acid
molecule and with (iii) at least one type of a nucleotide having an
energy transfer acceptor moiety, so as to incorporate the
nucleotide into the nucleic acid molecule thereby generating the
energy transfer signal.
[0110] Provided herein are methods for generating an energy
transfer signal comprising the steps of: contacting (i) a
polymerase having altered nucleotide incorporation kinetics and
linked to an energy transfer donor moiety with (ii) a nucleic acid
molecule and with (iii) at least one type of a hexaphosphate
nucleotide having an energy transfer acceptor moiety, so as to
incorporate the hexaphosphate nucleotide into the nucleic acid
molecule thereby generating the energy transfer signal.
[0111] Provided herein are methods for generating an energy
transfer signal comprising the steps of: contacting (i) a
polymerase having altered nucleotide incorporation kinetics and
linked to an energy transfer donor moiety with (ii) a target
nucleic acid molecule which is base-paired with a polymerization
initiation site having a 3' OH group and with (iii) at least one
type of a nucleotide having an energy transfer acceptor moiety, so
as to incorporate the nucleotide onto the 3' OH group thereby
generating the energy transfer signal.
[0112] Provided herein are methods for generating an energy
transfer signal comprising the steps of: contacting (i) a
polymerase having altered nucleotide incorporation kinetics and
linked to an energy transfer donor moiety with (ii) a target
nucleic acid molecule which is base-paired with a polymerization
initiation site having a 3' OH group and with (iii) at least one
type of a hexaphosphate nucleotide having an energy transfer
acceptor moiety, so as to incorporate the hexaphosphate nucleotide
onto the 3' OH group thereby generating the energy transfer
signal.
[0113] Provided herein are methods for generating an energy
transfer signal comprising the steps of: contacting (i) a
polymerase having an energy transfer donor nanoparticle with (ii) a
nucleic acid molecule and with (iii) at least one type of a
nucleotide having an energy transfer acceptor moiety, so as to
incorporate the nucleotide into the nucleic acid molecule thereby
generating the energy transfer signal.
[0114] Provided herein are methods for generating an energy
transfer signal comprising the steps of: contacting (i) a
polymerase having an energy transfer donor nanoparticle with (ii) a
nucleic acid molecule and with (iii) at least one type of a
hexaphosphate nucleotide having an energy transfer acceptor moiety,
so as to incorporate the hexaphosphate nucleotide into the nucleic
acid molecule thereby generating the energy transfer signal.
[0115] Provided herein are methods for generating an energy
transfer signal comprising the steps of: contacting (i) a
polymerase having an energy transfer donor nanoparticle with (ii) a
target nucleic acid molecule which is base-paired with a
polymerization initiation site having a 3' OH group and with (iii)
at least one type of a nucleotide having an energy transfer
acceptor moiety, so as to incorporate the nucleotide onto the 3' OH
group thereby generating the energy transfer signal.
[0116] Provided herein are methods for generating an energy
transfer signal comprising the steps of: contacting (i) a
polymerase having an energy transfer donor nanoparticle with (ii) a
target nucleic acid molecule which is base-paired with a
polymerization initiation site having a 3' OH group and with (iii)
at least one type of a hexaphosphate nucleotide having an energy
transfer acceptor moiety, so as to incorporate the hexaphosphate
nucleotide onto the 3' OH group thereby generating the energy
transfer signal.
[0117] Provided herein are methods for generating an energy
transfer signal comprising the steps of: contacting (i) a
polymerase according to any one of SEQ ID NO:1-3 and linked to an
energy transfer donor moiety with (ii) a nucleic acid molecule and
with (iii) at least one type of a hexaphosphate nucleotide linked
to an energy transfer acceptor moiety, so as to incorporate the
hexaphosphate nucleotide into the nucleic acid molecule thereby
generating the energy transfer signal.
Detecting the Energy Transfer Signal
[0118] In one embodiment, additional steps can be conducted to
detect the energy transfer signal or the change in the energy
transfer signal. The additional steps comprise: (a) exciting the
energy transfer donor moiety with an excitation source; and (b)
detecting the energy transfer signal or a change in the energy
transfer signal from the energy transfer donor moiety and the
energy transfer acceptor moiety that are in close proximity to each
other.
[0119] In one embodiment, the excitation source can be
electromagnetic energy. In another embodiment, the excitation
source can be a laser. In another embodiment, the energy transfer
signal or the change in the energy transfer signal is a FRET
signal. In yet another embodiment, the energy transfer signal or
the change in the energy transfer signal can be optically
detectable.
Identifying the Incorporated Nucleotide
[0120] In another embodiment, additional steps can be conducted to
identify the incorporated nucleotide. The additional steps
comprise: (a) exciting the energy transfer donor moiety with an
excitation source; (b) detecting the energy transfer signal or a
change in the energy transfer signal from the energy transfer donor
moiety and the energy transfer acceptor moiety that are in close
proximity to each other; and (c) identifying the energy transfer
signal or the change in the energy transfer signal from the energy
transfer accepter moiety.
[0121] In one embodiment, the excitation source can be
electromagnetic energy. In another embodiment, the excitation
source can be a laser. In another embodiment, the energy transfer
signal or the change in the energy transfer signal is a FRET
signal. In yet another embodiment, the energy transfer signal or
the change in the energy transfer signal can be optically
detectable.
Embodiments of Methods for Generating an Energy Transfer
Signal:
Nucleotide Incorporation:
[0122] In methods for generating an energy transfer signal, in one
embodiment, the energy transfer donor and acceptor moieties can
fluoresce in response to exposure to an excitation source, such as
electromagnetic radiation. These fluorescence responses can be an
energy transfer signal. In another embodiment, the energy transfer
acceptor moiety can fluoresce in response to energy transferred
from a proximal excited energy transfer donor moiety. These
fluorescence responses can be an energy transfer signal. The
proximal distance between the donor and acceptor moieties that
accommodates energy transfer can be dependent upon the particular
donor/acceptor pair. The proximal distance between the donor and
acceptor moieties can be about 1-20 nm, or about 1-10 nm, or about
1-5 nm, or about 5-10 nm. In another embodiment, the energy
transfer signal generated by proximity of the donor moiety to the
acceptor moiety can remain unchanged. In another embodiment, the
energy transfer signal generated by proximity of the donor moiety
to the acceptor moiety results in changes in the energy transfer
signal. In another embodiment, the changes in the energy transfer
signals from the donor or acceptor moiety can include changes in
the: intensity of the signal; duration of the signal; wavelength of
the signal; amplitude of the signal; polarization state of the
signal; duration between the signals; and/or rate of the change in
intensity, duration, wavelength or amplitude. In another
embodiment, the change in the energy transfer signal can include a
change in the ratio of the change of the energy transfer donor
signal relative to change of the energy transfer acceptor signals.
In another embodiment, the energy transfer signal from the donor
can increase or decrease. In another embodiment, the energy
transfer signal from the acceptor can increase or decrease. In
another embodiment, the energy transfer signal associated with
nucleotide incorporation includes: a decrease in the donor signal
when the donor is proximal to the acceptor; an increase in the
acceptor signal when the acceptor is proximal to the donor; an
increase in the donor signal when the distance between the donor
and acceptor increases; and/or a decrease in the acceptor signal
when the distance between the donor and acceptor increases.
[0123] In one embodiment, the detecting the energy transfer signal
can be performed using confocal laser scanning microscopy, Total
Internal Reflection (TIR), Total Internal Reflection Fluorescence
(TIRF), near-field scanning microscopy, far-field confocal
microscopy, wide-field epi-illumination, light scattering, dark
field microscopy, photoconversion, wide field fluorescence, single
and/or multi-photon excitation, spectral wavelength discrimination,
evanescent wave illumination, scanning two-photon, scanning wide
field two-photon, Nipkow spinning disc, and/or multi-foci
multi-photon.
[0124] In practicing the nucleotide binding and/or nucleotide
incorporation methods, non-desirable fluorescent signals can come
from sources including background and/or noise. In one embodiment,
the energy transfer signals can be distinguished from the
non-desirable fluorescent signals by measuring, analyzing and
characterizing attributes of all fluorescent signals generated by
the nucleotide incorporation reaction. In one embodiment,
attributes of the energy transfer signal that can permit
distinction from the non-desirable fluorescent signals can include:
duration; wavelength; amplitude; photon count; and/or the rate of
change of the duration, wavelength, amplitude; and/or photon count.
In one embodiment, the identifying the energy transfer signal,
includes measuring, analyzing and characterizing attributes of:
duration; wavelength; amplitude; photon count and/or the rate of
change of the duration, wavelength, amplitude; and/or photon count.
In one embodiment, identifying the energy transfer signal can be
used to identify the incorporated nucleotide.
[0125] In one embodiment, the nucleic acid molecule can be DNA, RNA
or DNA/RNA.
[0126] In one embodiment, the polymerase has an active site. The
nucleotide can bind the active site. In another embodiment, the
polymerase can be a DNA-dependent or RNA-dependent polymerase, or a
reverse transcriptase. In another embodiment, the polymerase having
altered nucleotide binding and/or nucleotide incorporation kinetics
can improve distinction between productive and non-productive
binding events. In another embodiment, the altered nucleotide
binding kinetics and/or altered nucleotide incorporation kinetics
can include altered kinetics for: polymerase binding to the target
molecule; polymerase binding to the nucleotide; polymerase
catalyzing nucleotide incorporation; the polymerase cleaving the
nucleotide and forming a cleavage product; and/or the polymerase
releasing the cleavage product. In another embodiment, the
polymerase can be linked to an energy transfer donor moiety to form
a conjugate. In another embodiment, the polymerase component of the
conjugate can be enzymatically active. In another embodiment, the
polymerase has altered kinetics for nucleotide binding and/or
nucleotide incorporation used in combination with labeled
nucleotides having six or more phosphate groups (or substituted
phosphate groups), which improve distinction between productive and
non-productive binding events. In another embodiment, the
polymerase can have improved photo-stability. The polymerase can be
a Phi29-like polymerase, including Phi29 or B103 polymerase. The
polymerase can be a mutant polymerase. The polymerase can be a B103
polymerase according to any one of SEQ ID NOS:1-5.
[0127] In one embodiment, the energy transfer donor moiety can be a
nanoparticle or a fluorescent dye. The nanoparticle can be about
1-20 nm in its largest dimensions. The nanoparticle can be a
core/shell nanoparticle. The nanoparticle can include a core
comprising semiconductor material(s). The core can include
materials (including binary, ternary and quaternary mixtures
thereof), from: Groups II-VI of the periodic table, including ZnS,
ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, HgTe, MgTe; Groups III-V,
including GaN, GaP, GaAs, GaSb, InN, InP, InAs, InSb, AlAs, AlP,
AlSb, AlS; and/or Group IV, including Ge, Si, Pb. The nanoparticle
can include at least one shell surrounding the core. The shell can
include semiconductor material(s). The nanoparticle can include an
inner shell and an outer shell. The shell can include materials
(including binary, ternary and quaternary mixtures thereof)
comprising: ZnO, ZnS, ZnSe, ZnTe, CdO, CdS, CdSe, CdTe, MgS, MgSe,
GaAs, GaN, GaP, GaAs, GaSb, HgO, HgS, HgSe, HgTe, InAs, InN, InP,
InSb, AlAs, AlN, AlP, or AlSb. In one embodiment, the nanoparticle
comprises a core having CdSe. In another embodiment, the
nanoparticle comprises an inner shell having CdS. In another
embodiment, the nanoparticle comprises an outer shell having ZnS.
The outermost surface of the core or shell can be coated with
tightly associated ligands which are not removed by ordinary
solvation. In some embodiments, the nanoparticle can have a layer
of ligands on its surface which can further be cross-linked to each
other. In some embodiments, the nanoparticle can have other or
additional surface coatings which can modify the properties of the
particle, for example, increasing or decreasing solubility in water
or other solvents. The nanoparticle can be water dispersible. The
nanoparticle can be a non-blinking nanoparticle. The nanoparticle
can be photo-stable. The nanoparticle may not interfere with
polymerase activity, including polymerase binding to the target
molecule, polymerase binding to the nucleotide, polymerase
catalyzing nucleotide incorporation, or the polymerase cleaving the
nucleotide and/or releasing the cleavage product.
[0128] In one embodiment, the target nucleic acid molecule can be
DNA or RNA or DNA/RNA molecule. In another embodiment, the target
nucleic acid molecule is a single nucleic acid molecule. In another
embodiment, the target nucleic acid molecule (e.g., target
molecule) is base-paired with a polymerization initiation site. In
another embodiment, the polymerization initiation site is a
terminal 3'OH of a primer molecule or of a self-primed target
molecule. In another embodiment, the polymerization initiation site
is a 3'OH within a gap or nick. In another embodiment, the target
nucleic acid molecule and/or the polymerization initiation site is
immobilized to a solid surface. In another embodiment, the target
nucleic acid molecule is a linear or circular nucleic acid
molecule.
[0129] In one embodiment, the at least one type of nucleotide can
include 3-10 phosphate groups or substituted phosphate groups, or a
combination of phosphate groups and substituted phosphate groups.
The nucleotide can include a terminal phosphate group or terminal
substituted phosphate group which can be linked to the energy
transfer acceptor moiety. The nucleotide can include the energy
transfer acceptor moiety which is linked the base, sugar, or any
phosphate group or substituted phosphate group. The nucleotide can
be adenosine, guanosine, cytosine, thymidine, uridine, or any other
type of nucleotide.
[0130] In one embodiment, the energy transfer acceptor moiety can
be a fluorescent dye. The energy transfer acceptor moiety and the
energy transfer donor moiety can be capable of energy transfer.
[0131] In one embodiment, more than one type of nucleotide can be
contacted with the polymerase. Each of the different types of
nucleotides can be linked to the same or to different types of
energy transfer acceptor moieties, or any combination of the same
or different types of acceptor moieties.
Methods for Incorporating Nucleotides
[0132] The methods, compositions, systems and kits disclosed herein
can be used for incorporating nucleotides. Provided herein are
methods for incorporating a nucleotide, comprising: contacting (i)
a polymerase having altered nucleotide incorporation kinetics and
linked to an energy transfer donor moiety with (ii) a nucleic acid
molecule and with (iii) at least one type of a nucleotide having an
energy transfer acceptor moiety, so as to incorporate the
nucleotide into the nucleic acid molecule. In one embodiment, the
nucleic acid molecule includes a polymerization initiation site
having a 3' OH group.
[0133] Provided herein are methods for incorporating a nucleotide
comprising the steps of: contacting (i) a polymerase having altered
nucleotide incorporation kinetics and linked to an energy transfer
donor moiety with (ii) a nucleic acid molecule and with (iii) at
least one type of a nucleotide having an energy transfer acceptor
moiety, so as to incorporate the nucleotide into the nucleic acid
molecule.
[0134] Provided herein are methods for incorporating a nucleotide
comprising the steps of: contacting (i) a polymerase having altered
nucleotide incorporation kinetics and linked to an energy transfer
donor moiety with (ii) a nucleic acid molecule and with (iii) at
least one type of a hexaphosphate nucleotide having an energy
transfer acceptor moiety, so as to incorporate the hexaphosphate
nucleotide into the nucleic acid molecule.
[0135] Provided herein are methods for incorporating a nucleotide
comprising the steps of: contacting (i) a polymerase having altered
nucleotide incorporation kinetics and linked to an energy transfer
donor moiety with (ii) a target nucleic acid molecule which is
base-paired with a polymerization initiation site having a 3' OH
group and with (iii) at least one type of a nucleotide having an
energy transfer acceptor moiety, so as to incorporate the
nucleotide onto the 3'0H group.
[0136] Provided herein are methods for incorporating a nucleotide
comprising the steps of: contacting (i) a polymerase having altered
nucleotide incorporation kinetics and linked to an energy transfer
donor moiety with (ii) a target nucleic acid molecule which is
base-paired with a polymerization initiation site having a 3' OH
group and with (iii) at least one type of a hexaphosphate
nucleotide having an energy transfer acceptor moiety, so as to
incorporate the hexaphosphate nucleotide onto the 3'OH group.
[0137] Provided herein are methods for incorporating a nucleotide,
comprising the steps of: contacting (i) a polymerase including an
energy transfer donor nanoparticle with (ii) a nucleic acid
molecule and with (iii) at least one type of a nucleotide having an
energy transfer acceptor moiety, so as to incorporate the
nucleotide into the nucleic acid molecule.
[0138] Provided herein are methods for incorporating a nucleotide
comprising the steps of: contacting (i) a polymerase including an
energy transfer donor nanoparticle with (ii) a nucleic acid
molecule and with (iii) at least one type of a hexaphosphate
phosphate nucleotide having an energy transfer acceptor moiety, so
as to incorporate the hexaphosphate nucleotide into the nucleic
acid molecule.
[0139] Provided herein are methods for incorporating a nucleotide
comprising the steps of: contacting (i) a polymerase including an
energy transfer donor nanoparticle with (ii) a target nucleic acid
molecule which is base-paired with a polymerization initiation site
having a 3' OH group and with (iii) at least one type of a
nucleotide having an energy transfer acceptor moiety, so as to
incorporate the nucleotide onto the 3'0H group.
[0140] Provided herein are methods for incorporating a nucleotide
comprising the steps of: contacting (i) a polymerase including an
energy transfer donor nanoparticle with (ii) a target nucleic acid
molecule which is base-paired with a polymerization initiation site
having a 3' OH group and with (iii) at least one type of a
hexaphosphate nucleotide having an energy transfer acceptor moiety,
so as to incorporate the hexaphosphate nucleotide onto the 3'OH
group.
[0141] Provided herein are methods for conducting a plurality of
nucleotide incorporation reactions (e.g., arrays), each nucleotide
incorporation reaction comprises the steps of: contacting (i) a
polymerase having altered nucleotide incorporation kinetics and
linked to an energy transfer donor moiety with (ii) a nucleic acid
molecule and with (iii) at least one type of a nucleotide having an
energy transfer acceptor moiety, so as to incorporate the
nucleotide into the nucleic acid molecule.
[0142] Provided herein are methods for conducting a plurality of
nucleotide incorporation reactions (e.g., arrays), each nucleotide
incorporation reaction comprises the steps of: contacting (i) a
polymerase including an energy transfer donor nanoparticle with
(ii) a nucleic acid molecule and with (iii) at least one type of a
nucleotide having an energy transfer acceptor moiety, so as to
incorporate the nucleotide into the nucleic acid molecule.
[0143] Provided herein are methods for successively incorporating
nucleotides comprising the steps of: contacting (i) a polymerase
having altered nucleotide incorporation kinetics and linked to an
energy transfer donor moiety with (ii) a nucleic acid molecule and
with (iii) a plurality of more than one type of a nucleotide each
type of nucleotide having a different type of energy transfer
acceptor moiety, so as to successively incorporate the nucleotides
into the nucleic acid molecule.
[0144] Provided herein are methods for successively incorporating
nucleotides comprising the steps of: contacting (i) a polymerase
including an energy transfer donor nanoparticle with (ii) a nucleic
acid molecule and with (iii) a plurality of more than one type of a
nucleotide each type of nucleotide having a different type of
energy transfer acceptor moiety, so as to successively incorporate
the nucleotides into the nucleic acid molecule.
[0145] Provided herein are methods for successively incorporating
nucleotides, comprising the steps of: contacting (i) a mutant
polymerase having altered nucleotide incorporation kinetics and
linked to an energy transfer donor moiety with (ii) a target DNA
molecule which is base-paired with a polymerization initiation site
having a 3' OH group and with (iii) a plurality of more than one
type of a hexaphosphate nucleotide each type of hexaphosphate
nucleotide having a different type of fluorescent dye acceptor, so
as to successively incorporate the hexaphosphate nucleotides onto
the 3' OH group.
[0146] Provided herein are methods for incorporating a nucleotide
comprising the steps of: contacting (i) a polymerase according to
any one of SEQ ID NO:1-3 and linked to an energy transfer donor
moiety with (ii) a nucleic acid molecule (iii) at least one type of
a hexaphosphate nucleotide linked to an energy transfer acceptor
moiety, so as to incorporate the hexaphosphate nucleotide into the
nucleic acid molecule.
[0147] Provided herein are methods for incorporating a nucleotide
comprising the steps of: contacting (i) a polymerase according to
any one of SEQ ID NO:1-3 and linked to an energy transfer donor
moiety with (ii) a nucleic acid molecule which is base-paired with
a polymerization initiation site having a terminal 3' OH group and
with (iii) at least one type of a hexaphosphate nucleotide linked
to an energy transfer acceptor moiety, so as to incorporate the
hexaphosphate nucleotide onto the 3'OH group.
[0148] Provided herein are methods for successively incorporating
nucleotides comprising the steps of: contacting (i) a polymerase
according to any one of SEQ ID NO:1-3 and linked to an energy
transfer donor moiety with (ii) a nucleic acid molecule and with
(iii) a plurality of more than one type of a hexaphosphate
nucleotide each type of hexaphosphate nucleotide linked to a
different type of energy transfer acceptor moiety, so as to
successively incorporate the hexaphosphate nucleotides into the
nucleic acid molecule.
[0149] Provided herein are methods for conducting a plurality of
nucleotide incorporation reactions, each nucleotide incorporation
reaction comprises the steps of: contacting (i) a polymerase
according to any one of SEQ ID NO:1-3 and linked to an energy
transfer donor moiety with (ii) a nucleic acid molecule and with
(iii) at least one type of a hexaphosphate nucleotide linked to an
energy transfer acceptor moiety, so as to incorporate the
hexaphosphate nucleotide into the nucleic acid molecule.
[0150] Provided herein are methods for successively incorporating
nucleotides comprising the steps of: contacting (i) a polymerase
according to any one of SEQ ID NO:1-3 and linked to an energy
transfer donor nanoparticle with (ii) a nucleic acid molecule and
with (iii) a plurality of more than one type of a hexaphosphate
nucleotide each type of hexaphosphate nucleotide linked to a
different type of energy transfer acceptor moiety, so as to
successively incorporate the hexaphosphate nucleotides into the
nucleic acid molecule.
[0151] Provided herein are methods for conducting a plurality of
nucleotide incorporation reactions, each nucleotide incorporation
reaction comprises the steps of: contacting (i) a polymerase
according to any one of SEQ ID NO:1-3 and linked to an energy
transfer donor nanoparticle with (ii) a nucleic acid molecule and
with (iii) at least one type of a hexaphosphate nucleotide linked
to an energy transfer acceptor moiety, so as to incorporate the
hexaphosphate nucleotide into the nucleic acid molecule.
Detecting Nucleotide Incorporation
[0152] In one embodiment, additional steps can be conducted to
detect nucleotide incorporation. The additional steps comprise: (a)
exciting the energy transfer donor moiety with an excitation
source; and (b) detecting the energy transfer signal or a change in
the energy transfer signal from the incorporated nucleotide whereby
the energy transfer donor moiety and the energy transfer acceptor
moiety are located in close proximity to each other.
[0153] In one embodiment, the excitation source can be
electromagnetic energy. In another embodiment, the excitation
source can be light. In another embodiment, the energy transfer
signal or the change in the energy transfer signal can be a FRET
signal. In yet another embodiment, the energy transfer signal or
the change in the energy transfer signal can be spectrally or
optically detectable.
Identifying the Incorporated Nucleotide
[0154] In another embodiment, additional steps can be conducted to
identify the incorporated nucleotide. The additional steps
comprise: (a) exciting the energy transfer donor moiety with an
excitation source; (b) detecting the energy transfer signal or a
change in the energy transfer signal from the incorporated
nucleotide whereby the energy transfer donor moiety and the energy
transfer acceptor moiety are located in close proximity to each
other; and (c) identifying the energy transfer signal or the change
in the energy transfer signal from the incorporated nucleotide.
[0155] In one embodiment, the excitation source can be
electromagnetic energy. In another embodiment, the excitation
source can be light. In another embodiment, the energy transfer
signal or the change in the energy transfer signal can be a FRET
signal. In yet another embodiment, the energy transfer signal or
the change in the energy transfer signal can be spectrally or
optically detectable.
Embodiments of Methods for Incorporating Nucleotides
[0156] In methods for generating an energy transfer signal, in one
embodiment, the energy transfer donor and acceptor moieties can
fluoresce in response to exposure to an excitation source, such as
electromagnetic radiation. These fluorescence responses can be an
energy transfer signal. In another embodiment, the energy transfer
acceptor moiety can fluoresce in response to energy transferred
from a proximal excited energy transfer donor moiety. These
fluorescence responses can be an energy transfer signal. The
proximal distance between the donor and acceptor moieties that
accommodates energy transfer can be dependent upon the particular
donor/acceptor pair. The proximal distance between the donor and
acceptor moieties can be about 1-20 nm, or about 1-10 nm, or about
1-5 nm, or about 5-10 nm. In another embodiment, the energy
transfer signal generated by proximity of the donor moiety to the
acceptor moiety can remain unchanged. In another embodiment, the
energy transfer signal generated by proximity of the donor moiety
to the acceptor moiety results in changes in the energy transfer
signal. In another embodiment, the changes in the energy transfer
signals from the donor or acceptor moiety can include changes in
the: intensity of the signal; duration of the signal; wavelength of
the signal; amplitude of the signal; polarization state of the
signal; duration between the signals; and/or rate of the change in
intensity, duration, wavelength or amplitude. In another
embodiment, the change in the energy transfer signal can include a
change in the ratio of the change of the energy transfer donor
signal relative to change of the energy transfer acceptor signals.
In another embodiment, the energy transfer signal from the donor
can increase or decrease. In another embodiment, the energy
transfer signal from the acceptor can increase or decrease. In
another embodiment, the energy transfer signal associated with
nucleotide incorporation includes: a decrease in the donor signal
when the donor is proximal to the acceptor; an increase in the
acceptor signal when the acceptor is proximal to the donor; an
increase in the donor signal when the distance between the donor
and acceptor increases; and/or a decrease in the acceptor signal
when the distance between the donor and acceptor increases.
[0157] In one embodiment, the detecting the energy transfer signal
can be performed using confocal laser scanning microscopy, Total
Internal Reflection (TIR), Total Internal Reflection Fluorescence
(TIRF), near-field scanning microscopy, far-field confocal
microscopy, wide-field epi-illumination, light scattering, dark
field microscopy, photoconversion, wide field fluorescence, single
and/or multi-photon excitation, spectral wavelength discrimination,
evanescent wave illumination, scanning two-photon, scanning wide
field two-photon, Nipkow spinning disc, and/or multi-foci
multi-photon.
[0158] In practicing the nucleotide binding and/or nucleotide
incorporation methods, non-desirable fluorescent signals can come
from sources including background and/or noise. In one embodiment,
the energy transfer signals can be distinguished from the
non-desirable fluorescent signals by measuring, analyzing and
characterizing attributes of all fluorescent signals generated by
the nucleotide incorporation reaction.
[0159] In one embodiment, attributes of the energy transfer signal
that can permit distinction from the non-desirable fluorescent
signals can include: duration; wavelength; amplitude; photon count;
and/or the rate of change of the duration, wavelength, amplitude;
and/or photon count. In one embodiment, the identifying the energy
transfer signal, includes measuring, analyzing and characterizing
attributes of: duration; wavelength; amplitude; photon count and/or
the rate of change of the duration, wavelength, amplitude; and/or
photon count. In one embodiment, identifying the energy transfer
signal can be used to identify the incorporated nucleotide.
[0160] In one embodiment, the nucleic acid molecule can be DNA, RNA
or DNA/RNA.
[0161] In one embodiment, the polymerase has an active site. The
nucleotide can bind the active site. In another embodiment, the
polymerase can be a DNA-dependent or RNA-dependent polymerase, or a
reverse transcriptase. In another embodiment, the polymerase having
altered nucleotide binding and/or nucleotide incorporation kinetics
can improve distinction between productive and non-productive
binding events. In another embodiment, the altered nucleotide
binding kinetics and/or altered nucleotide incorporation kinetics
can include altered kinetics for: polymerase binding to the target
molecule; polymerase binding to the nucleotide; polymerase
catalyzing nucleotide incorporation; the polymerase cleaving the
nucleotide and forming a cleavage product; and/or the polymerase
releasing the cleavage product. In another embodiment, the
polymerase can be linked to an energy transfer donor moiety to form
a conjugate. In another embodiment, the polymerase component of the
conjugate can be enzymatically active. In another embodiment, the
polymerase has altered kinetics for nucleotide binding and/or
nucleotide incorporation used in combination with labeled
nucleotides having six or more phosphate groups (or substituted
phosphate groups), which improve distinction between productive and
non-productive binding events. In another embodiment, the
polymerase can have improved photo-stability. The polymerase can be
a Phi29-like polymerase, including Phi29 or B103 polymerase. The
polymerase can be a mutant polymerase. The polymerase can be a B103
polymerase according to any one of SEQ ID NOS:1-5.
[0162] In one embodiment, the energy transfer donor moiety can be a
nanoparticle or a fluorescent dye. The nanoparticle can be about
1-20 nm in its largest dimensions. The nanoparticle can be a
core/shell nanoparticle. The nanoparticle can include a core
comprising semiconductor material(s). The core can include
materials (including binary, ternary and quaternary mixtures
thereof), from: Groups II-VI of the periodic table, including ZnS,
ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, HgTe, MgTe; Groups III-V,
including GaN, GaP, GaAs, GaSb, InN, InP, InAs, InSb, AlAs, AlP,
AlSb, AlS; and/or Group IV, including Ge, Si, Pb. The nanoparticle
can include at least one shell surrounding the core. The shell can
include semiconductor material(s). The nanoparticle can include an
inner shell and an outer shell. The shell can include materials
(including binary, ternary and quaternary mixtures thereof)
comprising: ZnO, ZnS, ZnSe, ZnTe, CdO, CdS, CdSe, CdTe, MgS, MgSe,
GaAs, GaN, GaP, GaAs, GaSb, HgO, HgS, HgSe, HgTe, InAs, InN, InP,
InSb, AlAs, AlN, AlP, or AlSb. In one embodiment, the nanoparticle
comprises a core having CdSe. In another embodiment, the
nanoparticle comprises an inner shell having CdS. In another
embodiment, the nanoparticle comprises an outer shell having ZnS.
The outermost surface of the core or shell can be coated with
tightly associated ligands which are not removed by ordinary
solvation. In some embodiments, the nanoparticle can have a layer
of ligands on its surface which can further be cross-linked to each
other. In some embodiments, the nanoparticle can have other or
additional surface coatings which can modify the properties of the
particle, for example, increasing or decreasing solubility in water
or other solvents. The nanoparticle can be water dispersible. The
nanoparticle can be a non-blinking nanoparticle. The nanoparticle
can be photo-stable. The nanoparticle may not interfere with
polymerase activity, including polymerase binding to the target
molecule, polymerase binding to the nucleotide, polymerase
catalyzing nucleotide incorporation, or the polymerase cleaving the
nucleotide and/or releasing the cleavage product.
[0163] In one embodiment, the target nucleic acid molecule can be
DNA or RNA or DNA/RNA molecule. In another embodiment, the target
nucleic acid molecule is a single nucleic acid molecule. In another
embodiment, the target nucleic acid molecule (e.g., target
molecule) is base-paired with a polymerization initiation site. In
another embodiment, the polymerization initiation site is a
terminal 3'OH of a primer molecule or of a self-primed target
molecule. In another embodiment, the polymerization initiation site
is a 3'OH within a gap or nick. In another embodiment, the target
nucleic acid molecule and/or the polymerization initiation site is
immobilized to a solid surface. In another embodiment, the target
nucleic acid molecule is a linear or circular nucleic acid
molecule.
[0164] In one embodiment, the at least one type of nucleotide can
include 3-10 phosphate groups or substituted phosphate groups, or a
combination of phosphate groups and substituted phosphate groups.
The nucleotide can include a terminal phosphate group or terminal
substituted phosphate group which can be linked to the energy
transfer acceptor moiety. The nucleotide can include the energy
transfer acceptor moiety which is linked the base, sugar, or any
phosphate group or substituted phosphate group. The nucleotide can
be adenosine, guanosine, cytosine, thymidine, uridine, or any other
type of nucleotide.
[0165] In one embodiment, the energy transfer acceptor moiety can
be a fluorescent dye. The energy transfer acceptor moiety and the
energy transfer donor moiety can be capable of energy transfer.
[0166] In one embodiment, more than one type of nucleotide can be
contacted with the polymerase. Each of the different types of
nucleotides can be linked to the same or to different types of
energy transfer acceptor moieties, or any combination of the same
or different types of acceptor moieties.
[0167] In another embodiment, a plurality of one or more different
types of nucleotides can be included in the nucleotide
incorporation reaction to permit successive nucleotide
incorporation.
Compositions and Systems
[0168] Provided herein are compositions and systems, comprising a
DNA-dependent polymerase having properties which offer advantages
over other DNA-dependent polymerase which are traditionally used
for nucleotide polymerization reactions.
[0169] For example, the compositions and systems comprise a
DNA-dependent polymerase having altered kinetics for nucleotide
binding and/or nucleotide incorporation for improved distinction
between productive and non-productive nucleotide binding events. In
another example, the compositions and systems comprise a
DNA-dependent polymerase which can polymerize nucleotides having 4,
5, 6, or more phosphate groups. In another example, the
compositions and systems comprise a DNA-dependent polymerase having
improved photo-stability when exposed to electromagnetic energy
(e.g., exposed to light during the nucleotide incorporation
reactions). In another example, the compositions and systems
comprise a DNA-dependent polymerase which is enzymatically stable
and retains enzymatic activity when linked to an energy transfer
moiety. In yet another example, the compositions and systems
comprise a DNA-dependent polymerase according to SEQ ID NOS:1, 2 or
3.
[0170] The compositions comprise an energy transfer moiety linked
to a DNA-dependent polymerase having altered kinetics for
nucleotide binding and/or nucleotide incorporation for improved
distinction between productive and non-productive nucleotide
binding events. For example, the compositions comprise a
nanoparticle or fluorescent dye linked to DNA-dependent polymerase
having altered kinetics for nucleotide binding and/or nucleotide
incorporation. In yet another example, the compositions comprise an
energy transfer donor (e.g., nanoparticle or fluorescent dye)
linked to DNA-dependent polymerase according to SEQ ID NOS:1, 2 or
3.
[0171] The compositions comprise an energy transfer moiety linked
to a DNA-dependent polymerase having altered kinetics for
nucleotide binding and/or nucleotide incorporation and the
polymerase is bound to a target nucleic acid molecule. In one
embodiment, the compositions comprises an energy transfer moiety
linked to a DNA-dependent polymerase having altered kinetics for
nucleotide binding and/or nucleotide incorporation and the
polymerase is bound to a target nucleic acid molecule which is
base-paired with a polymerization initiation site having a 3'OH
group. In another embodiment, the compositions comprises an energy
transfer moiety linked to a DNA-dependent polymerase according to
SEQ ID NOS:1, 2 or 3, and the polymerase is bound to a target
nucleic acid molecule. In another embodiment, the compositions
comprises an energy transfer moiety linked to a DNA-dependent
polymerase according to SEQ ID NOS:1, 2 or 3, and the polymerase is
bound to a target nucleic acid molecule which is base-paired with a
polymerization initiation site having a 3'0H group.
[0172] Embodiments of the compositions include: the target molecule
can be base-paired with a polymerization initiation site having a
3' OH group; the target molecule can be base-paired with a nucleic
acid primer; the target molecule can be immobilized; the nucleic
acid primer molecule can be immobilized; and/or the target and
primer molecules can be immobilized.
[0173] Provided herein are systems, comprising a DNA-dependent
polymerase having properties which offer advantages over other
DNA-dependent polymerase which are traditionally used for
nucleotide polymerization reactions.
[0174] For example, the systems comprise an energy transfer moiety
linked to a DNA-dependent polymerase having altered kinetics for
nucleotide binding and/or nucleotide incorporation.
[0175] In another example, the systems comprise a nanoparticle or
fluorescent dye linked to a DNA-dependent polymerase having altered
kinetics for nucleotide binding and/or nucleotide
incorporation.
[0176] In another example, the systems comprise an energy transfer
moiety (e.g., nanoparticle or fluorescent dye) linked to a
DNA-dependent polymerase having altered kinetics for nucleotide
binding and/or nucleotide incorporation and the polymerase is bound
to a target nucleic acid molecule.
[0177] In another example, the systems comprise an energy transfer
moiety (e.g., nanoparticle or fluorescent dye) linked to a
DNA-dependent polymerase having altered kinetics for nucleotide
binding and/or nucleotide incorporation and the polymerase is bound
to a target nucleic acid molecule which is base-paired with a
polymerization initiation site having a 3'OH group.
[0178] In another example, the systems comprise an energy transfer
moiety (e.g., nanoparticle or fluorescent dye) linked to a
DNA-dependent polymerase having altered kinetics for nucleotide
binding and/or nucleotide incorporation and the polymerase is bound
to a target nucleic acid molecule which is base-paired with a
nucleic acid primer molecule.
[0179] In another example, the systems comprise (i) an energy
transfer moiety (e.g., nanoparticle or fluorescent dye) linked to a
DNA-dependent polymerase having altered kinetics for nucleotide
binding and/or nucleotide incorporation and the polymerase is bound
to a target nucleic acid molecule and (ii) a nucleotide linked to
an energy transfer moiety.
[0180] In another example, the systems comprise (i) an energy
transfer moiety (e.g., nanoparticle or fluorescent dye) linked to a
DNA-dependent polymerase having altered kinetics for nucleotide
binding and/or nucleotide incorporation and the polymerase is bound
to a target nucleic acid molecule which is base-paired with a
polymerization initiation site having a 3'OH group and (ii) a
nucleotide linked to an energy transfer moiety.
[0181] In another example, the systems comprise (i) an energy
transfer moiety (e.g., nanoparticle or fluorescent dye) linked to a
DNA-dependent polymerase having altered kinetics for nucleotide
binding and/or nucleotide incorporation and the polymerase is bound
to a target nucleic acid molecule which is base-paired with a
nucleic acid primer and (ii) a nucleotide linked to an energy
transfer moiety.
[0182] Embodiments of the systems include: the DNA-dependent
polymerase having altered kinetics for nucleotide binding and/or
nucleotide incorporation can be any of SEQ ID NO:1, 2, or 3; the
target molecule can be base-paired with a polymerization initiation
site having a 3' OH group; the target molecule can be base-paired
with a nucleic acid primer; the target molecule can be immobilized;
the nucleic acid primer molecule can be immobilized; the target and
primer molecules can be immobilized; the energy transfer moiety
which is linked to the nucleotide can be an energy transfer
acceptor moiety (e.g., fluorescent dye).
Reagent Exchange Methods
[0183] Provided herein are compositions, systems, methods, and
kits, for exchanging (e.g., replacing) the reagents for nucleotide
binding or nucleotide incorporation reactions with fresh reagents
on the same target nucleic acid molecule.
[0184] The methods for exchanging the reagents can be used for:
re-sequencing at least a portion of the same nucleic acid molecule;
or replacing any reagent used to practice nucleotide binding and/or
nucleotide incorporation with functional reagents to permit
continuing the nucleotide incorporation reaction on the same
nucleic acid molecule; or performing nucleotide binding or
nucleotide incorporation reactions and switching to reactions
having different nucleotide binding and/or nucleotide incorporation
reaction properties on the same nucleic acid molecule.
[0185] The reagents which can be exchanged include any reagent
which is used in a nucleotide binding or nucleotide incorporation
reaction, including but not limited to any type of: target
molecule; primer; polymerization initiation site; polymerase;
nucleotides (e.g., hydrolyzable, non-hydrolyzable,
chain-terminating, or labeled or non-labeled nucleotides); the
synthesized strand; compounds which reduce photo-damage; buffers;
salts; co-factors; divalent cations; and chelating agents. The
fresh reagents can be the same or different types of reagents
compared to the old reagents.
[0186] For example, one round of a reagent exchange reaction can be
conducted using three types of nucleotides (e.g., A, G, and C)
labeled with a different type of energy transfer acceptor dye, and
another different type of nucleotide (e.g., T) can be unlabeled. In
one embodiment, the A nucleotides can be labeled with dye type 1, G
nucleotides can be labeled with dye type 2, and C nucleotides can
be labeled with dye type 3. In a second round, the reagent exchange
reaction can be conducted using three types of nucleotides (e.g.,
G, C, and T) labeled with a different type of energy transfer
acceptor dye, and another different type of nucleotide (e.g., A)
can be unlabeled. In a third round, the reagent exchange reaction
can be conducted using three types of nucleotides (e.g., C, T, and
A) labeled with a different type of energy transfer acceptor dye,
and another different type of nucleotide (e.g., G) can be
unlabeled. In a fourth round, the reagent exchange reaction can be
conducted using three types of nucleotides (e.g., T, A, and G)
labeled with a different type of energy transfer acceptor dye, and
another different type of nucleotide (e.g., C) can be unlabeled.
The first, second, third, and fourth rounds of reagent exchange
reactions can be conducted in any order, and in any combination. In
any of the rounds of reagent exchange reactions, the different
types of nucleotides can be linked to the same or different type of
energy transfer dye.
[0187] In another example, multiple rounds of reagent exchange
reactions can be conducted using four types of nucleotides (e.g.,
A, G, C, and T) each labeled with a different type of energy
transfer acceptor dye in each round. In one embodiment, in round
one, the A nucleotides can be labeled with dye type 1, G labeled
with dye type 2, C labeled with dye type 3, and T labeled with dye
type 4. In a subsequent round, the reagent exchange reaction can be
conducted using A labeled with dye type 2, G labeled with dye type
3, C labeled with dye type 4, and T labeled with dye type 1. One
skilled in the art will readily recognize that many combinations
are possible.
[0188] In one aspect, the reagent exchange methods can be used to
sequence the same target nucleic acid molecule 1, 2, 3, 4, or 5
times, or up to 10 times, or up to 25 times, or up to 50 times, or
more than 50 times. For example, errors in detecting and/or
identifying the incorporated nucleotides may necessitate
re-sequencing the same target molecule. The errors can arise when a
non-reporting nucleotide (e.g., which is linked to a non-reporting
energy transfer acceptor dye) is incorporated but does not emit a
detectable signal. The same target molecule can be sequenced one or
more times to provide redundant nucleotide sequence information.
The reagent exchange methods can be used to sequence the strand
which is synthesized during a nucleotide incorporation reaction.
The synthesized strand can be sequenced 1, 2, 3, 4, or 5 times, or
up to 10 times, or up to 25 times, or up to 50 times, or more than
50 times, to provide redundant nucleotide sequence information. The
same target molecule, or synthesized strand, can be re-sequenced
using exchanged primers having sequences which are the same or a
different from the sequence of the old primers. Sequencing the same
target molecule multiple times, and/or sequencing the same
synthesized strand multiple times, can provide multiple data sets
of sequence information which can be aligned and compared. In one
embodiment, the alignment can be used to deduce a consensus
sequence of the target molecule or the synthesized strand. The
alignment can be used to provide multi-fold coverage of the
nucleotides which are contained within the target molecule or
synthesized strand.
[0189] The reagent exchange methods can be used to replace inactive
polymerases and/or non-functional nucleotides or energy transfer
moieties, with fresh polymerase, nucleotides, and/or other
reagents, in order to continue the nucleotide incorporation
reaction on the same target molecule or synthesized strand. For
example, fresh polymerase, nucleotides, and/or reagents can be
added to the immobilized target/primer molecules to permit
continuation of the nucleotide incorporation reaction on the same
target or synthesized molecule.
[0190] The reagent exchange methods can be used to replace the
reagents in an on-going nucleotide binding or incorporation
reaction, in order to switch to a different type of nucleotide
binding or nucleotide incorporation reaction on the same target or
synthesized molecule. For example, the first nucleotide
incorporation reaction can be conducted using a polymerase,
nucleotides, and other reagents, which exhibit certain properties,
such as: nucleotide fidelity; rate of nucleotide incorporation;
processivity; strand displacement; kinetics of nucleotide binding,
catalysis, release of the cleavage product, and/or polymerase
translocation; exonuclease activity; and/or activity at certain
temperatures. The reagents (e.g., polymerases and/or nucleotides)
can be exchanged with different reagents to conduct a nucleotide
incorporation reaction which exhibits different nucleotide
incorporation properties (on the same target molecule or on the
same synthesized strand).
[0191] The reagent exchange methods can be practiced using any type
of nucleotide binding or nucleotide incorporation reactions,
including but not limited to: the energy transfer methods disclosed
herein; any type of discontinuous reactions (e.g., synchronous
nucleotide incorporation methods described in: (U.S. Ser. No.
61/184,774, filed on Jun. 5, 2009; U.S. Ser. No. 61/242,762, filed
on Sep. 15, 2009; and U.S. Ser. No. 61/180,811, filed on May 22,
2009; U.S. Ser. No. 61/295,533, filed on Jan. 15, 2010); and any
type of continuous reactions (e.g., asynchronous nucleotide
incorporation methods as described in: (U.S. Ser. No. 61/077,090,
filed on Jun. 30, 2008; U.S. Ser. No. 61/089,497, filed on Aug. 15,
2008; U.S. Ser. No. 61/090,346, filed on Aug. 20, 2008; PCT
application No. PCT/US09/049,324, filed on Jun. 30, 2009; U.S. Ser.
No. 61/164,324, filed on Mar. 27, 2009; and U.S. Ser. No.
61/263,974, filed on Nov. 24, 2009; U.S. Ser. Nos. 61/289,388;
61/293,616; 61/299,917; 61/307,356).
[0192] The reagent exchange methods can be practiced using any type
of format using an immobilized: primer; target molecule;
synthesized strand; and/or polymerase. The reagent exchange methods
can be practiced on a single target nucleic acid molecule, or on
random or organized arrays of single nucleic acid molecules, and
using any type of solid surface (U.S. Ser. No. 61/220,174, filed on
Jun. 24, 2009; and U.S. Ser. No. 61/245,248, filed on Sep. 23,
2009; U.S. Ser. No. 61/302,475). The target molecules and
synthesized strands can be genomic, recombinant, DNA, RNA,
double-stranded, or single-stranded nucleic acid molecules. The
target nucleic acid molecules can be linear or circular. The target
nucleic acid molecules can be self-priming molecules or can be
associated with primer molecules. The target nucleic acid molecules
can be immobilized using any method, including the methods depicted
in any of FIGS. 2-8.
[0193] Provided herein are reagent exchange methods, where the
existing target molecule, synthesized strand, primer, polymerase,
nucleotides, and/or other reagents, can be removed in a manner
which does not remove the immobilized target molecule, primer, or
synthesized strand. In some embodiments, the primer, target
molecule, or synthesized strand can be removed. Methods for
removing the components include physical, chemical, and/or
enzymatic methods.
[0194] The polymerase can be inactivated and/or removed using
physical, chemical, and/or enzymatic method, in any combination and
in any order. For example, the polymerase can be deactivated using
elevated temperatures, such as 45-80.degree. C., for about 30
seconds to 10 minutes. In another example, the polymerase can be
removed from the target molecule or synthesized strand using a
protein-degrading enzyme, such as proteinase-K. In another example,
the polymerase can be removed from the target molecule or
synthesized strand using compounds known to disrupt protein
complexes, where the compounds include detergents (e.g., N-lauroyl
sarcosine, SDS), chaotropic salt (e.g., guanidinium hydrochloride),
lithium sulfate, and EDTA.
[0195] Any combination of capture molecule, primer, target
molecule, and/or synthesized strand, can be dissociated (e.g.,
denatured) from each other using physical, chemical, and/or
enzymatic methods, in any combination and in any order. For
example, the target molecule/synthesized strand duplex can be
denatured using elevated temperatures, such as about 75-100.degree.
C. (e.g., without formamide) or about 45-90.degree. C. (e.g., with
formamide). In another example, the target molecule or synthesized
strand can be degraded using a nucleic acid degrading enzyme, such
as a 5'.fwdarw.3' or 3'.fwdarw.5' exonuclease (e.g., exonuclease
III, T7 gene 6 exonuclease, exonuclease I). In yet another example,
the target molecule or synthesized strand can be denatured using
any compound known to dissociate double-stranded nucleic acid
molecules, such as any combination of: formamide, urea, DMSO,
alkali conditions (e.g., NaOH at about 0.01-0.3 M, or about
0.05-0.1 M; e.g., elevated pH of about 7-12), or low salt or
very-low salt conditions (e.g., about less than 0.001-0.3 mM
cationic conditions), or water.
[0196] In practicing the reagent exchange methods, the target
molecule, synthesized strand, polymerase, primer, capture molecule,
or any reagent, can be removed using fluid flow, washing, and/or
aspiration. The target molecule, primer molecule, synthesized
strand, or capture molecule can be operably linked to the solid
surface in a manner which withstands flowing, washing, aspirating,
and changes in salt, temperature, chemical, enzymatic, and/or pH
conditions. A fresh supply of polymerase, nucleotides, reagents,
primer molecules, splinter molecules, and/or adaptor molecules, can
be added to the immobilized nucleic acid molecules. The polymerase
(e.g., donor-labeled) and nucleotides (e.g., and acceptor-labeled)
can be added to the immobilized nucleic acid molecules under
conditions which are suitable for nucleotide binding and/or
nucleotide incorporation to occur. The fresh polymerase,
nucleotides, and reagents, can be the same or different from the
old polymerase, nucleotides, and/or reagents.
[0197] In the following embodiments (e.g., FIGS. 2-8), the "N" can
be any nucleotide base, and the "I" can be a universal base such as
inosine.
[0198] In one embodiment, a target molecule can be ligated to an
immobilized capture molecule using a splinter oligonucleotide
(which can hybridize to the target molecule and capture
oligonucleotide) and enzymes for ligation and/or nucleotide
polymerization (e.g., T4 ligase and T4 DNA polymerase,
respectively) (see FIG. 2). A primer can be annealed to the
immobilized target molecule, and a synthesized strand can be
produced using a polymerase and nucleotides. Physical, chemical,
and/or enzymatic conditions can be used to remove the synthesized
strand, polymerase, and nucleotides. The remaining target molecule
can be contacted with fresh reagents to permit re-sequencing the
same target molecule. FIG. 2 depicts re-sequencing the same target
molecule, in a direction away from the solid surface. A "two-pass"
method for re-sequencing the same nucleic acid molecule has been
described (Harris, et al., 2008 Science 320:106-109, and supporting
online material).
[0199] In another embodiment, a polynucleotide tail (e.g., poly-A,
-G, -C, or -T) can be added to a target molecule, for example using
a terminal transferase enzyme (TdT in FIG. 3). The tailed target
molecule can be ligated to an immobilized capture molecule using a
splinter oligonucleotide (which can hybridize to the target
molecule and capture oligonucleotide) and enzymes for ligation
and/or nucleotide polymerization (e.g., T4 ligase and T4 DNA
polymerase, respectively). A primer can be annealed to the
immobilized target molecule, and a synthesized strand can be
produced using a polymerase and nucleotides. Physical, chemical,
and/or enzymatic conditions can be used to remove the synthesized
strand, polymerase, and nucleotides. The remaining target molecule
can be contacted with fresh reagents to permit re-sequencing the
same target molecule. FIG. 3 depicts re-sequencing the same target
molecule, in a direction away from the solid surface.
[0200] In yet another embodiment, a target molecule can be ligated
to an immobilized hairpin capture molecule, where a portion of the
capture molecule can hybridize to the target molecule (see FIG. 4).
The target molecule can be ligated to the hairpin capture molecule
using enzymes for ligation and/or nucleotide polymerization (e.g.,
T4 ligase and T4 DNA polymerase, respectively). The hairpin adaptor
molecule can include a recognition sequence for cleavage (scission)
by an endonuclease enzyme. For example, the recognition sequence
can be an RNA portion which can be 3-6 nt in length, to form a
DNA/RNA hybrid. The RNA portion can be 4 nt in length. The RNA
portion can include purines (A and G) in any order. The RNA portion
of the RNA/DNA duplex can be a substrate for cleavage by an
endoribonuclease (e.g., RNase H). In another example, the
recognition sequence can be an AP site (apurinic/apyrimidinic)
having a THF substrate (tetrahydrofuran) which can be cleaved by an
AP endonuclease. In another example, the recognition sequence can
include nucleotide analogs (e.g., 8-oxo-7,8-dihydroguanine,
8-oxoguanine, or 8-hydroxyguanine) which can be cleaved by DNA
glycosylase OGG1. In yet another example, the recognition sequence
can include any sequence which can be cleaved by a nicking enzyme.
After scission, a primer can be annealed to the target molecule,
and a synthesized strand can be produced using a polymerase and
nucleotides. Physical, chemical, and/or enzymatic conditions can be
used to remove the synthesized strand, polymerase, and nucleotides.
The remaining target molecule can be contacted with fresh reagents
to permit re-sequencing the same target molecule. FIG. 4 depicts
re-sequencing the same target molecule, in a direction away from
the solid surface.
[0201] In yet another embodiment, the 5' end of a target molecule
can be ligated to an adaptor molecule using T4 ligase (FIG. 5A).
The adaptor molecule can be annealed with a primer having a blocked
3' end (FIG. 5A). The target molecule can be reacted with terminal
transferase to add a poly-nucleotide tail (e.g., poly-A, -G, -C, or
-T) (TdT in FIG. 5A). The tailed target molecule can be captured by
an immobilized oligonucleotide (FIG. 5B). The immobilized
oligonucleotide can be used to produce a synthesized strand, using
a polymerase and nucleotides (FIG. 5B). Physical, chemical, and/or
enzymatic conditions can be used to remove the target strand,
polymerase, and nucleotides. A primer can be annealed to the
remaining synthesized strand. A newly synthesized strand can be
produced using a polymerase and nucleotides. Physical, chemical,
and/or enzymatic conditions can be used to remove the newly
synthesized strand, polymerase, and nucleotides. The remaining
synthesized strand can be contacted with fresh reagents to permit
re-sequencing the same synthesized strand. FIGS. 5A and B depict
re-sequencing the same synthesized strand, in a direction towards
the solid surface.
[0202] In yet another embodiment, the target molecule can be
reacted with terminal transferase to add a poly-nucleotide tail
(e.g., poly-A, -G, -C, or -T) (TdT in FIG. 6A). The tailed target
molecule can be captured by an immobilized capture oligonucleotide
(FIG. 6A). The immobilized capture oligonucleotide can be used to
generate a synthesized strand, using a polymerase and nucleotides
(FIG. 6B). The 3' end of the synthesized strand can be ligated to
an adaptor molecule. Physical, chemical, and/or enzymatic
conditions can be used to remove the target molecule, polymerase,
and nucleotides. The 3' end of the remaining synthesized strand can
be annealed to a primer. A newly synthesized strand can be
generated with a polymerase and nucleotides. Physical, chemical,
and/or enzymatic conditions can be used to remove the newly
synthesized strand, polymerase, and nucleotides. The remaining
synthesized strand can be contacted with fresh reagents to permit
re-sequencing the same synthesized strand. FIGS. 6A and B depict
re-sequencing the same synthesized strand, in a direction towards
the solid surface.
[0203] In yet another embodiment, the target molecule can be
reacted with terminal transferase to add a poly-nucleotide tail
(e.g., poly-A, -G, -C, or -T) (TdT in FIG. 7). The tailed target
molecule can be circularized. The circularized target molecule can
be captured by an immobilized oligonucleotide. The 3' end of the
capture oligonucleotide can be used to generate a synthesized
strand using a polymerase and nucleotides, in a rolling circle
replication mode. A strand-displacement DNA polymerase can be used
for the rolling circle replication.
[0204] In another embodiment, stem-loop adaptor molecules can be
ligated to both ends of a double-stranded target molecule using T4
ligase (FIG. 8) to produce a closed-ended molecule. The resulting
molecule can be captured by an immobilized oligonucleotide via
complementary sequences in one of the stem-loop adaptor molecules.
The immobilized capture oligonucleotide can be used as a primer to
generate the synthesized strand, using a polymerase and
nucleotides.
Nucleotides
[0205] The methods, compositions, systems and kits disclosed herein
can include nucleotides. The nucleotides can be linked with at
least one energy transfer moiety (FIG. 1). The energy transfer
moiety can be an energy transfer acceptor or donor moiety. The
different types of nucleotides (e.g., adenosine, thymidine,
cytidine, guanosine, and uridine) can be labeled with a different
type energy transfer acceptor or donor moiety so that the
detectable signals (e.g., energy transfer signals) from each of the
different types nucleotides can be distinguishable to permit base
identity. In one embodiment, the different types of nucleotides
(e.g., adenosine, thymidine, cytidine, guanosine, and uridine) can
be labeled with a different type of energy transfer acceptor moiety
so that the detectable signals (e.g., energy transfer signals) from
each of the different types nucleotides can be distinguishable to
permit base identity. The nucleotides can be labeled in a way that
does not interfere with the events of nucleotide polymerization.
For example the attached energy transfer acceptor moiety does not
interfere with: nucleotide binding; nucleotide incorporation;
cleavage of the nucleotide; or release of the cleavage product. See
for example, U.S. Ser. No. 61/164,091, Ronald Graham, concurrently
filed Mar. 27, 2009. See for example U.S. Pat. Nos. 7,041,812,
7,052,839, 7,125,671, and 7,223,541; U.S. Pub. Nos. 2007/0072196
and 2008/0091005; Sood et al., 2005, J. Am. Chem. Soc.
127:2394-2395; Arzumanov et al., 1996, J. Biol. Chem.
271:24389-24394; and Kumar et al., 2005, Nucleosides, Nucleotides
& Nucleic Acids, 24(5):401-408.
[0206] In one aspect, the energy transfer acceptor moiety may be
linked to any position of the nucleotide. For example, the energy
transfer acceptor moiety can be linked to any phosphate group (or
substituted phosphate group), the sugar or the base. In another
example, the energy transfer moiety can be linked to any phosphate
group (or substituted phosphate group) which is released as part of
a phosphate cleavage product upon incorporation. In yet another
example, the energy transfer acceptor moiety can be linked to the
terminal phosphate group (or substituted phosphate group). In
another aspect, the nucleotide may be linked with an additional
energy transfer acceptor moiety, so that the nucleotide is attached
with two or more energy transfer acceptor moieties. The additional
energy transfer acceptor moiety can be the same or different as the
first energy transfer acceptor moiety. In one embodiment, the
energy transfer acceptor moiety can be a FRET acceptor moiety.
[0207] In one aspect, the nucleotide may be linked with a reporter
moiety which is not an energy transfer moiety. For example, the
reporter moiety can be a fluorophore.
[0208] In one aspect, the energy transfer acceptor moieties and/or
the reporter moiety can be attached to the nucleotide via a linear
or branched linker moiety. An intervening linker moiety can connect
the energy transfer acceptor moieties with each other and/or to the
reporter moiety, in any combination of linking arrangements.
[0209] In another aspect, the nucleotides comprise a sugar moiety,
base moiety, and at least three, four, five, six, seven, eight,
nine, ten, or more phosphate groups (or substituted phosphate
groups) linked to the sugar moiety by an ester or phosphoramide
linkage. The phosphates can be linked to the 3' or 5' C of the
sugar moiety.
[0210] In one aspect, different linkers can be used to operably
link the different nucleotides (e.g., A, G, C, T or U) to the
energy transfer moieties or reporter moieties. For example,
adenosine nucleotide can be attached to one type of energy transfer
moiety using one type of linker, and guanosine nucleotide can be
linked to a different type of energy transfer moiety using a
different type of linker. In another example, adenosine nucleotide
can be attached to one type of energy transfer moiety using one
type of linker, and the other types of nucleotides can be attached
to different types of energy transfer moieties using the same type
of linker. One skilled in the art will appreciate that many
different combinations of nucleotides, energy transfer moieties,
and linkers are possible.
[0211] In one aspect, the distance between the nucleotide and the
energy transfer moiety can be altered. For example, the linker
length and/or number of phosphate groups (or substitute phosphate
groups) can lengthen or shorten the distance from the sugar moiety
to the energy transfer moiety. In another example, the distance
between the nucleotide and the energy transfer moiety can differ
for each type of nucleotide (e.g., A, G, C , T or U).
[0212] In another aspect, the number of energy transfer moieties
which are linked to the different types of nucleotides (e.g., A, G,
C, T or U) can be the same or different. For example: A can have
one dye, and G, C, and T have two; A can have one dye, C has two, G
has three, and T has four; A can have one dye, C and G have two,
and T has four. One skilled in the art will recognize that many
different combinations are possible.
[0213] In another aspect, the concentration of the labeled
nucleotides used to conduct the nucleotide binding or nucleotide
incorporation reactions, or the concentration included in the
systems or kits, can be about 0.0001 nM-1 .mu.M, or about 0.0001
nM-0.001 nM, or about 0.001 nM-0.01 nM, or about 0.01 nM-0.1 nM, or
about 0.1 nM-1.0 nM, or about 1 nM-25 nM, or about 25 nM-50 nM, or
about 50 nM-75 nM, or about 75 nM-100 nM, or about 100 nM-200 nM,
or about 200 nM-500 nM, or about 500 nM-750 nM, or about 750
nM-1000 nM, or about 0.1 .mu.M-20 .mu.M, or about 20 .mu.M-50
.mu.M, or about 50 .mu.M-75 .mu.M, or about 75 .mu.M-100 .mu.M, or
about 100 .mu.M-200.1 .mu.M, or about 200 .mu.M-500 .mu.M, or about
500 .mu.M-750 .mu.M, or about 750 .mu.M-1000 .mu.M.
[0214] In another aspect, the concentration of the different types
of labeled nucleotides, which are used to conduct the nucleotide
binding or incorporation reaction, can be the same or different
from each other.
Sugar Moieties
[0215] The nucleotides typically comprise suitable sugar moieties,
such as carbocyclic moieties (Ferraro and Gotor 2000 Chem. Rev.
100: 4319-48), acyclic moieties (Martinez, et al., 1999 Nucleic
Acids Research 27: 1271-1274; Martinez, et al., 1997 Bioorganic
& Medicinal Chemistry Letters vol. 7: 3013-3016), and other
suitable sugar moieties (Joeng, et al., 1993 J. Med. Chem. 36:
2627-2638; Kim, et al., 1993 J. Med. Chem. 36: 30-7; Eschenmosser
1999 Science 284:2118-2124.; and U.S. Pat. No. 5,558,991). The
sugar moiety may be selected from the following: ribosyl,
2'-deoxyribosyl, 3'-deoxyribosyl, 2',3'-dideoxyribosyl,
2',3'-didehydrodideoxyribosyl, 2'-alkoxyribosyl, 2'-azidoribosyl,
2'-aminoribosyl, 2'-fluororibosyl, 2'-mercaptoriboxyl,
2'-alkylthioribosyl, 3'-alkoxyribosyl, 3'-azidoribosyl,
3'-aminoribosyl, 3'-fluororibosyl, 3'-mercaptoriboxyl,
3'-alkylthioribosyl carbocyclic, acyclic and other modified sugars.
In one aspect, the 3'-position has a hydroxyl group, for
strand/chain elongation.
Base Moieties
[0216] The nucleotides typically comprise a hetero cyclic base
which includes substituted or unsubstituted nitrogen-containing
parent heteroaromatic ring which is commonly found in nucleic
acids, including naturally-occurring, substituted, modified, or
engineered variants. The base is capable of forming Watson-Crick
and/or Hoogstein hydrogen bonds with an appropriate complementary
base. Exemplary bases include, but are not limited to, purines and
pyrimidines such as: 2-aminopurine, 2,6-diaminopurine, adenine (A),
ethenoadenine, N.sup.6-.DELTA..sup.2-isopentenyladenine (61A),
N.sup.6-.DELTA..sup.2-isopentenyl-2-methylthioadenine (2 ms6iA),
N.sup.6-methyladenine, guanine (G), isoguanine,
N.sup.2-dimethylguanine (dmG), 7-methylguanine (7mG),
2-thiopyrimidine, 6-thioguanine (6sG), hypoxanthine and
O.sup.6-methylguanine; 7-deaza-purines such as 7-deazaadenine
(7-deaza-A) and 7-deazaguanine (7-deaza-G); pyrimidines such as
cytosine (C), 5-propynylcytosine, isocytosine, thymine (T),
4-thiothymine (4sT), 5,6-dihydrothymine, O.sup.4-methylthymine,
uracil (U), 4-thiouracil (4sU) and 5,6-dihydrouracil
(dihydrouracil; D); indoles such as nitroindole and 4-methylindole;
pyrroles such as nitropyrrole; nebularine; inosines;
hydroxymethylcytosines; 5-methycytosines; base (Y); as well as
methylated, glycosylated, and acylated base moieties; and the like.
Additional exemplary bases can be found in Fasman, 1989, in:
Practical Handbook of Biochemistry and Molecular Biology, pp.
385-394, CRC Press, Boca Raton, Fla., and the references cited
therein.
Phosphate Groups
[0217] The nucleotides typically comprise phosphate groups which
can be linked to the 2', 3' and/or 5' position of the sugar moiety.
The phosphate groups include analogs, such as phosphoramidate,
phosphorothioate, phosphorodithioate, and O-methylphosphoroamidite
groups. In one embodiment, at least one of the phosphate groups can
be substituted with a fluoro and/or chloro group. The phosphate
groups can be linked to the sugar moiety by an ester or
phosphoramide linkage. Typically, the nucleotide comprises three,
four, five, six, seven, eight, nine, ten, or more phosphate groups
linked to the 5' position of the sugar moiety.
Non-Hydrolyzable Nucleotides
[0218] The methods, compositions, systems and kits disclosed herein
can include non-hydrolyzable nucleotides. The nucleotide binding
and nucleotide incorporation methods can be practiced using
incorporatable nucleotides and non-hydrolyzable nucleotides. In the
presence of the incorporatable nucleotides (e.g., labeled), the
non-hydrolyzable nucleotides (e.g., non-labeled) can compete for
the polymerase binding site to permit distinction between the
complementary and non-complementary nucleotides, or for
distinguishing between productive and non-productive binding
events. In the nucleotide incorporation reaction, the presence of
the non-hydrolyzable nucleotides can alter the length of time,
frequency, and/or duration of the binding of the labeled
incorporatable nucleotides to the polymerase.
[0219] The non-hydrolyzable nucleotides can be non-labeled or can
be linked to a reporter moiety (e.g., energy transfer moiety). The
labeled non-hydrolyzable nucleotides can be linked to a reporter
moiety at any position, such as the sugar, base, or any phosphate
(or substituted phosphate group). For example, the non-hydrolyzable
nucleotides can have the general structure:
R.sub.11--(--P).sub.n--S--B
[0220] Where B can be a base moiety, such as a hetero cyclic base
which includes substituted or unsubstituted nitrogen-containing
heteroaromatic ring. Where S can be a sugar moiety, such as a
ribosyl, riboxyl, or glucosyl group. Where n can be 1-10, or more.
Where P can be one or more substituted or unsubstituted phosphate
or phosphonate groups. Where R.sub.11, if included, can be a
reporter moiety (e.g., a fluorescent dye). In one embodiment, the
non-hydrolyzable nucleotide having multiple phosphate or
phosphonate groups, the linkage between the phosphate or
phosphonate groups can be non-hydrolyzable by the polymerase. The
non-hydrolyzable linkages include, but are not limited to, amino,
alkyl, methyl, and thio groups. The phosphate or phosphonate
portion of the non-hydrolyzable nucleotide can have the general
structure:
##STR00001##
[0221] Where B can be a base moiety and S can be a sugar moiety.
Where any one of the R.sub.1-R.sub.7 groups can render the
nucleotide non-hydrolyzable by a polymerase. Where the sugar C5
position can be CH.sub.2, CH.sub.2O, CH.dbd., CHR, or CH.sub.2
CH.sub.2. Where the R.sub.1 group can be O, S, CH.dbd., CH(CN), or
NH. Where the R.sub.2, R.sub.3, and R.sub.4, groups can
independently be O, BH.sub.3, or SH. Where the R.sub.5 and R.sub.6
groups can independently be an amino, alkyl, methyl, thio group, or
CHF, CF.sub.2, CHBr, CCl.sub.2, O--O, or --C.ident.C--. Where the
R.sub.7 group can be oxygen, or one or more additional phosphate or
phosphonate groups, or can be a reporter moiety. Where R.sub.8 can
be SH, BH.sub.3, CH.sub.3, NH.sub.2, or a phenyl group or phenyl
ring. Where R.sub.9 can be SH. Where R.sub.10 can be CH.sub.3,
N.sub.3CH.sub.2CH.sub.2, NH.sub.2, ANS, N.sub.3, MeO, SH, Ph, F,
PhNH, PhO, or RS (where Ph can be a phenyl group or phenyl ring,
and F can be a fluorine atom or group). The substituted groups can
be in the S or R configuration.
[0222] The non-hydrolyzable nucleotides can be alpha-phosphate
modified nucleotides, alpha-beta nucleotides, beta-phosphate
modified nucleotides, beta-gamma nucleotides, gamma-phosphate
modified nucleotides, caged nucleotides, or di-nucleotides.
[0223] Many examples of non-hydrolyzable nucleotides are known
(Rienitz 1985 Nucleic Acids Research 13:5685-5695), including
commercially-available ones from Jena Bioscience (Jena,
Germany).
Polymerases
[0224] The compositions, methods, systems and kits disclosed herein
involve the use of one or more polymerases. In some embodiments,
the polymerase incorporates one or more nucleotides into a nucleic
acid molecule.
[0225] In some embodiments, the polymerase provided herein can
offer unexpected advantages over polymerases that are traditionally
used for nucleotide polymerization reactions. In some embodiments,
the polymerases can be enzymatically active when conjugated to an
energy transfer moiety (e.g., donor moiety). In some embodiments,
the polymerases have altered kinetics for nucleotide binding and/or
nucleotide incorporation which improve distinction between
productive and non-productive nucleotide binding events. In some
embodiments, the polymerases having altered kinetics for nucleotide
binding and/or nucleotide incorporation can be used in combination
with labeled nucleotides having six or more phosphate groups (or
substituted phosphate groups), which improves distinction between
productive and non-productive binding events. In some embodiments,
the polymerases have improved photo-stability compared to
polymerases traditionally used for nucleotide polymerization.
Examples of polymerases having altered kinetics for nucleotide
binding and/or nucleotide incorporation include B103 polymerases
disclosed in U.S. Ser. Nos. 61/242,771, 61/293,618, and any one of
SEQ ID NOS:1-5.
[0226] In some embodiments, the polymerase can be unlabeled.
Alternatively, the polymerase can be linked to one or more reporter
moiety. In some embodiments, the reporter moiety comprises at least
one energy transfer moiety.
[0227] The polymerase may be linked with at least one energy
transfer donor or acceptor moiety. One or more energy transfer
donor or acceptor moiety can be linked to the polymerase at the
amino end or carboxyl end or may be inserted at any site
therebetween. Optionally, the energy transfer donor or acceptor
moiety can be attached to the polymerase in a manner which does not
significantly interfere with the nucleotide binding activity, or
with the nucleotide incorporation activity of the polymerase. In
such embodiments, the energy transfer donor or acceptor moiety is
attached to the polymerase in a manner that does not significantly
interfere with polymerase activity.
[0228] In one aspect, a single energy transfer donor or acceptor
moiety can be linked to more than one polymerase and the attachment
can be at the amino end or carboxyl end or may be inserted within
the polymerase.
[0229] In another aspect, a single energy transfer donor or
acceptor moiety can be linked to one polymerase.
[0230] In one aspect, the energy transfer donor moiety can be a
nanoparticle (e.g., a fluorescent nanoparticle) or a fluorescent
dye. The polymerase, which can be linked to the nanoparticle or
fluorescent dye, typically retains one or more activities that are
characteristic of the polymerase, e.g., polymerase activity,
exonuclease activity, nucleotide binding, and the like.
[0231] In one aspect, the polymerases can be replicases,
DNA-dependent polymerases, primases, RNA-dependent polymerases
(including RNA-dependent DNA polymerases such as, for example,
reverse transcriptases), strand-displacement polymerases, or
thermo-stable polymerases. In another aspect, the polymerase can be
any Family A or B type polymerase. Many types of Family A (e.g., E.
coli Pol I), B (e.g., E. coli Pol II), C (e.g., E. coli Pol III), D
(e.g., Euryarchaeotic Pol II), X (e.g., human Pol beta), and Y
(e.g., E. coli UmuC/DinB and eukaryotic RAD30/xeroderma pigmentosum
variants) polymerases are described in Rothwell and Watsman 2005
Advances in Protein Chemistry 71:401-440.
[0232] In yet another aspect, the polymerases can be isolated from
a cell, or generated using recombinant DNA technology or chemical
synthesis methods. In another aspect, the polymerases can be
expressed in prokaryote, eukaryote, viral, or phage organisms. In
another aspect, the polymerases can be post-translationally
modified proteins or fragments thereof.
[0233] In one aspect, the polymerase can be a recombinant protein
which is produced by a suitable expression vector/host cell system.
The polymerases can be encoded by suitable recombinant expression
vectors carrying inserted nucleotide sequences of the polymerases.
The polymerase sequence can be linked to a suitable expression
vector. The polymerase sequence can be inserted in-frame into the
suitable expression vector. The suitable expression vector can
replicate in a phage host, or a prokaryotic or eukaryotic host
cell. The suitable expression vector can replicate autonomously in
the host cell, or can be inserted into the host cell's genome and
be replicated as part of the host genome. The suitable expression
vector can carry a selectable marker which confers resistance to
drugs (e.g., kanamycin, ampicillin, tetracycline, chloramphenicol,
or the like), or confers a nutrient requirement. The suitable
expression vector can have one or more restriction sites for
inserting the nucleic acid molecule of interest. The suitable
expression vector can include expression control sequences for
regulating transcription and/or translation of the encoded
sequence. The expression control sequences can include: promoters
(e.g., inducible or constitutive), enhancers, transcription
terminators, and secretion signals. The expression vector can be a
plasmid, cosmid, or phage vector. The expression vector can enter a
host cell which can replicate the vector, produce an RNA transcript
of the inserted sequence, and/or produce protein encoded by the
inserted sequence. The recombinant polymerase can include an
affinity tag for enrichment or purification, including a poly-amino
acid tag (e.g., poly His tag), GST, and/or HA sequence tag. Methods
for preparing suitable recombinant expression vectors and
expressing the RNA and/or protein encoded by the inserted sequences
are well known (Sambrook et al, Molecular Cloning (1989)).
[0234] The polymerases may be DNA polymerases and include without
limitation bacterial DNA polymerases, prokaryotic DNA polymerase,
eukaryotic DNA polymerases, archaeal DNA polymerases, viral DNA
polymerases and phage DNA polymerases. The polymerase can be a
commercially available polymerase.
[0235] In some embodiments, the polymerase can be a DNA polymerase
and include without limitation bacterial DNA polymerases,
eukaryotic DNA polymerases, archaeal DNA polymerases, viral DNA
polymerases and phage DNA polymerases.
[0236] Suitable bacterial DNA polymerase include without limitation
E. coli DNA polymerases I, II and III, IV and V, the Klenow
fragment of E. coli DNA polymerase, Clostridium stercorarium (Cst)
DNA polymerase, Clostridium thermocellum (Cth) DNA polymerase and
Sulfolobus solfataricus (Sso) DNA polymerase.
[0237] Suitable eukaryotic DNA polymerases include without
limitation the DNA polymerases .alpha., .delta., .epsilon., .eta.,
.zeta., .gamma., .beta., .sigma., .lamda., .mu., .tau., and
.kappa., as well as the Rev1 polymerase (terminal deoxycytidyl
transferase) and terminal deoxynucleotidyl transferase (TdT).
[0238] Suitable viral and/or phage DNA polymerases include without
limitation T4 DNA polymerase, T5 DNA polymerase, T7 DNA polymerase,
Phi-15 DNA polymerase, Phi-29 DNA polymerase (see, e.g., U.S. Pat.
No. 5,198,543; also referred to variously as .PHI.29 polymerase,
phi29 polymerase, phi 29 polymerase, Phi 29 polymerase, and Phi29
polymerase); .PHI.15 polymerase (also referred to herein as Phi-15
polymerase); .PHI.21 polymerase (Phi-21 polymerase); PZA
polymerase; PZE polymerase, PRD1 polymerase; Nf polymerase; M2Y
polymerase; SF5 polymerase; f1 DNA polymerase, Cp-1 polymerase;
Cp-5 polymerase; Cp-7 polymerase; PR4 polymerase; PR5 polymerase;
PR722 polymerase; L17 polymerase; M13 DNA polymerase, RB69 DNA
polymerase, G1 polymerase; GA-1 polymerase, BS32 polymerase; B103
polymerase; BA103 polymerase, a polymerase obtained from any phi-29
like phage or derivatives thereof, etc. See, e.g., U.S. Pat. No.
5,576,204, filed Feb. 11, 1993; U.S. Pat. Appl. No. 2007/0196846,
published Aug. 23, 2007.
[0239] Suitable archaeal DNA polymerases include without limitation
the thermostable and/or thermophilic DNA polymerases such as, for
example, DNA polymerases isolated from Thermus aquaticus (Taq) DNA
polymerase, Thermus filiformis (Tfi) DNA polymerase, Thermococcus
zilligi (Tzi) DNA polymerase, Thermus thermophilus (Tth) DNA
polymerase, Thermus flavus (Tfl) DNA polymerase, Pyrococcus woesei
(Pwo) DNA polymerase, Pyrococcus furiosus (Pfu) DNA polymerase as
well as Turbo Pfu DNA polymerase, Thermococcus litoralis (Tli) DNA
polymerase or Vent DNA polymerase, Pyrococcus sp. GB-D polymerase,
"Deep Vent" DNA polymerase, New England Biolabs), Thermotoga
maritima (Tma) DNA polymerase, Bacillus stearothermophilus (Bst)
DNA polymerase, Pyrococcus Kodakaraensis (KOD) DNA polymerase, Pfx
DNA polymerase, Thermococcus sp. JDF-3 (JDF-3) DNA polymerase,
Thermococcus gorgonarius (Tgo) DNA polymerase, Thermococcus
acidophilium DNA polymerase; Sulfolobus acidocaldarius DNA
polymerase; Thermococcus sp. 9.degree. N-7 DNA polymerase;
Thermococcus sp. NA1; Pyrodictium occultum DNA polymerase;
Methanococcus voltae DNA polymerase; Methanococcus
thermoautotrophicum DNA polymerase; Methanococcus jannaschii DNA
polymerase; Desulfurococcus strain TOK DNA polymerase (D. Tok Pol);
Pyrococcus abyssi DNA polymerase; Pyrococcus horikoshii DNA
polymerase; Pyrococcus islandicum DNA polymerase; Thermococcus
fumicolans DNA polymerase; Aeropyrum pernix DNA polymerase; the
heterodimeric DNA polymerase DP1/DP2, etc.
[0240] Suitable RNA polymerases include, without limitation, T3,
T5, T7, and SP6 RNA polymerases.
[0241] Suitable reverse transcriptases include without limitation
reverse transcriptases from HIV, HTLV-I, HTLV-II, FeLV, FIV, SIV,
AMV, MMTV and MoMuLV, as well as the commercially available
"Superscript" reverse transcriptases, (Life Technologies Corp.,
Carlsbad, Calif.) and telomerases.
[0242] In some embodiments, the polymerase is selected from the
group consisting of: Phi-29 DNA polymerase, a variant of Phi-29 DNA
polymerase, B103 DNA polymerase and a variant of B103 DNA
polymerase.
[0243] In another aspect, the polymerases can include one or more
mutations that improve the performance of the polymerase in the
particular biological assay of interest. The mutations can include
amino acid substitutions, insertions, or deletions.
Selecting a Polymerase
[0244] The selection of the polymerase for use in the disclosed
methods can be based on the desired polymerase behavior in the
particular biological assay of interest. For example, the
polymerase can be selected to exhibit enhanced or reduced activity
in a particular assay, or enhanced or reduced interaction with one
or more particular substrates.
[0245] For example, in some embodiments the polymerase is selected
based on the polymerization kinetics of the polymerase either in
unconjugated form or when linked to a reporter moiety (labeled
polymerase conjugate). For example, the polymerase can be a
polymerase having altered nucleotide binding and/or altered
nucleotide incorporation kinetics which are selected on the basis
of kinetic behavior relating to nucleotide binding (e.g.,
association), nucleotide dissociation (intact nucleotide),
nucleotide fidelity, nucleotide incorporation (e.g., catalysis),
and/or release of the cleavage product. The selected polymerase can
be wild-type or mutant.
[0246] In one embodiment, polymerases may be selected that retain
the ability to selectively bind complementary nucleotides. In
another embodiment, the polymerases may be selected which exhibit a
modulated rate (faster or slower) of nucleotide association or
dissociation. In another embodiment, the polymerases may be
selected which exhibit a reduced rate of nucleotide incorporation
activity (e.g., catalysis) and/or a reduced rate of dissociation of
the cleavage product and/or a reduced rate of polymerase
translocation (after nucleotide incorporation). Some modified
polymerases which exhibit nucleotide binding and a reduced rate of
nucleotide incorporation have been described (Rank, U.S. published
patent application No. 2008/0108082; Hanzel, U.S. published patent
application No. 2007/0196846).
[0247] In polymerases from different classes (including
DNA-dependent polymerases), an active-site lysine can interact with
the phosphate groups of a nucleoside triphosphate molecule bound to
the active site. The lysine residue has been shown to protonate the
pyrophosphate leaving-group upon nucleotidyl transfer. Mutant
polymerases having this lysine substituted with leucine, arginine,
histidine or other amino acids, exhibit greatly reduced nucleotide
incorporation rates (Castro, et al., 2009 Nature Structural and
Molecular Biology 16:212-218). One skilled in the art can use amino
acid alignment and/or comparison of crystal structures of
polymerases as a guide to determine which lysine residue to replace
with alternative amino acids. The sequences of Phi29 (SEQ ID
NOS:6-12), RB69 (SEQ ID NO:13), B103 (SEQ ID NOS:1-5), and Klenow
fragment can be used as the basis for selecting the amino acid
residues to be modified (for B103 polymerase, see Hendricks, et
al., U.S. Ser. No. 61/242,771, filed on Sep. 15, 2009, or U.S. Ser.
No. 61/293,618, filed on Jan. 8, 2010). In one embodiment, a
modified phi29 polymerase can include lysine at position 379 and/or
383 substituted with leucine, arginine or histidine.
[0248] In other embodiments, the polymerase can be selected based
on the combination of the polymerase and nucleotides, and the
reaction conditions, to be used for the nucleotide binding and/or
nucleotide incorporation reactions. For example, certain
polymerases in combination with nucleotides which comprise 3, 4, 5,
6, 7, 8, 9, 10 or more phosphate groups can be selected for
performing the disclosed methods. In another example, certain
polymerases in combination with nucleotides which are linked to an
energy transfer moiety can be selected for performing the
nucleotide incorporation methods.
[0249] The polymerases, nucleotides, and reaction conditions, can
be screened for their suitability for use in the nucleotide binding
and/or nucleotide incorporation methods, using well known screening
techniques. For example, the suitable polymerase may be capable of
binding nucleotides and/or incorporating nucleotides. For example,
the reaction kinetics for nucleotide binding, association,
incorporation, and/or dissociation rates, can be determined using
rapid kinetics techniques (e.g., stopped-flow or quench flow
techniques). Using stopped-flow or quench flow techniques, the
binding kinetics of a nucleotide can be estimated by calculating
the 1/k.sub.d value. Stopped-flow techniques which analyze
absorption and/or fluorescence spectroscopy properties of the
nucleotide binding, incorporation, or dissociation rates to a
polymerase are well known in the art (Kumar and Patel 1997
Biochemistry 36:13954-13962; Tsai and Johnson 2006 Biochemistry
45:9675-9687; Hanzel, U.S. published patent application No.
2007/0196846). Other methods include quench flow (Johnson 1986
Methods Enzymology 134:677-705), time-gated fluorescence decay time
measurements (Korlach, U.S. Pat. No. 7,485,424), plate-based assays
(Clark, U.S. published patent application No. 2009/0176233), and
X-ray crystal structure analysis (Berman 2007 EMBO Journal
26:3494). Nucleotide incorporation by a polymerase can also be
analyzed by gel separation of the primer extension products. In one
embodiment, stopped-flow techniques can be used to screen and
select combinations of nucleotides with polymerases having a
t.sub.po1 value (e.g., 1/k.sub.po1) which is less than a t.sub.-1
(e.g., 1/k.sub.-1) value. Stopped-flow techniques for measuring
t.sub.po1 (MP Roettger 2008 Biochemistry 47:9718-9727; M Bakhtina
2009 Biochemistry 48:3197-320) and t.sub.-1 (M Bakhtina 2009
Biochemistry 48:3197-3208) are known in the art.
[0250] For example, some phi29 or B103 (SEQ ID NOS:1, 2, or 3)
polymerases (wild-type or mutant) exhibit t.sub.po1 values which
are less than t.sub.-1 values, when reacted with tetraphosphate,
pentaphosphate or hexaphosphate nucleotides. These polymerases can
offer improvements in distinguishing between productive and
non-productive nucleotide binding events compared to other
polymerases. In another embodiment, polymerases can be modified by
binding it to a chemical compound or an antibody, in order to
inhibit nucleotide incorporation.
[0251] In some embodiments, the selection of the polymerase may be
determined by the level of processivity desired for conducting
nucleotide incorporation or polymerization reactions. The
polymerase processivity can be gauged by the number of nucleotides
incorporated for a single binding event between the polymerase and
the target molecule base-paired with the polymerization initiation
site. For example, the processivity level of the polymerase may be
about 1, 5, 10, 20, 25, 50, 100, 250, 500, 750, 1000, 2000, 5000,
or 10,000 or more nucleotides incorporated with a single binding
event. Processivity levels typically correlate with read lengths of
a polymerase. Optionally, the polymerase can be selected to retain
the desired level of processivity when conjugated to a reporter
moiety.
[0252] The selection of the polymerase may be determined by the
level of fidelity desired, such as the error rate per nucleotide
incorporation. The fidelity of a polymerase may be partly
determined by the 3'.fwdarw.5' exonuclease activity associated with
a DNA polymerase. The fidelity of a DNA polymerase may be measured
using assays well known in the art (Lundburg et al., 1991 Gene,
108:1-6). The error rate of the polymerase can be one error per
about 100, or about 250, or about 500, or about 1000, or about 1500
incorporated nucleotides. In some embodiments, the polymerase is
selected to exhibit high fidelity. Such high-fidelity polymerases
include those exhibiting error rates typically of about
5.times.10.sup.-6 per base pair or lower.
[0253] In some embodiments, the selection of the polymerase may be
determined by the rate of nucleotide incorporation such as about
one nucleotide per 2-5 seconds, or about one nucleotide per second,
or about 5 nucleotides per second, or about 10 nucleotides per
second, or about 20 nucleotides per second, or about 30 nucleotides
per second, or more than 40 nucleotides per second, or more than
50-100 per second, or more than 100 per second. In one embodiment,
polymerases exhibiting reduced nucleotide incorporation rates
include mutant phi29 polymerase having lysine substituted with
leucine, arginine, histidine or other amino acids (Castro 2009
Nature Structural and Molecular Biology 16:212-218).
[0254] In some embodiments, the polymerase can be selected to
exhibit either reduced or enhanced rates of nucleotide
incorporation when reacted with nucleotides linked at the terminal
phosphate group with an energy transfer acceptor.
[0255] In some embodiments, the polymerase can be selected to
exhibit either reduced or enhanced nucleotide binding times for a
particular nucleotide of interest. In some embodiments, the
nucleotide binding time of the selected polymerase for the
particular labeled nucleotide of interest can be between about 20
msec and about 300 msec, typically between about 55 msec and about
100 msec. In some embodiments, the nucleotide binding time of the
selected polymerase for the particular labeled nucleotide of
interest can be between about 1.5 and about 4 times the nucleotide
binding time of the corresponding wild-type polymerase for the
labeled nucleotide. These polymerases can offer improvements in
distinguishing between productive and non-productive nucleotide
binding events compared to other polymerases.
[0256] In some embodiments, the polymerase can be selected,
mutated, modified, evolved or otherwise engineered to exhibit
either reduced or enhanced entry of nucleotides, particularly
labeled nucleotides, into the polymerase active site. These
polymerases can offer improvements in distinguishing between
productive and non-productive nucleotide binding events compared to
other polymerases.
[0257] In some embodiments, the polymerase can be selected to
exhibit a reduced K.sub.sub for a substrate, particularly a labeled
nucleotide. In some embodiments, the polymerase can comprise one or
more mutations resulting in altered K.sub.cat/K.sub.sub and/or
V.sub.max/K.sub.sub for a particular labeled nucleotide. In some
embodiments, the K.sub.cat/K.sub.sub, the V.sub.max/K.sub.sub, or
both, are increased compared to the wild type polymerase.
[0258] In one embodiment, mutant polymerases having altered
nucleotide binding kinetics and/or altered nucleotide incorporation
kinetics can be selected for use in the nucleotide incorporation
methods. The altered kinetics for nucleotide binding and/or for
nucleotide incorporation include: polymerase binding to the target
molecule; polymerase binding to the nucleotide; polymerase
catalyzing nucleotide incorporation; the polymerase cleaving the
phosphate group or substituted phosphate group; and/or the
polymerase releasing the cleavage product. These polymerases can
offer improvements in distinguishing between productive and
non-productive nucleotide binding events compared to other
polymerases.
[0259] In one embodiment, the selected polymerases can have
improved photo-stability compared to polymerases traditionally used
in nucleotide polymerization reactions. The desirable polymerases
can remain enzymatically active during and/or after exposure to
electromagnetic energy (e.g., light). For example, the desirable
polymerase can retain a level of enzymatic activity, and/or be
enzymatically active for a greater length of time, compared to
polymerases traditionally used in nucleotide polymerization
reactions after exposure to electromagnetic energy. Methods for
measuring enzymatic activity are well known in the art.
[0260] In one embodiment, the selected polymerase can be
enzymatically active when conjugated to an energy transfer moiety
(e.g., nanoparticle or fluorescent dye). The selected polymerase,
as part of a polymerase-energy transfer moiety conjugate, can
polymerize nucleotides. For example, various forms of B103
polymerase (SEQ ID NOS: 1, 2, and 3) retain enzymatic activity when
linked to a nanoparticle or fluorescent dye. Conjugates having
these types of selected polymerases offer advantages over other
polymerases which may lose most or all enzymatic activity when
linked to an energy transfer moiety.
[0261] In some embodiments, the polymerase can be a deletion mutant
which retains nucleotide polymerization activity but lacks the
3'.fwdarw.5' or 5'.fwdarw.3' exonuclease activity (SEQ ID
NOS:1-12). For example, mutant phi29 polymerases having
exonuclease-minus activity, or reduced exonuclease activity, can
optionally comprise the amino acid sequence of SEQ ID NO:7-12 and
further comprise one or more amino acid substitutions at positions
selected from the group consisting of: 12, 14, 15, 62, 66, 165 and
169 (wherein the numbering is relative to the amino acid sequence
of wild type phi29 according to SEQ ID NO:6). In some embodiments,
the polymerase is a phi29 polymerase comprising the amino acid
sequence of SEQ ID NO:6 and one or more of the following amino acid
substitutions: D12A, E14I, E14A, T15I, N62D, D66A, Y165F, Y165C,
and D169A, wherein the numbering is relative to SEQ ID NO:6.
[0262] In one embodiment, the mutant phi29 polymerases include one
or more amino acid mutations at positions selected from the group
consisting of: 132, 135, 250, 266, 332, 342, 368, 371, 375, 379,
380, 383, 387, 390, 458, 478, 480, 484, 486 and 512, wherein the
numbering is relative to the amino acid sequence of SEQ ID NO:6. In
some embodiments, the phi29 polymerase can comprise an amino acid
deletion, wherein the deletion includes some of all of the amino
acids spanning positions 306 to 311 (relative to the numbering in
SEQ ID NO:6).
[0263] In one embodiment, the mutant phi29 polymerase includes one
or more amino acid mutations selected from the group consisting of:
K132A, K135A, K135D, K135E, V250A, V250C, Y266F, D332Y, L342G,
T368D, T368E, T368F, K370A, K371E, T372D, T372E, T372R, T372K,
E375A, E375F, E375H, E375K, E375Q, E375R, E375S, E375W, E375Y,
K379A, Q380A, K383E, K383H, K383L, K383R, N387Y, Y390F, D458N,
K478D, K478E, K478R, L480K, L480R, A484E, E486A, E486D, K512A
K512D, K512E, K512R, K512Y, K371E/K383E/N387Y/D458N, Y266F/Y390F,
Y266F/Y390F/K379A/Q380A, K379A/Q380A, E375Y/Q380A/K383R,
E375Y/Q380A/K383H, E375Y/Q380A/K383L, E375Y/Q380A/V250A,
E375Y/Q380A/V250C, E375Y/K512Y/T368F, E375Y/K512Y/T368F/A484E,
K379A/E375Y, K379A/K383R, K379A/K383H, K379A/K383L, K379A/Q380A,
V250A/K379A, V250A/K379A/Q380A, V250C/K379A/Q380A, K132A/K379A and
deletion of some or all of the amino acid residues spanning R306 to
K311, wherein the numbering is relative to the amino acid sequence
of SEQ ID NO:6.
[0264] Without being bound to any particular theory, it is thought
that the domain comprising amino acid residues 304-314 of the amino
acid sequence of SEQ ID NO: 6 (Phi-29 polymerase), or homologs
thereof, can reduce or otherwise interfere with DNA initiation
and/or elongation by inhibiting access to the Phi-29 polymerase
active site, and that this region must be displaced in order to
allow access to the active site. See, e.g., Kamtekar et al., "The
.PHI.29 DNA polymerase:protein primer structure suggests a model
for the initiation to elongation transition", EMBO J., 25:1335-1343
(2005).
[0265] In another embodiment, the polymerase can be a B103
polymerase comprising the amino acid sequence of SEQ ID NOS:1-5.
The B103 polymerase can optionally include one or more mutations
that reduce the exonuclease activity of the polymerase. Optionally,
such mutations can include any one or a combination of mutations at
the following amino acid positions: 2, 9, 11, 12, 14, 15, 58, 59,
63, 162, 166, 377 and 385, wherein the numbering is relative to SEQ
ID NOS:1 or 2. In some embodiments, the B103 polymerase can
optionally comprise the amino acid sequence of SEQ ID NOS:1 or 2,
and further comprise one or more amino acid substitutions selected
from the group consisting of: D9A, E11A, E11I, T12I, H58R, N59D,
D63A, Y162F, Y162C, D166A, Q377A and S385G, wherein the numbering
is relative to SEQ ID NOS:1 or 2.
[0266] In some embodiments, the B103 polymerase can optionally the
amino acid sequence of SEQ ID NOS:1 or 2, and further comprise one
or more amino acid substitutions selected from the group consisting
of (in single letter amino acid code): H370G, H370T, H370S, H370K,
H370R, H370A, H370Q, H370 W, H370Y, H370F, E371G, E371H, E371T,
E3715, E371K, E371R, E371A, E371Q, E371W, E371Y, E371F, K372G,
K372E, K372T, K372S, K372R, K372A, K372Q, K372W, K372Y, K372F,
K380E, K380T, K380S, K380R, K380A, K380Q, K380W, K380Y, K380F,
D507H, D507G, D507E, D507T, D5075, D507R, D507A, D507R, D507Q,
D507W, D507Y, D507F, K509H, K509G, K509D, K509R, K509E, K509T,
K5095, K509R, K509A, K509Q, K509W, K509Y and K509F, wherein the
numbering is relative to the sequence shown in SEQ ID NOS:1 or 2.
The B103 polymerase can optionally further comprise the amino acid
sequence of any of the polymerases disclosed by Hendricks, in U.S.
Ser. No. 61/242,771, filed on Sep. 15, 2009, or U.S. Ser. No.
61/293,618, filed on Jan. 8, 2010.
[0267] Polymerases having desirable properties, including those
having altered nucleotide binding and/or nucleotide incorporation
kinetics, having improved photo-stability, and/or having improved
enzymatic activity when conjugated to an energy transfer moiety,
include polymerases according to SEQ ID NOS:1-5.
TABLE-US-00001 SEQ ID NO: 1
MPRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYKIGNSLDEFMQWVME
IQADLYFHNLKFDGAFIVNWLEHHGFKWSNEGLPNTYNTIISKMGQWYMI
DICFGYKGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHAERP
VGHEITPEEYEYIKNAIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILS
TKKFNKVFPKLSLPMDKEIRRAYRGGFTWLNDKYKEKEIGEGMVFDVNSL
YPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQ
IKKNPFFKGNEYLKNSGAEPVELYLTNVDLELIQEHYEMYNVEYIDGFKF
REKTGLFKEFIDKWTYVKTHEKGAKKQLAKLMLNSLYGKFASNPDVTGKV
PYLKEDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRII
YCDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQD
IYAKEVDGKLIECSPDEATTTKFSVKCAGMTDTIKKKVTFDNFRVGFSST
GKPKPVQVNGGVVLVDSVFTIK SEQ ID NO: 2
MPRKMFSCDFETTTKLDDCRVWAYGYMEIGNLDNYKIGNSLDEFMQWVME
IQADLYFHNLKFDGAFIVNWLEHHGFKWSNEGLPNTYNTIISKMGQWYMI
DICFGYKGKRKLHTVIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHAERP
VGHEITPEEYEYIKNDIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILS
TKKFNKVFPKLSLPMDKEIRRAYRGGFTWLNDKYKEKEIGEGMVFDVNSL
YPSQMYSRPLPYGAPIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQ
IKKNPFFKGNEYLKNSGAEPVELYLTNVDLELIQEHYEMYNVEYIDGFKF
REKTGLFKEFIDKWTYVKTHEKGAKKQLAKLMLNSLYGKFASNPDVTGKV
PYLKEDGSLGFRVGDEEYKDPVYTPMGVFITAWARFTTITAAQACYDRII
YCDTDSIHLTGTEVPEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQD
IYAKEVDGKLIECSPDEATTTKFSVKCAGMTDTIKKKVTFDNFRVGFSST
GKPKPVQVNGGVVLVDSVFTIK SEQ ID NO: 3
MSHHHHHHSMSGLNDIFEAQKIEWHEGAPGARGSKHMPRKMFSCDFETTT
KLDDCRVWAYGYMEIGNLDNYKIGNSLDEFMQWVMEIQADLYFHNLKFDG
AFIVNWLEHHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHT
VIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHAERPVGHEITPEEYEYIK
NAIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLP
MDKEIRRAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPYGA
PIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLK
NSGAEPVELYLTNVDLELIQEHYEMYNVEYIDGFKFREKTGLFKEFIDKW
TYVKTREKGAKKQLAKLMLNSLYGKFASNPDVTGKVPYLKEDGSLGFRVG
DEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEV
PEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYAKEVDGKLIECS
PDEATTTKFSVKCAGMTDTIKKKVTFDNFRVGFSSTGKPKPVQVNGGVVL VDSVFTIK SEQ ID
NO: 4 MNHLVHHHHHHIEGRHMELGTLEGSMKHMPRKMFSCDFETTTKLDDCRVW
AYGYMEIGNLDNYKIGNSLDEFMQWVMEIQADLYFHNLKFDGAFIVNWLE
HHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHTVIYDSLKK
LPFPVKKIAKDFQLPLLKGDIDYHAERPVGHEITPEEYEYIKNDIEIIAR
ALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLPMDKEIRRA
YRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPYGAPIVFQGKY
EKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLKNSGAEPVE
LYLTNVDLELIQEHYEMYNVEYIDGFKFREKTGLFKEFIDKWTYVKTHEK
GAKKQLAKLMLNSLYGKFASNPDVTGKVPYLKEDGSLGFRVGDEEYKDPV
YTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEVPEIIKDIV
DPKKLGYWAHESTFKRAKYLRQKTYIQDIYAKEVDGKLIECSPDEATTTK
FSVKCAGMTDTIKKKVTFDNFRVGFSSTGKPKPVQVNGGVVLVDSVFTIK SEQ ID NO: 5
MSHHHHHHSMSGLNDIFEAQKIEWHEGAPGARGSKHMPRKMFSCDFETTT
KLDDCRVWAYGYMEIGNLDNYKIGNSLDEFMQWVMEIQADLYFHNLKFDG
AFIVNWLEHHGFKWSNEGLPNTYNTIISKMGQWYMIDICFGYKGKRKLHT
VIYDSLKKLPFPVKKIAKDFQLPLLKGDIDYHAERPVGHEITPEEYEYIK
NAIEIIARALDIQFKQGLDRMTAGSDSLKGFKDILSTKKFNKVFPKLSLP
MDKEIRRAYRGGFTWLNDKYKEKEIGEGMVFDVNSLYPSQMYSRPLPYGA
PIVFQGKYEKDEQYPLYIQRIRFEFELKEGYIPTIQIKKNPFFKGNEYLK
NSGAEPVELYLTNVDLELIQEHYEMYNVEYIDGFKFREKTGLFKEFIDKW
TYVKTHEKGAKKQLAKLMLNSLYGKFASNPDVTGKVPYLKEDGSLGFRVG
DEEYKDPVYTPMGVFITAWARFTTITAAQACYDRIIYCDTDSIHLTGTEV
PEIIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYAKEVDGKLIECS
PDEATTTKFSVKCAGMTDTIKKKVTFDNFRVGFSSTGKPKPVQVNGGVVL VDSVFTIK SEQ ID
NO: 6 MKHMPRKMYSCDFETTTKVEDCRVWAYGYMNIEDHSEYKIGNSLDEFMAW
VLKVQADLYFHNLKFDGAFIINWLERNGFKWSADGLPNTYNTIISRMGQW
YMIDICLGYKGKRKIHTVIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHK
ERPVGYKITPEEYAYIKNDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKD
IITIKKFKKVFPTLSLGLDKEVRYAYRGGFTWLNDRFKEKEIGEGMVFDV
NSLYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIP
TIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISG
LKFKATTGLFKDFIDKWTYIKTTSEGAIKQLAKLMLNSLYGKFASNPDVT
GKVPYLKENGALGFRLGEEETKDPVYTPMGVFITAWARYTTITAAQACYD
RIIYCDTDSIHLTGTEIPDVIKDIVDPKKLGYWAHESTFKRAKYLRQKTY
IQDIYMKEVDGKLVEGSPDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGF
SRKMKPKPVQVPGGVVLVDDTFTIK SEQ ID NO: 7
MGLRRASLHHLLGGGGSGGGGSAAAGSAARKMYSCDFETTTKVEDCRVWA
YGYMNIEDHSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIINWLER
NGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIHTVIYDSLKKL
PFPVKKIAKDFKLTVLKGDIDYHKERPVGYKITPEEYAYIKNDIQIIAEA
LLIQFKQGLDRMTAGSDSLKGFKDIITIKKFKKVFPTLSLGLDKEVRYAY
RGGFTWLNDRFKEKEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYV
WDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADL
WLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSEG
AIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVY
TPMGVFITAWARYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVD
PKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLVEGSPDDYTDIKF
SVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK SEQ ID NO: 8
MHHHHHHLLGGGGSGGGGSAAAGSAARKMYSCDFETTTKVEDCRVWAYGY
MNIEDHSEYKIGNSLDEFMAWVLKVQADLYFHNLKFDGAFIINWLERNGF
KWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIHTVIYDSLKKLPFP
VKKIAKDFKLTVLKGDIDYHKERPVGYKITPEEYAYIKNAIQIIAEALLI
QFKQGLDRMTAGSDSLKGFKDIITIKKFKKVFPTLSLGLDKEVRYAYRGG
FTWLNDRFKEKEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDE
DYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIADLWLS
NVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSEGAIK
QLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPVYTPM
GVFITAWARYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKK
LGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLVEGSPDDYTDIKFSVK
CAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK SEQ ID NO: 9
MNHLVHHHHHHIEGRHMELGTLEGSMKHMPRKMYSCAFETTTKVEDCRVW
AYGYMNIEDHSEYKIGNSLDEFMAWVLKVQADLYFHNLKFAGAFIINWLE
RNGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIHTVIYDSLKK
LPFPVKKIAKDFKLTVLKGDIDYHKERPVGYKITPEEYAYIKNDIQIIAE
ALLIQFKQGLDRMTAGSDSLKGFKDIITIKKFKKVFPILSLGLDKEVRYA
YRGGFTWLNDRFKEKEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKY
VWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIAD
LWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSE
GAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPV
YTPMGVFITAWARYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIV
DPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLVEGSPDDYTDIK
FSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK SEQ ID NO: 10
MNHLVHHHHHHIEGRHMELGTLEGSMKHMPRKMYSCAFETTTKVEDCRVW
AYGYMNIEDHSEYKIGNSLDEFMAWVLKVQADLYFHNLKFAGAFIINWLE
RNGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIHTVIYDSLKK
LPFPVKKIAKDFKLTVLKGDIDYHKERPVGYKITPEEYAYIKNDIQIIAE
ALLIQFKQGLDRMTAGSDSLKGFKDIITIKKFKKVFPILSLGLDKEVRYA
YRGGFTWLNDRFKEKEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKY
VWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIAD
LWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSE
GAIKALAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPV
YTPMGVFITAWARYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIV
DPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLVEGSPDDYTDIK
FSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK SEQ ID NO: 11
MNHLVHHHHHHIEGRHMELGTLEGSMKHMPRKMYSCAFETTTKVEDCRVW
AYGYMNIEDHSEYKIGNSLDEFMAWVLKVQADLYFHNLKFAGAFIINWLE
RNGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIHTVIYDSLKK
LPFPVKKIAKDFKLTVLKGDIDYHKERPVGYKITPEEYAYIKNDIQIIAE
ALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLDKEVRYA
YRGGFTWLNDRFKEKEIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKY
VWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLKSSGGEIAD
LWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKWTYIKTTSE
GAIKQLAKLMLNGLYGKFASNPDVTGKVPYLKENGALGFRLGEEETKDPV
YTPMGVFITAWARYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIV
DPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLVEGSPDDYTDIK
FSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIK SEQ ID NO: 12
MSHHHHHHSMSGLNDIFEAQKIEWHEGAPGARGSKHMPRKMYSCAFETTT
KVEDCRVWAYGYMNIEDHSEYKIGNSLDEFMAWVLKVQADLYFHNLKFAG
AFIINWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIHT
VIYDSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYKITPEEYAYIK
NDIQIIAEALLIQFKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLG
LDKEVRYAYRGGFTWLNDRFKEKEIGEGMVFDVNSLYPAQMYSRLLPYGE
PIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIPTIQIKRSRFYKGNEYLK
SSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLFKDFIDKW
TYIKTTSEGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLG
EEETKDPVYTPMGVFITAWARYTTITAAQACYDRIIYCDTDSIHLTGTEI
PDVIKDIVDPKKLGYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLVEGS
PDDYTDIKFSVKCAGMTDKIKKEVTFENFKVGFSRKMKPKPVQVPGGVVL VDDTFTIK SEQ ID
NO: 13 MKEFYLTVEQIGDSIFERYIDSNGRERTREVEYKPSLFAHCPESQATKYF
DIYGKPCTRKLFANMRDASQWIKRMEDIGLEALGMDDFKLAYLSDTYNYE
IKYDHTKIRVANFDIEVTSPDGFPEPSQAKHPIDAITHYDSIDDRFYVFD
LLNSPYGNVEEWSIEIAAKLQEQGGDEVPSEIIDKIIYMPFDNEKELLME
YLNFWQQKTPVILTGWNVESFDIPYVYNRIKNIFGESTAKRLSPHRKTRV
KVIENMYGSREIITLFGISVLDYIDLYKKFSFTNQPSYSLDYISEFELNV
GKLKYDGPISKLRESNHQRYISYNIIDVYRVLQIDAKRQFINLSLDMGYY
AKIQIQSVFSPIKTWDAIIFNSLKEQNKVIPQGRSHPVQPYPGAFVKEPI
PNRYKYVMSFDLTSLYPSIIRQVNISPETIAGTFKVAPLHDYINAVAERP
SDVYSCSPNGMMYYKDRDGVVPTEITKVFNQRKEHKGYMLAAQRNGEIIK
EALHNPNLSVDEPLDVDYRFDFSDEIKEKIKKLSAKSLNEMLFRAQRTEV
AGMTAQINRKLLINSLYGALGNVWFRYYDLRNATAITTFGQMALQWIERK
VNEYLNEVCGTEGEAFVLYGDTDSIYVSADKIIDKVGESKFRDTNHWVDF
LDKFARERMEPAIDRGFREMCEYMNNKQHLMFMDREAIAGPPLGSKGIGG
FWTGKKRYALNVWDMEGTRYAEPKLKIMGLETQKSSTPKAVQKALKECIR
RMLQEGEESLQEYFKEFEKEFRQLNYISIASVSSANKIAKYDVGGFPGPK
CPFHIRGILTYNRAIKGNIDAPQVVEGEKVYVLPLREGNPFGDKCIAWPS
GTEITDLIKDDVLHWMDYTVLLEKTFIKPLEGFTSAAKLDYEKKASLFDM FDF
Fusion Proteins
[0268] In one aspect, the polymerase can be a fusion protein
comprising the amino acid sequence of a nucleic acid-dependent
polymerase (the polymerase portion) linked to the amino acid
sequence of a second enzyme or a biologically active fragment
thereof (the second enzyme portion). The second enzyme portion of
the fusion protein may be linked to the amino or carboxyl end of
the polymerase portion, or may be inserted within the polymerase
portion. The polymerase portion of the fusion protein may be linked
to the amino or carboxyl end of the second enzyme portion, or may
be inserted within the second enzyme portion. In some embodiments,
the polymerase and second enzyme portions can be linked to each
other in a manner which does not significantly interfere with
polymerase activity of the fusion or with the ability of the fusion
to bind nucleotides, or does not significantly interfere with the
activity of the second enzyme portion. In the fusion protein, the
polymerase portion or the second enzyme portions can be linked with
at least one energy transfer donor moiety. The fusion protein can
be a recombinant protein having a polymerase portion and a second
enzyme portion. In some embodiments, the fusion protein can include
a polymerase portion chemically linked to the second enzyme
portion.
Evolved Polymerases
[0269] The polymerase can be a modified polymerase having certain
desired characteristics, such as an evolved polymerase selected
from a directed or non-directed molecular evolution procedure. The
evolved polymerase can exhibit modulated characteristics or
functions, such as changes in: affinity, specificity, or binding
rates for substrates (e.g., target molecules, polymerization
initiation sites, or nucleotides); binding stability to the
substrates (e.g., target molecules, polymerization initiation
sites, or nucleotides); nucleotide incorporation rate; nucleotide
permissiveness; exonuclease activity (e.g., 3'.fwdarw.5' or
5'.fwdarw.3'); rate of extension; processivity; fidelity;
stability; or sensitivity and/or requirement for temperature,
chemicals (e.g., DTT), salts, metals, pH, or electromagnetic energy
(e.g., excitation or emitted energy). Many examples of evolved
polymerases having altered functions or activities can be found in
U.S. provisional patent application No. 61/020,995, filed Jan. 14,
2008.
[0270] Methods for creating and selecting proteins and enzymes
having the desired characteristics are known in the art, and
include: oligonucleotide-directed mutagenesis in which a short
sequence is replaced with a mutagenized oligonucleotide;
error-prone polymerase chain reaction in which low-fidelity
polymerization conditions are used to introduce point mutations
randomly across a sequence up to about 1 kb in length (R. C.
Caldwell, et al., 1992 PCR Methods and Applications 2:28-33; H.
Gramm, et al., 1992 Proc. Natl. Acad. Sci. USA 89:3576-3580); and
cassette mutagenesis in which a portion of a sequence is replaced
with a partially randomized sequence (A. R. Oliphant, et al., 1986
Gene 44:177-183; J. D. Hermes, et al., 1990 Proc. Natl. Acad. Sci.
USA 87:696-700; A. Arkin and D. C. Youvan 1992 Proc. Natl. Acad.
Sci. USA 89:7811-7815; E. R. Goldman and D. C. Youvan 1992
Bio/Technology 10:1557-1561; Delagrave et al., 1993 Protein
Engineering 6: 327-331; Delagrave et al., 1993 Bio/Technology 11:
1548-155); and domain shuffling.
[0271] Methods for creating evolved antibody and antibody-like
polypeptides can be adapted for creating evolved polymerases, and
include applied molecular evolution formats in which an
evolutionary design algorithm is applied to achieve specific mutant
characteristics. Many library formats can be used for evolving
polymerases including: phage libraries (J. K. Scott and G. P. Smith
1990 Science 249:386-390; S. E. Cwirla, et al. 1990 Proc. Natl.
Acad. Sci. USA 87:6378-6382; J. McCafferty, et al. 1990 Nature
348:552-554) and lad (M. G. Cull, et al., 1992 Proc. Natl. Acad.
Sci. USA 89:1865-1869).
[0272] Another adaptable method for evolving polymerases employs
recombination (crossing-over) to create the mutagenized
polypeptides, such as recombination between two different plasmid
libraries (Caren et al. 1994 Bio/Technology 12: 517-520), or
homologous recombination to create a hybrid gene sequence
(Calogero, et al., 1992 FEMS Microbiology Lett. 97: 41-44; Galizzi
et al., WO91/01087). Another recombination method utilizes host
cells with defective mismatch repair enzymes (Radman et al.,
WO90/07576). Other methods for evolving polymerases include random
fragmentation, shuffling, and re-assembly to create mutagenized
polypeptides (published application No. U.S. 2008/0261833,
Stemmer). Adapting these mutagenesis procedures to generate evolved
polymerases is well within the skill of the art.
[0273] In some embodiments, the polymerase can be fused with, or
otherwise engineered to include, DNA-binding or other domains from
other proteins that are capable of modulating DNA polymerase
activity. For example, fusion of suitable portions of the
Single-Stranded DNA Binding Protein (SSBP), thioredoxin and/or T7
DNA polymerase to bacterial or viral DNA polymerases has been shown
to enhance both the processivity and fidelity of the DNA
polymerase. Similarly, other groups have described efforts to
engineer polymerases so as to broaden their substrate range. See,
e.g., Ghadessy et al, Nat. Biotech., 22 (6):755-759 (2004).
Similarly, the conjugates of the present disclosure can optionally
comprise any polymerase engineered to provide suitable performance
characteristics, including for example a polymerase fused to intact
SSBP or fragments thereof, or to domains from other DNA-binding
proteins (such as the herpes simplex virus UL42 protein.)
[0274] In some embodiments, a blend of different conjugates, each
of which comprises a polymerase of unique sequence and
characteristics, can be used according to the methods described
herein. Use of such conjugate blends can additionally increase the
fidelity and processivity of DNA synthesis. For example, use of a
blend of processive and non-processive polymerases has been shown
to result in increased overall read length during DNA synthesis, as
described in U.S. Published App. No. 2004/0197800. Alternatively,
conjugates comprising polymerases of different affinities for
specific acceptor-labeled nucleotides can be used so as to achieve
efficient incorporation of all four nucleotides.
[0275] In one embodiment, the polymerase can be a mutant which
retains nucleotide polymerization activity but lacks the
3'.fwdarw.5' or 5'.fwdarw.3' exonuclease activity (SEQ ID
NOS:1-12). In another embodiment, the polymerase can be an
exonuclease minus mutant which is based on wild type phi29
polymerase (SEQ ID NO:6) (Blanco, U.S. Pat. Nos. 5,001,050,
5,198,543, and 5,576,204; and Hardin PCT/US2009/31027 with an
International filing date of Jan. 14, 2009) and comprising one or
more substitution mutations, including: D12A, D66A, D169A, H61R,
N62D, Q380A, and/or S388G, and any combination thereof.
[0276] In some embodiments, the polymerase can comprise the amino
acid sequence of any polymerase disclosed in U.S. Provisional
Application Nos. 61/242,771, filed on Sep. 15, 2009; 61/263,974,
filed on Nov. 24, 2009 and 61/299,919, filed on Jan. 29, 2010, or
any variant thereof.
Polymerases Linked with Energy Transfer Moieties
[0277] The polymerase (or polymerase fusion protein) may be linked
with at least one energy transfer donor moiety. In the polymerase
fusion protein, the energy transfer donor moiety can be attached to
the polymerase portion or to the second enzyme portion. One or more
energy transfer donor moieties can be linked to the polymerase (or
polymerase fusion protein) at the amino end or carboxyl end or may
be inserted in the interior of the polymerase (or fusion protein
sequence). The energy transfer donor moiety can be attached to the
fusion protein in a manner which does not interfere with the
nucleotide binding activity, or with the nucleotide incorporation
activity, or with the activity of the second enzyme.
[0278] In one aspect, a single energy transfer donor moiety can be
operably attached with more than one polymerase (or more than one
polymerase fusion protein) and the attachment can be at the amino
end or carboxyl end or may be inserted within the polymerase (or
fusion protein sequence).
[0279] In another aspect, a single energy transfer donor moiety can
be linked to one polymerase or polymerase fusion protein.
Target Nucleic Acid Molecules
[0280] The methods, compositions, systems and kits disclosed herein
can involve the use of target nucleic acid molecules. The target
nucleic acid molecule may be single or double-stranded molecules.
The target nucleic acid molecules can be linear or circular. The
target nucleic acid molecules may be DNA, RNA or hybrid DNA-RNA
molecules, DNA hairpins, DNA/RNA hybrids, or RNA hairpins. The
target nucleic acid molecules may be isolated in any form including
chromosomal, genomic, organellar (e.g., mitochondrial, chloroplast
or ribosomal), recombinant molecules, cloned, amplified, cDNA, RNA
such as precursor mRNA or mRNA, oligonucleotide, or any type of
nucleic acid library. The target nucleic acid molecules may be
isolated from any source including from: organisms such as
prokaryotes, eukaryotes (e.g., humans, plants and animals), fungus,
and viruses; cells; tissues; body fluids including blood, urine,
serum, lymph, tumor, saliva, anal and vaginal secretions, amniotic
samples, perspiration, and semen; environmental samples; culture
samples; or synthesized nucleic acid molecules prepared using
recombinant molecular biology or chemical synthesis methods.
[0281] The target nucleic acid molecules comprise
naturally-occurring nucleotides, nucleotide variants, or any
combination thereof. For example, the target molecules comprise
alternate backbones, including: phosphoramidate; phosphorothioate;
phosphorodithioate; O-methylphosphoroamidite linkages; and peptide
nucleic acid backbones and linkages. Other nucleic acids include
those with bicyclic structures including locked nucleic acids;
positive backbones; non-ionic backbones; and non-ribose
backbones.
[0282] The target nucleic acid molecules can carry a tag (e.g.,
His-tag), a polynucleotide tail (e.g., polynucleotide tail of A, G,
C, T, or U), or can be methylated. The target nucleic acid
molecules may be nicked, sheared, or treated with an enzyme such as
a restriction endonuclease or a nuclease. The target nucleic acid
molecules can be about 10-50 nucleotides, about 50-100 nucleotides,
about 100-250 nucleotides, about 250-500 nucleotides, or about
500-1000 nucleotides in length, or longer. The target nucleic acid
molecules may be linked to an energy transfer moiety (e.g., donor
or acceptor) or to a reporter moiety (e.g., dye) using methods well
known in the art.
[0283] The target nucleic acid molecules can have a nucleotide
sequence which has been previously determined or is unknown (e.g.,
de novo sequencing). The target molecule can be fragmented into
shorter pieces and/or modified for immobilization. Selection of the
fragmentation and modification technique may depend upon the
desired fragment sizes and subsequent preparation steps. Any
combination of fragmentation and/or modification techniques may be
practiced in any order.
Single- or Double-Stranded Nucleic Acid Molecules
[0284] The target molecules can be single-stranded nucleic acid
molecules which are isolated by denaturing double-stranded
molecules, or by chemically synthesizing single-stranded molecules.
The target molecules can be double-stranded nucleic acid molecules.
The single-stranded molecules can be isolated away from
double-stranded molecules by bead (e.g., magnetic, biotinylated, or
probe capture) attachment and enrichment procedures, CsCl gradient
centrifugation methods, gel electrophoresis (e.g., polyacrylamide),
or by capillary gel electrophoresis. The nucleic acid molecules can
be attached to the beads via covalent or non-covalent linkage.
Nucleic Acid Sample Preparation
[0285] The nucleic acid molecules, including the target molecules,
primers, and oligonucleotides, may be isolated and modified at
their ends and/or the interior of the molecules using well known
procedures, including: fragmentation, ligation, hybridization,
enzymatic, and/or chemical modification, conjugation with an energy
transfer (donor or acceptor) or reporter moiety, or any combination
of these procedures.
Nucleic Acid Molecules--Fragmentation
[0286] Techniques which fragment the nucleic acid molecules at
random or specific sites, or a combination of these techniques can
be used.
[0287] The nucleic acid molecules can be fragmented at random or
specific sites using any fragmentation procedures. The nucleic acid
molecules can be fragmented using mechanical force, including:
shear forces (e.g., small orifice or a needle); nebulization (S.
Surzycki 1990 In: "The International Conference on the Status and
Future of Research on the Human Genome. Human Genome II", San
Diego, Calif., pp. 51; and S. J. Surzycki, 2000 in: "Basic Methods
in Molecular Biology", New York, N.Y.: Springer-Verlag); or
sonication. For example, nucleic acid molecules can be fragmented
by sonicating in a COVARIS (e.g., Models S2, E210, or AFA).
[0288] The nucleic acid molecules can be chemically fragmented
using, for example: acid-catalyzed hydrolysis of the backbone and
cleavage with piperidine; internucleosomal DNA fragmentation using
a copper (II) complex of 1,10-phenanthroline (o-phenanthroline,
OP), CuII(OP).sub.2 in the presence of ascorbic acid (Shui Ying
Tsang 1996 Biochem. Journal 317:13-16).
[0289] The nucleic acid molecules can be enzymatically fragmented
using type I, II or II restriction endonucleases (N. E. Murray 2000
Microbiol. Mol. Biol. Rev. 64: 412-34; A. Pingoud and A. Jeltsch
2001 Nucleic Acids Res. 29: 3705-27; D. T. Dryden, et al., 2001
Nucleic Acids Res. 29: 3728-41; and A. Meisel, et al., 1992 Nature
355: 467-9). Enzymatic cleavage of DNA may include digestion using
various ribo- and deoxyribonucleases or glycosylases. The nucleic
acid molecules can be digested with DNase I or II. The nucleic acid
fragments can be generated by enzymatically copying an RNA
template. Fragments can be generated using processive enzymatic
degradation (e.g., S1 nuclease). The enzymatic reactions can be
conducted in the presence or absence of salts (e.g., Mg.sup.2+,
Mn.sup.2+, and/or Ca.sup.2+), and the pH and temperature conditions
can be varied according to the desired rate of reaction and
results, as is well known in the art.
Modified Nucleic Acid Molecules
[0290] The 5' or 3' overhang ends of a nucleic acid molecule can be
converted to blunt-ends using a "fill-in" procedure (e.g., dNTPS
and DNA polymerase, Klenow, or Pfu or T4 polymerase) or using
exonuclease procedure to digest away the protruding end.
[0291] The nucleic acid molecule ends can be ligated to one or more
oligonucleotides using DNA ligase or RNA ligase. The nucleic acid
molecules can be hybridized to one or more oligonucleotides. The
oligonucleotides can serve as linkers, adaptors, bridges, clamps,
anchors, or capture oligonucleotides.
[0292] The oligonucleotides can be ligation-ready, having over-hang
ends which can be ligated to the ends of the target molecules. The
ligation-ready oligonucleotides can be used to circularize the
target molecules.
[0293] A pair of oligonucleotides can include complementary
sequences for hybridization. These paired oligonucleotides can be
used as end-ligated oligonucleotides to permit circularization of
the target molecule. These paired oligonucleotides can be used to
hybridize to capture probes immobilized on a surface.
[0294] The oligonucleotides can include sequences which are: enzyme
recognition sequences (e.g., restriction endonuclease recognition
sites, DNA or RNA polymerase recognition sites); hybridization
sites; or can include a detachable portion.
[0295] The oligonucleotide can be linked to a protein-binding
molecule such as biotin or streptavidin.
[0296] The oligonucleotides can be 4-20 nt/bp in length, or 20-40
nt/bp in length, or 40-60 nt/bp in length, or longer.
Enzymatic and Chemical Modifications
[0297] The nucleic acid molecules can be methylated, for example,
to confer resistance to restriction enzyme digestion (e.g.,
EcoRI).
[0298] The nucleic acid molecule ends can be phosphorylated or
dephosphorylated.
[0299] A nick can be introduced into the nucleic acid molecules
using, for example DNase I. A pre-designed nick site can be
introduced in dsDNA using a double stranded probe, type II
restriction enzyme, ligase, and dephosphorylation (Fu Dong-Jing,
1997 Nucleic Acids Research 25:677-679).
[0300] A nick can be repaired using polymerase (e.g., DNA po1 I or
phi29), ligase (e.g., T4 ligase) and kinase (polynucleotide
kinase).
[0301] A poly tail can be added to the 3' end of the fragment using
terminal transferase (e.g., polyA, polyG, polyC, polyT, or
polyU).
[0302] The target nucleic acid molecule can include pre-existing
methylation sites. The target molecule can be modified using
bisulfite treatment (e.g., disodium bisulfite) to convert
unmethylated cytosines to uracils, which permits detection of
methylated cytosines using, for example, methylation specific
procedures (e.g., PCR or bisulfite genomic sequencing).
Size Selection
[0303] The nucleic acid molecules can be size selected, or the
desired nucleic acid molecules can separated from undesirable
molecules, using any art known methods, including gel
electrophoresis, size exclusion chromatography (e.g., spin
columns), sucrose sedimentation, or gradient centrifugation. Very
large nucleic acid molecules, including whole chromosomes, can be
size separated using pulsed-field gel electrophoresis (Schwartz and
Cantor 1984 Cell, 37: 67-75).
Amplification
[0304] The nucleic acid molecules can be amplified using methods,
including: polymerase chain reaction (PCR); ligation chain
reaction, which is sometimes referred to as oligonucleotide ligase
amplification (OLA); cycling probe technology (CPT); strand
displacement assay (SDA); transcription mediated amplification
(TMA); nucleic acid sequence based amplification (NASBA); rolling
circle amplification (RCA); and invasive cleavage technology.
Enrichment
[0305] Undesired compounds, or undesired fragments, can be removed
or separated from the desired target nucleic acid molecules to
facilitate enrichment of the desired target molecules. Enrichment
methods can be achieved using well known methods, including gel
electrophoresis, chromatography, or solid phase immobilization
(reversible or non-reversible). For example, AMPURE beads
(Agencourt) can bind DNA fragments but not bind unincorporated
nucleotides, free primers, DNA polymerases, and salts, thereby
facilitating enrichment of the desired DNA fragments.
Embodiments of the Target Molecule
[0306] In one embodiment, the target molecule can be a recombinant
DNA molecule which is a self-priming hairpin oligonucleotide. The
hairpin oligonucleotide can be linked at the 5' or 3' end, or
internally, to at least one molecule of a binding partner (e.g.,
biotin). The biotin molecule can be used to immobilize the hairpin
oligonucleotide to the surface (via avidin-like molecule), or for
attachment to a reporter moiety. The hairpin oligonucleotide can be
linked to at least one energy transfer moiety, such as a
fluorescent dye or a nanoparticle.
[0307] In another embodiment, a plurality of target nucleic acid
molecules can be linked to a solid surface (via the 5' or 3' end,
or via an internal site) to form a DNA curtain (see Greene, U.S.
published patent application No. 2008/0274905, published on Nov. 6,
2008; and Fazio, et al., 2008 Langmuir 24:10524-10531). The
nucleotide incorporation methods can be practiced on the DNA
curtain in an aqueous flowing condition.
Reporter Moieties
[0308] The methods, systems, compositions and kits disclosed herein
can involve the use of one or more reporter moieties which are
linked to the solid surfaces, nanoparticles, polymerases,
nucleotides, target nucleic acid molecules, primers, and/or
oligonucleotides.
[0309] The reporter moieties may be selected so that each absorbs
excitation radiation and/or emits fluorescence at a wavelength
distinguishable from the other reporter moieties to permit
monitoring the presence of different reporter moieties in the same
reaction. Two or more different reporter moieties can be selected
having spectrally distinct emission profiles, or having minimal
overlapping spectral emission profiles.
[0310] In one aspect, the signals (e.g., energy transfer signals)
from the different reporter moieties do not significantly overlap
or interfere, by quenching, colorimetric interference, or spectral
interference.
[0311] The chromophore moiety may be 5-bromo-4-chloro-3-indolyl
phosphate, 3-indoxyl phosphate, p-nitrophenyl phosphate,
.beta.-lactamase, peroxidase-based chemistry, and derivatives
thereof.
[0312] The chemiluminescent moiety may be a phosphatase-activated
1,2-dioxetane compound. The 1,2-dioxetane compound includes
disodium
2-chloro-5-(4-methoxyspiro[1,2-dioxetane-3,2'-(5-chloro-)tricyclo[3,3,1-1-
.sup.3,7]-decan]-1-yl)-1-phenyl phosphate (e.g., CDP-STAR),
chloroadamant-2'-ylidenemethoxyphenoxy phosphorylated dioxetane
(e.g., CSPD), and
3-(2'-spiroadamantane)-4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2-dioxetan-
e (e.g., AMPPD).
[0313] In some embodiments, the fluorescent moiety can optionally
include: rhodols; resorufins; coumarins; xanthenes; acridines;
fluoresceins; rhodamines; erythrins; cyanins; phthalaldehydes;
naphthylamines; fluorescamines; benzoxadiazoles; stilbenes;
pyrenes; indoles; borapolyazaindacenes; quinazolinones; eosin;
erythrosin; Malachite green; CY dyes (GE Biosciences), including
Cy3 (and its derivatives) and Cy5 (and its derivatives); DYOMICS
and DYLIGHT dyes (Dyomics) including DY-547, DY-630, DY-631,
DY-632, DY-633, DY-634, DY-635, DY-647, DY-649, DY-652, DY-678,
DY-680, DY-682, DY-701, DY-734, DY-752, DY-777 and DY-782; Lucifer
Yellow; CASCADE BLUE; TEXAS RED; BODIPY (boron-dipyrromethene)
(Molecular Probes) dyes including BODIPY 630/650 and BODIPY
650/670; ATTO dyes (Atto-Tec) including ATTO 390, ATTO 425, ATTO
465, ATTO 610 611X, ATTO 610 (N-succinimidyl ester), ATTO 635 (NHS
ester); ALEXA FLUORS including ALEXA FLUOR 633, ALEXA FLUOR 647,
ALEXA FLUOR 660, ALEXA FLUOR 700, ALEXA FLUOR 750, and ALEXA FLUOR
680 (Molecular Probes); DDAO
(7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one or any
derivatives thereof) (Molecular Probes); QUASAR dyes (Biosearch);
IRDYES dyes (LiCor) including IRDYE 700DX (NHS ester), IRDYE 800RS
(NHS ester) and IRDYE 800CW (NHS ester); EVOBLUE dyes (Evotech
Biosystems); JODA 4 dyes (Applied Biosystems); HILYTE dyes
(AnaSpec); MR121 and MR200 dyes (Roche); Hoechst dyes 33258 and
33242 (Invitrogen); FAIR OAKS RED (Molecular Devices); SUNNYVALE
RED (Molecular Devices); LIGHT CYCLER RED (Roche); EPOCH (Glen
Research) dyes including EPOCH REDMOND RED (phosphoramidate), EPOCH
YAKIMA YELLOW (phosphoramidate), EPOCH GIG HARBOR GREEN
(phosphoramidate); Tokyo green (M. Kamiya, et al., 2005 Angew.
Chem. Int. Ed. 44:5439-5441); and CF dyes including CF 647 and
CF555 (Biotium).
[0314] Quencher dyes may include: ATTO 540Q, ATTO 580Q, and ATTO
612Q (Atto-Tec); QSY dyes including QSY 7, QSY 9, QSY 21, and QSY
35 (Molecular Probes); and EPOCH ECLIPSE QUENCHER (phosphoramidate)
(Glen Research). The fluorescent moiety can be a
7-hydroxycoumarin-hemicyanine hybrid molecule which is a far-red
emitting dye (Richard 2008 Org. Lett. 10:4175-4178).
[0315] The fluorescent moiety may be a fluorescence-emitting metal
such as a lanthanide complex, including those of Europium and
Terbium.
[0316] A number of examples of fluorescent moieties are found in
PCT publication WO/2008/030115, and in Haugland, Molecular Probes
Handbook, (Eugene, Oreg.) 6th Edition; The Synthegen catalog
(Houston, Tex.), Lakowicz, Principles of Fluorescence Spectroscopy,
2nd Ed., Plenum Press New York (1999).
[0317] In one aspect, the reporter moieties can be energy transfer
moieties.
FRET
[0318] In some embodiments, the methods, compositions, systems and
kits disclosed herein can involve the use of one or more moieties
capable of undergoing energy transfer. Such energy transfer
moieties can include energy transfer donors and acceptors. The
energy transfer moieties can be linked to the solid surfaces,
nanoparticles, polymerases, nucleotides, target nucleic acid
molecules, primers, and/or oligonucleotides.
[0319] In one aspect, the energy transfer moiety can be an energy
transfer donor. For example, the energy transfer donor can be a
nanoparticle or an energy transfer donor moiety (e.g., fluorescent
dye). In another aspect, the energy transfer moiety can be an
energy transfer acceptor. For example, the energy transfer acceptor
can be an energy acceptor dye. In another aspect, the energy
transfer moiety can be a quencher moiety.
[0320] In one aspect, the energy transfer pair can be linked to the
same molecule. For example, the energy transfer donor and acceptor
pair can be linked to a single polymerase, which can provide
detection of conformational changes in the polymerase. In another
aspect, the donor and acceptor can be linked to different molecules
in any combination. For example, the donor can be linked to the
polymerase, target molecule, or primer molecule, and/or the
acceptor can be linked to the nucleotide, the target molecule, or
the primer molecule.
[0321] The energy transfer donor is capable of absorbing
electromagnetic energy (e.g., light) at a first wavelength and
emitting excitation energy in response. The energy acceptor is
capable of absorbing excitation energy emitted by the donor and
fluorescing at a second wavelength in response.
[0322] The donor and acceptor moieties can interact with each other
physically or optically in a manner which produces a detectable
signal (e.g., energy transfer signal) when the two moieties are in
proximity with each other. A proximity event includes two different
moieties (e.g., energy transfer donor and acceptor) approaching
each other, or associating with each other, or binding each
other.
[0323] The donor and acceptor moieties can transfer energy in
various modes, including: fluorescence resonance energy transfer
(FRET) (L. Stryer 1978 Ann. Rev. Biochem. 47: 819-846; Schneider,
U.S. Pat. No. 6,982,146; Hardin, U.S. Pat. No. 7,329,492; Hanzel
U.S. published patent application No. 2007/0196846), scintillation
proximity assays (SPA) (Hart and Greenwald 1979 Molecular
Immunology 16:265-267; U.S. Pat. No. 4,658,649), luminescence
resonance energy transfer (LRET) (G. Mathis 1995 Clin. Chem.
41:1391-1397), direct quenching (Tyagi et al, 1998 Nature
Biotechnology 16:49-53), chemiluminescence energy transfer (CRET)
(Campbell and Patel 1983 Biochem. Journal 216:185-194),
bioluminescence resonance energy transfer (BRET) (Y. Xu, et al.,
1999 Proc. Natl. Acad. Sci. 96:151-156), and excimer formation (J.
R. Lakowicz 1999 "Principles of Fluorescence Spectroscopy", Kluwer
Academic/Plenum Press, New York).
[0324] In one exemplary embodiment, the energy transfer moieties
can be a FRET donor/acceptor pair. FRET is a distance-dependent
radiationless transmission of excitation energy from a first
moiety, referred to as a donor moiety, to a second moiety, referred
to as an acceptor moiety. Typically, the efficiency of FRET energy
transmission is dependent on the inverse sixth-power of the
separation distance between the donor and acceptor, r. For a
typical donor-acceptor pair, r can vary between approximately
10-100 Angstroms. FRET is useful for investigating changes in
proximity between and/or within biological molecules. In some
embodiments, FRET efficiency may depend on donor-acceptor distance
r as 1/r.sup.6 or 1/r.sup.4. The efficiency of FRET energy transfer
can sometimes be dependent on energy transfer from a point to a
plane which varies by the fourth power of distance separation (E.
Jares-Erijman, et al., 2003 Nat. Biotechnol. 21:1387). The distance
where FRET efficiency is 50% is termed R.sub.0, also know as the
Forster distance. R.sub.0 is unique for each donor-acceptor
combination and may be about 1-20 nm, or about 1-10 nm, or about
1-5 nm, or about 5-10 nm. A change in fluorescence from a donor or
acceptor during a FRET event (e.g., increase or decrease in the
signal) can be an indication of proximity between the donor and
acceptor.
[0325] In biological applications, FRET can provide an on-off type
signal indicating when the donor and acceptor moieties are proximal
(e.g., within R.sub.0) of each other. Additional factors affecting
FRET efficiency include the quantum yield of the donor, the
extinction coefficient of the acceptor, and the degree of spectral
overlap between the donor and acceptor. Procedures are well known
for maximizing the FRET signal and detection by selecting high
yielding donors and high absorbing acceptors with the greatest
possible spectral overlap between the two (D. W. Piston and G. J.
Kremers 2007 Trends Biochem. Sci. 32:407). Resonance energy
transfer may be either an intermolecular or intramolecular event.
Thus, the spectral properties of the energy transfer pair as a
whole, change in some measurable way if the distance and/or
orientation between the moieties are altered.
[0326] The production of signals from FRET donors and acceptors can
be sensitive to the distance between donor and acceptor moieties,
the orientation of the donor and acceptor moieties, and/or a change
in the environment of one of the moieties (Deuschle et al. 2005
Protein Science 14: 2304-2314; Smith et al. 2005 Protein Science
14:64-73). For example, a nucleotide linked with a FRET moiety
(e.g., acceptor) may produce a detectable signal when it
approaches, associates with, or binds a polymerase linked to a FRET
moiety (e.g., donor). In another example, a FRET donor and acceptor
linked to one protein can emit a FRET signal upon conformational
change of the protein. Some FRET donor/acceptor pairs exhibit
changes in absorbance or emission in response to changes in their
environment, such as changes in pH, ionic strength, ionic type
(NO.sub.2. Ca.sup.+2, Mg.sup.+2, Zn.sup.+2, Na.sup.+, Cl.sup.-,
K.sup.+), oxygen saturation, and solvation polarity.
[0327] The FRET donor and/or acceptor may be a fluorophore,
luminophore, chemiluminophore, bioluminophore, or quencher (P.
Selvin 1995 Methods Enzymol 246:300-334; C. G. dos Remedios 1995 J.
Struct. Biol. 115:175-185; P. Wu and L. Brand 1994 Anal Biochem
218:1-13).
[0328] In some embodiments, the energy transfer moieties may not
undergo FRET, but may undergo other types of energy transfer with
each other, including luminescence resonance energy transfer,
bioluminescence resonance energy transfer, chemiluminescence
resonance energy transfer, and similar types of energy transfer not
strictly following the Forster's theory, such as the
non-overlapping energy transfer when non-overlapping acceptors are
utilized (Laitala and Hemmila 2005 Anal. Chem. 77: 1483-1487).
[0329] In one embodiment, the polymerase can be linked to an energy
transfer donor moiety. In another embodiment, the nucleotide can be
linked to an energy transfer acceptor moiety. For example, in one
embodiment the nucleotide comprises a polyphosphate chain and an
energy transfer moiety linked to the terminal phosphate group of
the polyphosphate chain. A change in a fluorescent signal can occur
when the labeled nucleotide is proximal to the labeled
polymerase.
[0330] In one embodiment, when an acceptor-labeled nucleotide is
proximal to a donor-labeled polymerase, the signal emitted by the
donor moiety decreases. In another embodiment, when the
acceptor-labeled nucleotide is proximal to the donor-labeled
polymerase, the signal emitted by the acceptor moiety increases. In
another embodiment, a decrease in donor signal and increase in
acceptor signal correlates with nucleotide binding to the
polymerase and/or correlates with polymerase-dependent nucleotide
incorporation.
Quenchers
[0331] The energy transfer moiety can be a FRET quencher.
Typically, quenchers have an absorption spectrum with large
extinction coefficients, however the quantum yield for quenchers is
reduced, such that the quencher emits little to no light upon
excitation. Quenching can be used to reduce the background
fluorescence, thereby enhancing the signal-to-noise ratio. In one
aspect, energy transferred from the donor may be absorbed by the
quencher which emits moderated (e.g., reduced) fluorescence. In
another aspect, the acceptor can be a non-fluorescent chromophore
which absorbs the energy transferred from the donor and emits heat
(e.g., the energy acceptor is a dark quencher).
[0332] For an example, a quencher can be used as an energy acceptor
with a nanoparticle donor in a FRET system, see I. L. Medintz, et
al., 2003 Nature Materials 2:630. One exemplary method involves the
use of quenchers in conjunction with reporters comprising
fluorescent reporter moieties. In this strategy, certain
nucleotides in the reaction mixture are labeled with a reporter
comprising a fluorescent label, while the remaining nucleotides are
labeled with one or more quenchers. Alternatively, each of the
nucleotides in the reaction mixture is labeled with one or more
quenchers. Discrimination of the nucleotide bases is based on the
wavelength and/or intensity of light emitted from the FRET
acceptor, as well as the intensity of light emitted from the FRET
donor. If no signal is detected from the FRET acceptor, a
corresponding reduction in light emission from the FRET donor
indicates incorporation of a nucleotide labeled with a quencher.
The degree of intensity reduction may be used to distinguish
between different quenchers.
[0333] Examples of fluorescent donors and non-fluorescent acceptor
(e.g., quencher) combinations have been developed for detection of
proteolysis (Matayoshi 1990 Science 247:954-958) and nucleic acid
hybridization (L. Morrison, in: Nonisotopic DNA Probe Techniques,
ed., L. Kricka, Academic Press, San Diego, (1992) pp. 31 1-352; S.
Tyagi 1998 Nat. Biotechnol. 16:49-53; S. Tyagi 1996 Nat.
Biotechnol. 14:947-8). FRET donors, acceptors and quenchers can be
moieties which absorb electromagnetic energy (e.g., light) at about
300-900 nm, or about 350-800 nm, or about 390-800 nm
Materials for Energy Transfer Moieties
[0334] Energy transfer donor and acceptor moieties can be made from
materials which typically fall into four general categories (see
the review in: K. E. Sapford, et al., 2006 Angew. Chem. Int. Ed.
45:4562-4588), including: (1) organic fluorescent dyes, dark
quenchers and polymers (e.g., dendrimers); (2) inorganic material
such as metals, metal chelates and semiconductors nanoparticles;
(3) biomolecules such as proteins and amino acids (e.g., green
fluorescent protein and derivatives thereof); and (4) enzymatically
catalyzed bioluminescent molecules. The material for making the
energy transfer donor and acceptor moieties can be selected from
the same or different categories.
[0335] The FRET donor and acceptor moieties which are organic
fluorescent dyes, quenchers or polymers can include traditional
dyes which emit in the UV, visible, or near-infrared region. The UV
emitting dyes include coumarin-, pyrene-, and naphthalene-related
compounds. The visible and near-infrared dyes include xanthene-,
fluorescein-, rhodol-, rhodamine-, and cyanine-related compounds.
The fluorescent dyes also includes DDAO
((7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one)),
resorufin, ALEXA FLUOR and BODIPY dyes (both Molecular Probes),
HILYTE Fluors (AnaSpec), ATTO dyes (Atto-Tec), DY dyes (Dyomics
GmbH), TAMRA (Perkin Elmer), tetramethylrhodamine (TMR), TEXAS RED,
DYLIGHT (Thermo Fisher Scientific), FAM (AnaSpec), JOE and ROX
(both Applied Biosystems), and Tokyo Green.
[0336] Additional fluorescent dyes which can be used as quenchers
includes: DNP, DABSYL, QSY (Molecular Probes), ATTO (Atto-Tec), BHQ
(Biosearch Technologies), QXL (AnaSpec), BBQ (Berry and Associates)
and CY5Q/7Q (Amersham Biosciences).
[0337] The FRET donor and acceptor moieties which comprise
inorganic materials include gold (e.g., quencher), silver, copper,
silicon, semiconductor nanoparticles, and fluorescence-emitting
metal such as a lanthanide complex, including those of Europium and
Terbium.
[0338] Suitable FRET donor/acceptor pairs include: FAM as the donor
and JOE, TAMRA, and ROX as the acceptor dyes. Other suitable pairs
include: CYA as the donor and R6G, TAMRA, and ROX as the donor
dyes. Other suitable donor/acceptor pairs include: a nanoparticle
as the donor, and ALEXA FLUORS dyes (e.g., 610, 647, 660, 680,
700). DYOMICS dyes, such as 634 and 734 can be used as energy
transfer acceptor dyes.
Nanoparticles
[0339] The methods, compositions, systems and kits disclosed herein
can involve the use of any suitable nanoparticles which can serve
as donor fluorophores in energy transfer reactions such as
FRET.
[0340] The nanoparticles can be attached to the solid surface or to
any component of the nucleotide incorporation or nucleotide
polymerization reactions in any combination (e.g., polymerases,
nucleotides, target nucleic acid molecules, primers, and/or
oligonucleotides).
[0341] "Nanoparticle" may refer to any particle with at least one
major dimension in the nanosize range. In general, nanoparticles
can be made from any suitable metal (e.g., noble metals,
semiconductors, etc.) and/or non-metal atoms. Nanoparticles can
have different shapes, each of which can have distinctive
properties including spatial distribution of the surface charge;
orientation dependence of polarization of the incident light wave;
and spatial extent of the electric field. The shapes include, but
are not limited to: spheres, rods, discs, triangles, nanorings,
nanoshells, tetrapods, nanowires, etc.
[0342] In one embodiment, the nanoparticle can be a core/shell
nanoparticle which typically comprises a core nanoparticle
surrounded by at least one shell. For example, the core/shell
nanoparticle can be surrounded by an inner and outer shell. In
another embodiment, the nanoparticle is a core nanoparticle which
has a core but no surrounding shell. The outmost surface of the
core or shell can be coated with tightly associated ligands which
are not removed by ordinary solvation.
[0343] Examples of a nanoparticle include a nanocrystal, such as a
core/shell nanocrystal, plus any associated organic ligands (which
are not removed by ordinary solvation) or other materials which may
coat the surface of the nanocrystal. In one embodiment, a
nanoparticle has at least one major dimension ranging from about 1
to about 1000 nm. In other embodiments, a nanoparticle has at least
one major dimension ranging from about 1 to about 20 nm, about 1 to
about 15 nm, about 1 to about 10 nm or about 1 to 5 nm.
[0344] In some embodiments, a nanoparticle can have a layer of
ligands on its surface which can further be cross-linked to each
other. In some embodiments, a nanoparticle can have other or
additional surface coatings which can modify the properties of the
particle, for example, increasing or decreasing solubility in water
or other solvents. Such layers on the surface are included in the
term `nanoparticle.`
[0345] In one embodiment, nanoparticle can refer to a nanocrystal
having a crystalline core, or to a core/shell nanocrystal, and may
be about 1 nm to about 100 nm in its largest dimension, about 1 nm
to about 20 nm, about 1 nm to about 15 nm, about 1 nm to about 10
nm or preferably about 5 nm to about 10 nm in its largest
dimension. Small nanoparticles are typically less than about 20 nm
in their largest dimension.
[0346] "Nanocrystal" as used herein can refer to a nanoparticle
made out of an inorganic substance that typically has an ordered
crystalline structure. It can refer to a nanocrystal having a
crystalline core (core nanocrystal) or to a core/shell
nanocrystal.
[0347] A core nanocrystal is a nanocrystal to which no shell has
been applied. Typically, it is a semiconductor nanocrystal that
includes a single semiconductor material. It can have a homogeneous
composition or its composition can vary with depth inside the
nanocrystal.
[0348] A core/shell nanocrystal is a nanocrystal that includes a
core nanocrystal and a shell disposed over the core nanocrystal.
Typically, the shell is a semiconductor shell that includes a
single semiconductor material. In some embodiments, the core and
the shell of a core/shell nanocrystal are composed of different
semiconductor materials, meaning that at least one atom type of a
binary semiconductor material of the core of a core/shell is
different from the atom types in the shell of the core/shell
nanocrystal.
[0349] The semiconductor nanocrystal core can be composed of a
semiconductor material (including binary, ternary and quaternary
mixtures thereof), from: Groups II-VI of the periodic table,
including ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, HgTe, MgTe;
Groups III-V, including GaN, GaP, GaAs, GaSb, InN, InP, InAs, InSb,
AlAs, AlP, AlSb, A1S; and/or Group IV, including Ge, Si, Pb.
[0350] The semiconductor nanocrystal shell can be composed of
materials (including binary, ternary and quaternary mixtures
thereof) comprising: ZnO, ZnS, ZnSe, ZnTe, CdO, CdS, CdSe, CdTe,
MgS, MgSe, GaAs, GaN, GaP, GaAs, GaSb, HgO, HgS, HgSe, HgTe, InAs,
InN, InP, InSb, AlAs, AlN, AlP, or AlSb.
[0351] Many types of nanocrystals are known, and any suitable
method for making a nanocrystal core and applying a shell to the
core may be employed. Nanocrystals can have a surface layer of
ligands to protect the nanocrystal from degradation in use or
during storage.
[0352] "Quantum dot" as used herein refers to a crystalline
nanoparticle made from a material which in the bulk is a
semiconductor or insulating material, which has a tunable
photophysical property in the near ultraviolet (UV) to far infrared
(IR) range.
[0353] "Water-soluble" or "water-dispersible" is used herein to
mean the item can be soluble or suspendable in an aqueous-based
solution, such as in water or water-based solutions or buffer
solutions, including those used in biological or molecular
detection systems as known by those skilled in the art. While
water-soluble nanoparticles are not truly `dissolved` in the sense
that term is used to describe individually solvated small
molecules, they are solvated (via hydrogen, electrostatic or other
suitable physical/chemical bonding) and suspended in solvents which
are compatible with their outer surface layer, thus a nanoparticle
which is readily dispersed in water is considered water-soluble or
water-dispersible. A water-soluble nanoparticle can also be
considered hydrophilic, since its surface is compatible with water
and with water solubility.
[0354] "Hydrophobic nanoparticle" as used herein refers to a
nanoparticle which is readily dispersed in or dissolved in a
water-immiscible solvent like hexanes, toluene, and the like. Such
nanoparticles are generally not readily dispersed in water.
[0355] "Hydrophilic" as used herein refers to a surface property of
a solid, or a bulk property of a liquid, where the solid or liquid
exhibits greater miscibility or solubility in a high-dielectric
medium than it does in a lower dielectric medium. By way of
example, a material which is more soluble in methanol than in a
hydrocarbon solvent such as decane would be considered
hydrophilic.
[0356] "Coordinating solvents" as used herein refers to a solvent
such as TDPA, OP, TOP, TOPO, carboxylic acids, and amines, which
are effective to coordinate to the surface of a nanocrystal.
`Coordinating solvents` also include phosphines, phosphine oxides,
phosphonic acids, phosphinic acids, amines, and carboxylic acids,
which are often used in growth media for nanocrystals, and which
form a coating or layer on the nanocrystal surface. Coordinating
solvents can exclude hydrocarbon solvents such as hexanes, toluene,
hexadecane, octadecene and the like, which do not have heteroatoms
that provide bonding pairs of electrons to coordinate with the
nanocrystal surface. Hydrocarbon solvents which do not contain
heteroatoms such as O, S, N or P to coordinate to a nanocrystal
surface are referred to herein as non-coordinating solvents. Note
that the term `solvent` is used in its ordinary way in these terms:
it refers to a medium which supports, dissolves or disperses
materials and reactions between them, but which does not ordinarily
participate in or become modified by the reactions of the reactant
materials. However, in certain instances, the solvent can be
modified by the reaction conditions. For example, TOP may be
oxidized to TOPO, or a carboxylic acid can be reduced to an
alcohol.
[0357] As used herein, the term "population" refers to a plurality
of nanoparticles having similar physical and/or optical properties.
`Population` can refer to a solution or structure with more than
one nanoparticle at a concentration suitable for single molecule
analysis. In some embodiments, the population can be monodisperse
and can exhibit less than at least 15% rms deviation in diameter of
the nanoparticles, and spectral emissions in a narrow range of no
greater than about 75 nm full width at half max (FWHM). In the
context of a solution, suspension, gel, plastic, or colloidal
dispersion of nanoparticles, the nature of the population can be
further characterized by the number of nanoparticles present, on
average, within a particular volume of the liquid or solid, or the
concentration. In a two-dimensional format such as an array of
nanoparticles adhered to a solid substrate, the concept of
concentration is less convenient than the related measure of
particle density, or the number of individual particles per
two-dimensional area. In this case, the maximum density would
typically be that obtained by packing particles
"shoulder-to-shoulder" in an array. The actual number of particles
in this case would vary due to the size of the particles--a given
array could contain a large number of small particles or a small
number of larger particles.
[0358] As used herein, the terms "moderate to high excitation"
refers to monochromatic illumination or excitation (e.g., laser
illumination) having a high power intensity sufficiently high such
that the absorbed photons per second for a given sample is between
about 200,000 and about 1,600,000.
[0359] In one aspect, the nanoparticle is a semiconductor
nanoparticle having size-dependent optical and electronic
properties. For example, the nanoparticle can emit a fluorescent
signal in response to excitation energy. The spectral emission of
the nanoparticle can be tunable to a desired energy by selecting
the particle size, size distribution, and/or composition of the
semiconductor nanoparticle. For example, depending on the
dimensions, the semiconductor nanoparticle can be a fluorescent
nanoparticle which emits light in the UV-visible-IR spectrum. The
shell material can have a bandgap greater than the bandgap of the
core material.
[0360] In one aspect, the nanoparticle is an energy transfer donor.
The nanoparticle can be excited by an electromagnetic source such
as a laser beam, multi-photon excitation, or electrical excitation.
The excitation wavelength can range between about 190 to about 800
nm including all values and ranges there in between. In some
embodiments, the nanoparticle can be excited by an energy source
having a wavelength of about 405 nm. In other embodiments, in
response to excitation, the nanoparticle can emit a fluorescent
signal at about 400-800 nm, or about 605 nm.
[0361] In one aspect, the nanoparticle can undergo Raman scattering
when subjected to an electromagnetic source (incident photon
source) such as a laser beam. The scattered photons have a
frequency that is different from the frequency of the incident
photons. As result, the wavelength of the scattered photons is
different than the incident photon source. In one embodiment, the
nanoparticle can be attached to a suitable tag or label to enhance
the detectability of the nanoparticle via Raman spectroscopy. The
associated tag can be fluorescent or nonfluorescent. Such
approaches can be advantageous in avoiding problems that can arise
in the context of fluorescent nanoparticles, such as photobleaching
and blinking. See, e.g., Sun et al., "Surface-Enhanced Raman
Scattering Based Nonfluorescent Probe for Multiplex DNA Detection",
Anal. Chem. 79(11):3981-3988 (2007)
[0362] In one aspect, the nanoparticle is comprised of a
multi-shell layered core which is achieved by a sequential shell
material deposition process, where one shell material is added at a
time, to provide a nanoparticle having a substantially uniform
shell of desired thickness which is substantially free of defects.
The nanoparticle can be prepared by sequential, controlled addition
of materials to build and/or applying layers of shell material to
the core. See e.g., U.S. PCT Application Serial No.
PCT/US09/061,951 which is incorporated herein by reference as if
set forth in full.
[0363] In another aspect, a method is provided for making a
nanoparticle comprising a core and a layered shell, where the shell
comprises at least one inner shell layer and at least one outer
shell layer. The method comprises the steps: (a) providing a
mixture comprising a core, at least one coordinating solvent; (b)
heating the mixture to a temperature suitable for formation of an
inner shell layer; (c) adding a first inner shell precursor
alternately with a second inner shell precursor in layer additions,
to form an inner shell layer which is a desired number of layers
thick; (d) heating the mixture to a temperature suitable for
formation of an outer shell layer; and (e) adding a first outer
shell precursor alternately with a second outer shell precursor in
layer additions, to form an outer shell layer which is a desired
number of layers thick. In one embodiment, if the coordinating
solvent of (a) is not amine, the method further comprises an amine
in (a).
[0364] In one aspect, at least one coordinating solvent comprises a
trialkylphosphine, a trialkylphosphine oxide, phosphonic acid, or a
mixture of these. In another aspect, at least one coordinating
solvent comprises trioctylphosphine (TOP), trioctylphosphine oxide
(TOPO), tetradecylphosphonic acid (TDPA), or a mixture of these. In
yet another aspect, the coordinating solvent comprises a primary or
secondary amine, for example, decylamine, hexadecylamine, or
dioctylamine.
[0365] In one aspect, the nanoparticle comprises a core comprising
CdSe. In another aspect, the nanoparticle shell can comprise YZ
wherein Y is Cd or Zn, and Z is S, or Se. In one embodiment, at
least one inner shell layer comprises CdS, and the at least one
outer shell layer comprises ZnS.
[0366] In one aspect, the first inner shell precursor is
Cd(OAc).sub.2 and the second inner shell precursor is
bis(trimethylsilyl)sulfide (TMS.sub.2S). In other aspects, the
first and second inner shell precursors are added as a solution in
trioctylphosphine (TOP). In other aspects, the first outer shell
precursor is diethylzinc (Et.sub.2Zn) and the second inner shell
precursor is dimethyl zinc (TMS.sub.2S). Sometimes, the first and
second outer shell precursors are added as a solution in
trioctylphosphine (TOP).
[0367] In one aspect, the nanoparticle can have ligands which coat
the surface. The ligand coating can comprise any suitable
compound(s) which provide surface functionality (e.g., changing
physicochemical properties, permitting binding and/or other
interaction with a biomolecule, etc.). In some embodiments, the
disclosed nanoparticle has a surface ligand coating (in direct
contact with the external shell layer) that adds various
functionalities which facilitate it being water-dispersible or
soluble in aqueous solutions. There are a number of suitable
surface coatings which can be employed to permit aqueous
dispersibility of the described nanoparticle. For example, the
nanoparticle(s) disclosed herein can comprise a core/shell
nanocrystal which is coated directly or indirectly with lipids,
phospholipids, fatty acids, polynucleic acids, polyethylene glycol
(PEG), primary antibodies, secondary antibodies, antibody
fragments, protein or nucleic acid based aptamers, biotin,
streptavidin, proteins, peptides, small organic molecules (e.g.,
ligands), organic or inorganic dyes, precious or noble metal
clusters. Specific examples of ligand coatings can include, but are
not limited to, amphiphilic polymer (AMP), bidentate thiols (i.e.,
DHLA), tridentate thiols, dipeptides, functionalized
organophosphorous compounds (e.g., phosphonic acids, phosphinic
acids), etc.
Non-Blinking Nanoparticles
[0368] Provided herein are nanoparticles which exhibit modulated,
reduced, or no intermittent (e.g., continuous, non-blinking)
fluorescence.
[0369] In one aspect, the nanoparticle or populations thereof
exhibit modulated, reduced or non-detectable intermittent (e.g.,
continuous, etc.) fluorescence properties. The nanoparticles can
have a stochastic blinking profile in a timescale which is shifted
to very rapid blinking or very slow or infrequent blinking relative
to a nanoparticle previously described in the art (conventional
nanoparticles are described in the art as having on-time fractions
of <0.2 in the best of conditions examined). For example, the
nanoparticles may blink on and off on a timescale which is too
rapid to be detected under the methods employed to study this
behavior.
[0370] In one aspect the nanoparticle or populations thereof are
photostable. The nanoparticles can exhibit a reduced or no
photobleaching with long exposure to moderate to high intensity
excitation source while maintaining a consistent spectral emission
pattern.
[0371] In one aspect, the nanoparticle or populations thereof have
a consistently high quantum yield. For example, the nanoparticles
can have a quantum yield greater than: about 10%, or about 20%, or
about 30%, or about 40%, or about 50%, or about 60%, or about 70%
or about 80%.
[0372] As used herein, fluorescence (or Forster) resonance energy
transfer (FRET) is a process by which a fluorophore (the donor) in
an excited state transfers its energy to a proximal molecule (the
acceptor) by nonradiative dipole-dipole interaction (Forster, T.
"Intermolecular Energy Migration and Fluorescence", Ann. Phys.,
2:55-75, 1948; Lakowicz, J. R., Principles of Fluorescence
Spectroscopy, 2nd ed. Plenum, New York. 367-394., 1999).
[0373] FRET efficiency (E) can be defined as the quantum yield of
the energy transfer transition, i.e. the fraction of energy
transfer event occurring per donor excitation event. It is a direct
measure of the fraction of photon energy absorbed by the donor
which is transferred to an acceptor, as expressed in Equation 1:
E=k.sub.ET/k.sub.f+k.sub.ET+Ek.sub.i, where k.sub.ET is the rate of
energy transfer, k.sub.f the radiative decay rate and the k.sub.i
are the rate constants of any other de-excitation pathway.
[0374] FRET efficiency E generally depends on the inverse of the
sixth power of the distance r(nm) between the two fluorophores
(i.e., donor and acceptor pair), as expressed in Equation 2: E=1/1+
(r/R.sub.0).sup.6.
[0375] The distance where FRET efficiency is at 50% is termed
R.sub.0, also know as the Forster distance. R.sub.0 can be unique
for each donor-acceptor combination and can range from between
about 5 nm to about 10 nm. Therefore, the FRET efficiency of a
donor (i.e., nanoparticle) describes the maximum theoretical
fraction of photon energy which is absorbed by the donor (i.e.,
nanoparticle) and which can then be transferred to a typical
organic dye (e.g., fluoresceins, rhodamines, cyanines, etc.).
[0376] In some embodiments, the disclosed nanoparticles are
relatively small (i.e., <15 nm) and thus may be particularly
well suited to be used as a donor or an acceptor in a FRET
reaction. That is, some embodiments of the disclosed nanoparticles
exhibit higher FRET efficiency than conventional nanoparticles and
thus are excellent partners (e.g., donors or acceptors) in a FRET
reaction.
[0377] "Quantum yield" as used herein refers to the emission
efficiency of a given fluorophore assessed by the number of times
which a defined event, e.g., light emission, occurs per photon
absorbed by the system. In other words, a higher quantum yield
indicates greater efficiency and thus greater brightness of the
described nanoparticle or populations thereof.
[0378] Any suitable method can be used to measure quantum yield. In
one example, quantum yield can be obtained using standard methods
such as those described in Casper et al (Casper, J. V.; Meyer, T.
J. J. Am. Chem. Soc. 1983, 105, 5583) and can be analyzed relative
to known fluorophores chosen as appropriate for maximal overlap
between standard emission and sample emission (e.g., fluorescein,
Rhodamine 6G, Rhodamine 101). Dilute solutions of the standard and
sample can be matched or nearly matched in optical density prior to
acquisition of absorbance and emission spectra for both. The
emission quantum yield (.PHI..sub.em) then can be determined
according to Equation 3:
.phi. em = .phi. em ' ( I I ' ) ( A ' A ) ##EQU00001##
[0379] where A and A' are the absorbances at the excitation
wavelength for the sample and the standard respectively and I and
I' are the integrated emission intensities for the sample and
standard respectively. In this case .PHI.'.sub.em can be the agreed
upon quantum yield for the standard.
[0380] Disclosed herein are fluorescent nanoparticles with superior
and robust properties which significantly expand the applications
in which nanoparticles are useful. These nanoparticles are superior
and surprisingly robust in that they are simultaneously stable,
bright, and sensitive to environmental stimuli. Moreover, the
disclosed nanoparticles have limited or no detectable blinking
(i.e., where the nanoparticle emits light non-intermittently when
subject to excitation), are highly photostable, have a consistently
high quantum yield, are small (e.g., .ltoreq.20 nm) and can act as
a donor which undergoes FRET with a suitable acceptor moiety (e.g.,
fluorescent dyes, etc.). The photostability of these nanoparticles
is reflected in their exhibiting reduced or no photobleaching
(i.e., fading) behavior when subjected to moderate to high
intensity excitation for at least about 20 minutes. Additionally,
the particles can remain substantially free from photo-induced
color shifting.
[0381] Put another way, the nanoparticles can maintain a consistent
spectral emission pattern (i.e., maintain the ability to fluoresce)
even when exposed to a large quantity of photons (i.e., moderate to
high intensity excitation) for a long period of time. This unique
combination of characteristics makes these types of nanoparticles
sensitive tools for single molecule analysis and other sensitive
high throughput applications. Moreover, these properties make the
nanoparticles particularly well suited for use as highly efficient
donor fluorophores in energy transfer reactions such as FRET
reactions (i.e., high FRET efficiency) or other reactions as well
as applications which require or are enhanced by greater response
to the environment.
[0382] Without being bound to a particular theory, blinking or
fluorescence intermittency may arise during the nanoparticle
charging process when an electron is temporarily lost to the
surrounding matrix (Auger ejection or charge tunneling) or captured
to surface-related trap states. The nanoparticle is "on" or
fluorescing when all of the electrons are intact and the particle
is "neutral" and the particle is "off" or dark when the electron is
lost and the particle is temporarily (or in some cases permanently)
charged. It is important to note that the complete suppression of
blinking may not necessarily be required and in some instances may
not be desirable. Blinking which occurs on a timescale much shorter
or much longer than the interrogation period for a particular assay
has relatively little impact on the performance of the system.
Thus, nanoparticles and nanoparticle populations having modulated
blinking properties, where blinking occurs on a very short or very
fast timescale relative to the assay interrogation periods are also
useful and fall within the scope of the present disclosure.
Localization of timescale or simply pushing timescale to one side
(e.g., to where the blinking is undetectable within the assay
system) can provide substantial benefit in application
development.
[0383] The blinking behavior of the nanoparticles described herein
can be analyzed and characterized by any suitable number of
parameters using suitable methodologies. The probability
distribution function of the "on" and "off" blinking time durations
(i.e., blinking behavior) can be determined using the form of an
inverse power law. A value, alpha (.alpha.) can be calculated,
wherein a represents an exponent in the power law. As the
percentage of the population which is non-blinking increases, the
value of .alpha..sub.on, theoretically approaches zero. In
conventional nanoparticle populations previously described,
.alpha..sub.on, typically ranges from about 1.5 to about 2.5, under
moderate to high excitation energy.
[0384] Most alpha calculations can use a predetermined threshold to
determine the "on" and "off" values of alpha-on and alpha-off
(i.e., .alpha..sub.on, and .alpha..sub.off). Typically, an alpha
estimator which calculates the on/off threshold for each dot
individually can be employed. The data can be represented by a plot
of signal versus frequency, and typically appears as a series of
Gaussian distributions around the "off state" and one or more "on
states." A log-log plot of frequency versus time for each period of
time that the dot is "on" provides a straight line having a slope
of .alpha..sub.on. The value of alpha-off (.alpha..sub.off) can be
similarly determined.
[0385] In a specific example (the "TIRF example"), the fluorescent
intermittency measurements can be made using a Total Internal
Reflection Fluorescence (TIRF) microscope fitted with a 60.times.
oil immersion objective lens, using a dual view with a longpass
filter on the acceptor side and a bandpass filter on the donor
side. Using the TIRF setup, the nanoparticles were imaged at 30 Hz
(33 ms), typically for 5 minutes, to produce a movie showing the
time and intensity of the emitted light for each individual spot
(corresponding to a single particle) within a binned frame which
was 33 ms long; the intensity for each binned frame can be
integrated. Each data set can be manually analyzed dot-by-dot, and
aggregates and other artifacts were excluded. From the edited
results, the following parameters can be calculated: alpha-on
(".alpha..sub.on"); alpha-off (".alpha..sub.off"); the percent on;
longest on/longest off; overlap scores; and the median values for
each of these parameters.
[0386] In some aspects, provided herein is a nanoparticle or
population thereof which has an .alpha..sub.on of less than about
1.5, .alpha..sub.on of less than about 1.4, .alpha..sub.on of less
than about 1.3, .alpha..sub.on of less than about 1.2, or an
.alpha..sub.on of less than about 1.1, under moderate to high
excitation energy. In some embodiments, at least about 50%, at
least about 60%, at least about 70%, at least about 80%, at least
about 90%, at least about 95%, at least about 98%, at least about
99% or more of the population has an .alpha..sub.on of less than
about 1.5, .alpha..sub.on of less than about 1.4, .alpha..sub.on of
less than about 1.3, .alpha..sub.on of less than about 1.2, or
.alpha..sub.on of less than about 1.1 for the time observed, under
moderate to high excitation energy. The observation time can be at
least about 5 minutes, at least about 10 minutes, at least about 15
minutes, at least about 30 minutes, at least about 45 minutes, at
least about 60 minutes, at least about 90 minutes, at least about
120 minutes or more under moderate to high excitation energy.
Compositions comprising such a nanoparticle and populations thereof
also are contemplated.
[0387] In some aspects, provided herein is a nanoparticle or a
population thereof having a stochastic blinking profile which is
either undetectable or rare (e.g., no more than 1-2 events during
the interrogation period) over an observed timescale. In this case,
"undetectable" encompasses the situation in which evidence might
exist for ultra-fast blinking on a timescale which is faster than
the binning timescale (e.g., dimming and brightening from bin to
bin) but there are no "off" events persisting for longer than the
bin time. Therefore, in some embodiments, a nanoparticle or
population thereof has a stochastic blinking profile which is
undetectable for at least about 50%, at least about 60%, at least
about 70%, at least about 80%, at least about 85%, at least about
90%, at least about 95%, at least about 98%, at least about 99% or
more of the time observed, under moderate to high excitation
energy. In other embodiments, at least about 50%, at least about
60%, at least about 70%, at least about 80%, at least about 90%, at
least about 95%, at least about 98%, at least about 99% or more of
the individual nanoparticles in a population have a stochastic
blinking on a timescale which is undetectable for the time
observed, under moderate to high excitation energy. The timescale
can be at least about 5 minutes, at least about 10 minutes, at
least about 15 minutes, at least about 30 minutes, at least about
45 minutes, at least about 60 minutes, at least about 90 minutes,
at least about 120 minutes or more under moderate to high
excitation energy.
[0388] In some aspects, the longest on and longest off values can
relate to the longest period of time a nanoparticle is observed to
be in either the "on" or the "off" state. In particular, the
longest on value can be important to determining the length of time
and amount of data which may be measured in a particular assay.
[0389] Thus, the blinking characteristics of the nanoparticles
herein can also be characterized by their on-time fraction, which
represents the (total on-time)/(total experiment time). Under the
TIRF example disclosed herein, the total on time can be determined
by the total number of frames "on" multiplied by 33 ms, and the
total experiment time is 5 minutes. For example, the blinking
properties of the disclosed nanoparticles or populations thereof
can be determined under continuous irradiation conditions using a
405 nm laser with an intensity of about 1 watt per cm.sup.2 during
an experimental window of at least 5 minutes.
[0390] On-time fractions can be used to characterize the blinking
behavior of a single nanoparticle or of a population of
nanoparticles. It is important to note that the on-time fraction
for a particular nanoparticle or population of nanoparticles is a
function of the specific conditions under which the percent of
blinking or "non-blinking" nanoparticles is determined.
[0391] In some aspects, provided herein is a nanoparticle or
population thereof having an on-time fraction of at least about
0.50, at least about 0.60, at least about 0.70, at least about
0.75, at least about 0.80, at least about 0.85, at least about
0.90, at least about 0.95, at least about 0.96, at least about
0.97, at least about 0.98, or at least about 0.99 or more, under
moderate to high excitation energy. In some embodiments, a
nanoparticle or populations thereof having a percent on-time of
about 98%, about 99% (i.e., on-time fraction of about 0.99) can be
considered to be "non-blinking," under moderate to high excitation
energy. In some embodiments, at least about 50%, at least about
60%, at least about 70%, at least about 80%, at least about 90%, at
least about 95%, at least about 98%, at least about 99%, or more of
the individual nanoparticles in a population of nanoparticles can
have an on-time fraction of at least about 0.50, at least about
0.60, at least about 0.70, at least about 0.75, at least about
0.80, at least about 0.85, at least about 0.90, at least about
0.95, at least about 0.96, at least about 0.97, at least about
0.98, or at least about 0.99 or more, under moderate to high
excitation energy. The on-times of the nanoparticles are typically
for at least about 5 minutes, at least about 10 minutes, at least
about 15 minutes, at least about 20 minutes, at least about 30
minutes, at least about 45 minutes, at least about 60 minutes, at
least about 70 minutes, at least about 80 minutes, at least about
90 minutes, at least about 120 minutes under moderate to high
intensity excitation of the nanoparticle or nanoparticle
population. Under one set of conditions, continuous irradiation
with 405 nm laser with an approximate intensity of 1 watt per
cm.sup.2 was used to determine the stochastic blinking profile.
[0392] In some embodiments, nanoparticles which have a stochastic
(i.e., random) blinking profile in a timescale which shifts from
very rapid blinking or very slow/infrequent blinking (relative to a
nanoparticle previously described in the art) can be considered to
have modulated blinking properties. In some embodiments, these
nanoparticles may blink on and off on a timescale which is too
rapid to be detected under the methods employed to study this
behavior. Thus, certain nanoparticles can effectively appear to be
"always on" or to have on-time fractions of about 0.99, when in
fact they flicker on and off at a rate too fast or too slow to be
detected. Such flickering has relatively little impact on the
performance of a system, and for practical purposes such
nanoparticles can be considered to be non-blinking.
[0393] In some instances, the disclosed nanoparticles and
populations thereof are not observed to blink off under the
analysis conditions, and such particles can be assessed as "always
on" (e.g., non-blinking). The percent of usable dots which are
"always on" can be a useful way to compare nanoparticles or
populations of nanoparticles. However, a determination of "always
on" may mean that the "off" time was insufficient to provide enough
a signal gap for accurate determination and thus the value in the
regime of particles is insufficient to calculate. Even these
"non-blinking" nanoparticles may flicker on and off on a timescale
which is not detected under the conditions used to assess blinking.
For example, certain particles may blink on a timescale which is
too fast to be detected, or they may blink very rarely, and, in
some embodiments, such particles may also be considered to be
"always-on" or non-blinking, as the terms are used herein.
[0394] In one aspect, provided herein is a nanoparticle or
population thereof which demonstrate some fluctuation in
fluorescence intensity. In some embodiments, the change in
fluorescence intensity for the nanoparticle is less than about 5%,
less than about 10%, less than about 20%, or less than about 25% of
the nanoparticle or populations thereof at its greatest intensity,
under moderate to high excitation energy. In some embodiments, such
changes in fluorescence intensity of less than about 5%, less than
about 10%, less than about 20%, or less than about 25% of the
highest intensity can occur in at least about 50%, at least about
60%, at least about 70%, at least about 80%, at least about 85%, at
least about 90%, at least about 95%, at least about 98%, at least
about 99% of the nanoparticles in the population, under moderate to
high excitation energy.
[0395] In some aspects, the nanoparticles with modulated, reduced
or no intermittent (e.g., continuous, non-blinking) fluorescence
provided herein can comprise of a core and a layered gradient
shell. In some embodiments, the nanoparticle(s) disclosed herein
can be comprised of a nanocrystal core (e.g., CdSe, etc.), at least
one inner (intermediate) shell layer (e.g., CdS, etc.), and at
least one outer (external) shell layer (e.g., ZnS, etc.). In some
embodiments, the inner and/or outer shell layers are each comprised
of two or more discrete monolayers of the same material. In some
embodiments, the largest dimension of the disclosed nanoparticle(s)
is less than about 15 nm. See for example, PCT Application Serial
No. PCT US/09/61951. See also PCT/US09/061,951 and PCT/US09/061,953
both filed on Oct. 23, 2009.
[0396] As discussed previously, the disclosed nanoparticles may be
particularly well suited for use as a donor or acceptor which
undergoes FRET with a suitable complementary partner (donor or
acceptor). A "FRET capable" nanoparticle refers to a nanoparticle
which can undergo a measurable FRET energy transfer event with a
donor or an acceptor moiety. In some embodiments, a FRET capable
nanoparticle is one which has at least about 25% efficiency in a
FRET reaction.
[0397] Thus, in one aspect, a FRET capable fluorescent nanoparticle
or population thereof with modulated, reduced or non intermittent
(e.g., continuous, etc.) fluorescence is provided. In some
embodiments, the nanoparticle is the donor in a FRET reaction. In
some embodiments, the nanoparticle is the acceptor in the FRET
reaction.
[0398] In some embodiments, the FRET capable non-blinking
fluorescent nanoparticle(s) disclosed herein can comprise a core
and a layered gradient shell. In some embodiments, the FRET capable
non-blinking nanoparticle(s) disclosed herein can be comprised of a
nanocrystal core (e.g., CdSe, etc.), at least one inner
(intermediate) shell layer (e.g., CdS, etc.), and at least one
outer (external) shell layer (e.g., ZnS, etc.). In some
embodiments, the inner and/or outer shell layers are each comprised
of two or more discrete monolayers of the same material. In some
embodiments, the largest dimension of the disclosed FRET capable
nanoparticle(s) is less than about 15 nm.
[0399] In some embodiments, the nanoparticle or population thereof
has a FRET efficiency of at least about 20%, at least about 40%, at
least about 50%, at least about 60%, at least about 70%, at least
about 80%, at least about 90%, at least about 95%, at least about
98%, at least about 99%, or greater.
[0400] In some embodiments, at least about 30%, at least about 40%,
at least about 50%, at least about 60% at least about 70%, at least
about 80%, at least about 90%, at least about 95%, at least about
98%, at least about 99% or more of the individual nanoparticles in
the population have a FRET efficiency of at least about 20%, at
least about 40%, at least about 50%, at least about 60%, at least
about 70%, at least about 80%, at least about 90%, at least about
95%, at least about 98%, at least about 99% or more.
[0401] In some embodiments, the FRET efficiency of the disclosed
nanoparticle or population thereof can be maintained for at least
about the first 10%, at least about the first 20%, at least about
the first 30%, at least about the first 40%, at least about the
first 50%, at least about the first 60%, at least about the first
70%, at least about the first 80%, at least about the first 90% or
more of the total emitted photons under conditions of moderate to
high excitation.
[0402] As discussed above, the nanoparticle(s) provided herein can
be considered to be surprisingly photostable. In particular, the
nanoparticle and populations described herein can be photostable
over an extended period of time while maintaining the ability to
effectively participate in energy transfer (i.e., FRET) reactions.
The disclosed nanoparticles can be stable under high intensity
conditions involving prolonged or continuous irradiation over an
extended period of time from a moderate to high excitation
source.
[0403] Thus, in one aspect, provided herein is a non-blinking
fluorescent nanoparticle and population thereof which is
photostable.
[0404] In some embodiments, the disclosed photostable nanoparticle
and population thereof can have an emitted light or energy
intensity sustained for at least about 10 minutes and does not
decrease by more than about 20% of maximal intensity achieved
during that time. Further, these nanoparticles and populations
thereof can have a wavelength spectrum of emitted light which does
not change more than about 10% upon prolonged or continuous
exposure to an appropriate energy source (e.g. irradiation).
[0405] In one embodiment, the photostable nanoparticles disclosed
herein can remain photostable under moderate to high intensity
excitation from at least about 10 minutes to about 2 hours. In
another embodiment, the photostable nanoparticles disclosed herein
can remain photostable under moderate to high intensity excitation
from at least about 10 minutes to about 10 hours. In still another
embodiment, the photostable nanoparticles disclosed herein can
remain photostable under moderate to high from about 10 minutes to
about 48 hours. However, it should be appreciated, that these are
just example photostable times for the disclosed nanoparticles, in
practice the nanoparticles can remain photostable for longer
periods of time depending on the particular application.
[0406] It should be appreciated that nanoparticles which are
photostable over longer timescales in combination with moderate to
high excitation energy sources are well suited for more sensitive
and broad-ranging applications such as the real-time monitoring of
single molecules involving FRET. That is, the nanoparticle and
population thereof described herein can be photostable over an
extended period of time while maintaining the ability to
effectively participate in energy transfer (i.e., FRET) reactions,
which makes the subject nanoparticles particularly useful for many
applications involving the real-time monitoring of single
molecules. As such, in some embodiments the photostable
nanoparticles disclosed herein have FRET efficiencies of at least
about 20%.
[0407] In some embodiments, the disclosed nanoparticles are stable
upon prolonged or continuous irradiation (under moderate to high
excitation rate) in which they do not exhibit significant
photo-bleaching on the timescales indicated. Photobleaching can
result from the photochemical destruction of a fluorophore (and can
be characterized by the nanoparticles losing the ability to produce
a fluorescent signal) by the light exposure or excitation source
used to stimulate the fluorescence. Photobleaching can complicate
the observation of fluorescent molecules in microscopy and the
interpretation of energy transfer reactions because the signals can
be destroyed or diminished increasingly as timescales for the
experiment increase or the energy intensity increases.
[0408] Photobleaching can be assessed by measuring the intensity of
the emitted light or energy for a nanoparticle or nanoparticle
population using any suitable method. In some embodiments, the
intensity of emitted light or energy from the disclosed
nanoparticle or population thereof does not decrease by more than
about 20% (and in some embodiments, not more than about 10%) upon
prolonged or continuous irradiation (under moderate to high
excitation rate). In some embodiments, the intensity of emitted
light from the disclosed nanoparticle or population thereof does
not decrease by more than about 20%, about 15%, about 10%, about 5%
or less upon irradiation from about 10 minutes, about 20 minutes,
about 30 minutes, about 45 minutes, about 60 minutes, about 90
minutes, about 2 hours, about 3 hours to about 4 hours, under
moderate to high excitation energy.
[0409] In some embodiments, the photostable nanoparticles provided
herein further demonstrate enhanced stability in which they exhibit
a reduction in or absence of spectral shifting during prolonged
excitation. In the conventional nanoparticles previously described
in the art, increased exposure to an excitation source--whether via
increase time or power--results in a spectral shift of the
wavelength emission wavelength profile of a nanoparticle and
populations thereof from a longer wavelength to an increasingly
shorter wavelength. Such spectral shifting of emission wavelength
represents a significant limitation as precise resolution of
emission spectra is required for applications which require rapid
detection, multi-color analysis, and the like. Shifting of any
significance then requires that the wavelength emissions used in an
assay be sufficiently separated to permit resolution, thus reducing
the number of colors available as well as increasing signal to
noise ratio to an unacceptable level as the initial spectral
profile cannot be relied upon once spectral shifting begins. Such
shifting may require shortened observation times or use of
fluorophores with widely separated emission spectra. The
nanoparticles provided herein have little to no spectral shift,
particularly over extended periods of excitation.
[0410] Wavelength emission spectra can be assessed by any suitable
method. For example, spectral characteristics of nanoparticles can
generally be monitored using any suitable light-measuring or
light-accumulating instrumentation. Examples of such
instrumentation are CCD (charge-coupled device) cameras, video
devices, CIT imaging, digital cameras mounted on a fluorescent
microscope, photomultipliers, fluorometers and luminometers,
microscopes of various configurations, and even the human eye. The
emission can be monitored continuously or at one or more discrete
time points. The photostability and sensitivity of nanoparticles
allow recording of changes in electrical potential over extended
periods of time.
[0411] Thus, in some embodiments, the photostable nanoparticle and
population thereof has a wavelength spectrum of emitted light which
does not change more than about 10% upon prolonged or continuous
exposure to an appropriate energy source (e.g. irradiation) over
about 4 minutes to about 10 minutes, under moderate to high
excitation energy. In some embodiments, the wavelength emission
spectra does not change more than about 5%, more than about 10%,
more than about 20% over 10 minutes, about 20 minutes, about 30
minutes, about 45 minutes, about 60 minutes, about 90 minutes,
about 2 hours, about 3 hours to about 4 hours.
[0412] It should be appreciated that there can be various other
objective indicia of nanoparticle photostability. For example, a
nanoparticle can be classified as photostable when the
nanoparticle, under moderate to high excitation, emits about
1,000,000 to about 100,000,000 photons or more preferably about
100,000,001 to about 100,000,000,000 photons or even more
preferably more than about 100,000,000,000 photons before becoming
non-emissive (i.e., bleached).
[0413] A nanoparticle with modulated, reduced or no fluorescent
intermittency (e.g., continuous, non-blinking, etc.); reduced or
absent spectral shifting; low to no photobleaching; high quantum
yield; and sufficient FRET efficiency can be of any suitable size.
Typically, it is sized to provide fluorescence in the UV-visible
portion of the electromagnetic spectrum as this range is convenient
for use in monitoring biological and biochemical events in relevant
media. The disclosed nanoparticle and population thereof can have
any combination of the properties described herein.
[0414] Thus, in some embodiments the nanoparticle or population
thereof has modulated or no blinking, are photostable (e.g.,
limited or no photobleaching, limited or no spectral shift), has
high quantum yield, have high FRET efficiency, has a diameter of
less than about 15 nm, is spherical or substantially spherical
shape, or any combination of all these properties as described
herein.
[0415] Likewise, in some embodiments, at least about 50%, at least
about 60%, at least about 70%, at least about 80%, at least about
90%, at least about 95%, at least about 98%, at least about 99%, or
more of the individual nanoparticles in a population of
nanoparticles have modulated or no blinking, are photostable (e.g.,
limited or no photobleaching, limited or no spectral shift), have
high quantum yield, have high FRET efficiency, have diameters of
less than about 15 nm, are spherical or substantially spherical
shape, or any combination of or all of these properties as
described herein.
[0416] In one aspect, the FRET capable, non-blinking and/or
photostable nanoparticle or population thereof provided herein has
a maximum diameter of less than about 20 nm. In some embodiments,
the nanoparticle(s) can be less than about 15 nm, less than about
10 nm, less than about 8 nm, less than about 6 nm, less than about
5 nm, less than about 4 nm, less than about 3 nm or less in its
largest diameter when measuring the core/shell structure. Any
suitable method may be used to determine the diameter of the
nanoparticle(s). The nanoparticle(s) provided herein can be grown
to the desired size using any of the methods disclosed herein. In
some embodiments, at least about 50%, at least about 60%, at least
about 70%, at least about 80%, at least about 90%, at least about
95%, at least about 98%, at least about 99%, or more of the
individual members of a population of nanoparticles have maximum
diameters (when measuring the core, core/shell or core/shell/ligand
structure) which are less than about 20 nm, less than about 15 nm,
less than about 10 nm, less than about 8 nm, less than about 6 nm,
less than about 5 nm, less than about 4 nm, less than about 3 nm or
less.
[0417] The FRET capable, non-blinking and/or photostable
nanoparticle(s) provided herein and populations thereof can be
spherical or substantially spherical. In some embodiments, a
substantially spherical nanoparticle can be one where any two
radius measurements do not differ by more than about 10%, about 8%,
about 5%, about 3% or less. In some embodiments, at least about
50%, at least about 60%, at least about 70%, at least about 80%, at
least about 90%, at least about 95%, at least about 98%, at least
about 99%, or more of the individual members of a population of
nanoparticles are spherical or substantially spherical.
[0418] Nanoparticles can be synthesized in shapes of different
complexity such as spheres, rods, discs, triangles, nanorings,
nanoshells, tetrapods, nanowires and so on. Each of these
geometries can have distinctive properties: spatial distribution of
the surface charge, orientation dependence of polarization of the
incident light wave, and spatial extent of the electric field. In
some embodiments, the nanoparticles are substantially spherical or
spheroidal.
[0419] For embodiments where the nanoparticle is not spherical or
spheroidal, e.g. rod-shaped, it may be from about 1 to about 15 nm,
from about 1 nm to about 10 nm, or 1 nm to about 5 nm in its
smallest dimension. In some such embodiments, the nanoparticles may
have a smallest dimension of about 0.5 nm, about 1 nm, about 2 nm,
about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 7 nm, about 8
nm, about 9 nm, about 10 nm, about 11 nm, about 12 nm, about 13 nm,
about 14 nm, about 15 nm, about 16 nm, about 17 nm, about 18 nm,
about 19 nm, about 20 nm, about 25 nm, about 30 nm, about 35 nm,
about 40 nm, about 45 nm, about 50 nm and ranges between any two of
these values.
[0420] The single-color preparation of the nanoparticles disclosed
herein can have individual nanoparticles which are of substantially
identical size and shape. Thus, in some embodiments, the size and
shape between the individual nanoparticles in a population of
nanoparticles vary by no more than about 20%, no more than about
15%, no more than about 10%, no more than about 8%, no more than
about 6%, no more than about 5%, no more than about 4%, no more
than about 3% or less in at least one measured dimension. In some
embodiments, disclosed herein is a population of nanoparticles,
where at least about 60%, at least about 70%, at least about 80%,
at least about 90%, at least about 95%, and ideally about 100% of
the particles are of the same size. Size deviation can be measured
as root mean square ("rms") of the diameter, with the population
having less than about 30% rms, preferably less than about 20% rms,
more preferably less than about 10% rms. Size deviation can be less
than about 10% rms, less than about 9% rms, less than about 8% rms,
less than about 7% rms, less than about 6% rms, less than about 5%
rms, less than about 3% rms, or ranges between any two of these
values. Such a collection of particles is sometimes referred to as
being a "monodisperse" population.
[0421] The color (emitted light) of a nanoparticle can be "tuned"
by varying the size and composition of the particle. Nanoparticles
as disclosed herein can absorb a wide spectrum of wavelengths, and
emit a relatively narrow wavelength of light. The excitation and
emission wavelengths are typically different, and non-overlapping.
The nanoparticles of a monodisperse population may be characterized
in that they produce a fluorescence emission having a relatively
narrow wavelength band. Examples of emission widths include less
than about 200 nm, less than about 175 nm, less than about 150 nm,
less than about 125 nm, less than about 100 nm, less than about 75
nm, less than about 60 nm, less than about 50 nm, less than about
40 nm, less than about 30 nm, less than about 20 nm, and less than
about 10 nm. In some embodiments, the width of emission is less
than about 60 nm full width at half maximum (FWHM), or less than
about 50 nm FWHM, and sometimes less than about 40 nm FWHM, less
than about 30 nm FWHM or less than about 20 nm FWHM. In some
embodiments, the emitted light preferably has a symmetrical
emission of wavelengths.
[0422] The emission maxima of the disclosed nanoparticle and
population thereof can generally be at any wavelength from about
200 nm to about 2,000 nm. Examples of emission maxima include about
200 nm, about 400 nm, about 600 nm, about 800 nm, about 1,000 nm,
about 1,200 nm, about 1,400 nm, about 1,600 nm, about 1,800 nm,
about 2,000 nm, and ranges between any two of these values.
[0423] As discussed previously, the disclosed nanoparticle or
populations thereof can comprise a core and a layered shell,
wherein the shell includes at least one inner (intermediate) shell
layer comprising a first shell material and at least one outer
(external) shell layer comprising a second shell material, and
wherein the layered shell is substantially uniform in coverage
around the core and is substantially free of defects.
[0424] Thus, in one aspect, the nanoparticle or population thereof
comprises a core (M.sup.1Y) and a layered shell, wherein the shell
comprises m inner shell monolayers comprising a first shell
material (M.sup.1X).sub.m and n outer shell monolayers comprising a
second shell material (M.sup.2X).sub.n, wherein M can be a metal
atom and X can be a non-metal atom, each of m and n is
independently an integer from 1 to 10, and the layered shell is
substantially uniform in coverage around the core and is
substantially free of defects. In specific embodiments, the sum of
m+n is 3-20, or 5-14, or 6-12, or 7-10.
[0425] In certain embodiments, the disclosed nanoparticles can
further comprise one or more additional shell layers between the at
least one inner shell layer and the at least one outer shell
layer.
[0426] In some embodiments, the nanoparticle core and population
thereof can have a first bandgap energy and the first shell
material can have a second bandgap energy, wherein the second
bandgap energy can be greater than the first bandgap energy.
[0427] In a further aspect, provided herein is a nanoparticle or
population thereof comprising a core and a layered shell, wherein
the shell comprises sequential monolayers comprising an alloyed
multi-component shell material of the form
M.sup.1.sub.xM.sup.2.sub.yX, where M.sup.1 and M.sup.2 can be metal
atoms and X can be a non metal atom, where the composition becomes
successively enriched in M.sup.2 as the monolayers of shell
material are deposited, where x and y represent the ratio of
M.sup.1 and M.sup.2 in the shell material, and wherein the
monolayered shell is substantially uniform in coverage around the
core and is substantially free of defects. In some embodiments, the
layered shell sometimes has about 3-20 monolayers of shell
material, sometimes about 5-14 monolayers of shell material,
sometimes about 6-12 monolayers of shell material, or sometimes
about 7-10 monolayers of shell material.
[0428] In one aspect, provided herein is a nanoparticle or
population thereof comprising a core and a layered shell having a
gradient potential, wherein the shell comprises at least one inner
shell layer and at least one outer shell layer, and wherein the
layered shell is substantially uniform in coverage around the core
and is substantially free of defects.
[0429] The layered shell may be engineered such that the sequential
monolayers are selected to provide a gradient potential from the
nanoparticle core to the outer surface of the nanoparticle shell.
The steepness of the potential gradient may vary depending on the
nature of the shell materials selected for each monolayer or group
of monolayers. For example, a nanoparticle comprising several
sequential monolayers of the same shell material may reduce the
potential through a series of steps, while a more continuous
gradient may be achievable through the use of sequential monolayers
of a multi-component alloyed shell material. In some embodiments,
both single component and multi-component shell materials may be
applied as different monolayers of a multi-layer shell on a
nanoparticle.
[0430] The nanoparticles can be synthesized as disclosed to the
desired size by sequential, controlled addition of materials to
build and/or apply monolayers of shell material to the core. This
is in contrast to conventional methods of adding shells where
materials (e.g., diethylzinc and bis(trimethylsilyl)sulfide) are
added together. Sequential addition permits the formation of thick
(e.g., >2 nm) relatively uniform individual shells (e.g.,
uniform size and depth) on a core. The layer additions generally
require the addition of an appropriate amount of the shell
precursors to form a single monolayer, based on the starting size
of the underlying core. This means that as each monolayer of shell
material is added, a new "core" size must be determined by taking
the previous "core" size and adding to it the thickness of
just-added shell monolayer. This leads to a slightly larger volume
of the following shell material needing to be added for each
subsequent monolayer of shell material being added.
[0431] Each monolayer of shell material can be independently
selected, and may be made up of a single component, or may comprise
a multi-component (e.g., alloyed, etc.) shell material. In some
embodiments, it is suitable to apply one or more sequential
monolayers of a first shell material, followed by one or more
sequential monolayers of a second shell material. This approach
allows the deposition of at least one inner shell layer of a
material having a bandgap and lattice size compatible with the
core, followed by the deposition of at least one outer shell layer
of a material having a bandgap and lattice size compatible with the
inner shell layer. In some embodiments, multiple sequential
monolayers of a single shell material can be applied to provide a
uniform shell of a desired number of monolayers of a single shell
material; in these embodiments, the first and second shell
materials are the same. In other embodiments, sequential monolayers
of an alloyed shell material are applied, where the ratio of the
components varies such that the composition becomes successively
enriched in one component of the multi-component mixture as the
successive monolayers of shell material are deposited.
[0432] In some embodiments, the layered shell can be about 3-20
monolayers of shell material thick, sometimes about 5-14 monolayers
of shell material thick, sometimes about 6-12 monolayers of shell
material thick or sometimes about 7-10 monolayers of shell material
thick. In some embodiments, at least one inner shell layer can be
comprised of about 3-5 monolayers, sometimes about 3-7 monolayers,
of the first shell material. In other embodiments, at least one
outer shell layer can be comprised of about 3-5 monolayers,
sometimes about 3-7 monolayers, of the second shell material. In
some embodiments, the inner shell layer can be at least 3
monolayers thick; in other embodiments, the outer shell layer can
be at least 3 monolayers thick. The individual monolayers can be
formed by the controlled, sequential addition of the layer
materials methods described herein. The monolayers may not always
be completely distinct as they may, in some embodiments, be a
latticing between the surfaces of contacting monolayers.
[0433] In certain embodiments, provided herein are nanoparticles
having a thick, uniform, layered shell, as described herein,
wherein the core comprises CdSe, the at least one inner shell layer
comprises CdS, and the at least one outer shell layer comprises
ZnS. In a particular embodiment, provided herein is a nanoparticle
or population thereof having a CdSe core and a layered shell
comprising 4CdS+3.5ZnS layers. In some embodiments, provided herein
is a nanoparticle which consists essentially of
CdSe/4CdS-3.5ZnS.
[0434] Also disclosed herein are methods of making a nanoparticle
and population thereof with modulated, reduced or no fluorescence
intermittency or "blinking". These nanoparticles can be small,
photostable, bright, highly FRET efficient or some combination
thereof. These nanoparticles can have a multi-shell layered core
achieved by a sequential shell material deposition process, whereby
one shell material is added at a time, to provide a nanoparticle
having a substantially uniform shell of desired thickness which is
substantially free of defects.
[0435] In one aspect, provided herein is a method for making a
nanoparticle or population thereof with modulated, reduced or no
fluorescence intermittency, comprising: providing a mixture
comprising a core and at least one coordinating solvent; adding a
first inner shell precursor alternately with a second inner shell
precursor in layer additions, to form an inner shell layer which is
a desired number of layers thick; and adding a first outer shell
precursor alternately with a second outer shell precursor in layer
additions, to form an outer shell layer which is a desired number
of layers thick. If the coordinating solvent of is not amine, the
method further comprises an amine in.
[0436] In some embodiments, the mixture can be heated to a
temperature which is suitable for shell formation before and/or
after every sequential addition of a shell precursor. In some
embodiments, the shell is substantially uniform in coverage around
the core and is substantially free of defects. In some embodiments,
the resulting nanoparticles have a diameter of less than about 15
nm. In other embodiments, the nanoparticles have a diameter of
between about 6 nm to about 10 nm. The nanoparticles made by this
method can have quantum yields greater than about 80%. The
nanoparticle made by this method can have on-time fractions (i.e.,
ratio of the time which nanoparticle emission is turned "on" when
the nanoparticle is excited) of greater than about 0.80 (under
moderate to high excitation energy).
[0437] In another aspect, provided herein is a method for making a
FRET capable nanoparticle and populations thereof with modulated,
reduced or no fluorescence intermittency, comprising: (a) providing
a mixture comprising a plurality of nanocrystal cores and at least
one coordinating solvent; (b) adding a first intermediate shell
precursor alternately with a second intermediate shell precursor in
layer additions to form an intermediate shell layer on each of the
plurality of nanocrystal cores, wherein the intermediate shell
layer is comprised of more than one monolayer; (c) adding a first
external shell precursor alternately with a second external shell
precursor in layer additions to form an external shell layer on
each of the plurality of nanocrystal cores, wherein the external
shell layer is disposed on top of the intermediate shell layer and
is comprised of more than one monolayer; (d) adding an aqueous
solution comprising a hydrophilic ligand; and (e) maintaining the
mixture under conditions which cause the plurality of nanocrystals
to migrate into an aqueous phase. If the coordinating solvent is
not an amine, at least one amine can be included in step (a). In
some embodiments, the resulting population of FRET capable
non-blinking nanoparticles has a .alpha..sub.on, value which is
less than about 1.4. In other embodiments, the resulting population
of FRET capable non-blinking nanoparticles has an on-time fraction
of least about 0.8 (under moderate to high excitation energy). In
some embodiments, the resulting population of FRET capable
non-blinking nanoparticles has diameters which are less than about
15 nm. In some embodiments, the resulting population of FRET
capable non-blinking nanoparticles has a FRET efficiency of at
least 20%. In some embodiments, the resulting population of FRET
capable non-blinking nanoparticles has a quantum yield of at least
about 40%.
[0438] In some embodiments, the methods disclosed above utilize a
one step or a two step ligand exchange process to replace the
hydrophobic ligands on the nanoparticles with hydrophilic ligands
to cause the plurality of nanocrystals to migrate into the aqueous
phase. See PCT Application Serial No. PCT/US09/053,018 and
PCT/US09/059,456 which are expressly incorporated herein by
reference as if set forth in full.
[0439] In another aspect, provided herein is a method for making a
FRET capable nanoparticle and populations thereof with modulated,
reduced or no fluorescence intermittency, comprising: providing a
mixture comprising a plurality of nanocrystal cores, functionalized
organophosphorous-based hydrophilic ligands and at least one
coordinating solvent; adding a first intermediate shell precursor
alternately with a second intermediate shell precursor in layer
additions to form an intermediate shell layer on each of the
plurality of nanocrystal cores; and adding a first external shell
precursor alternately with a second external shell precursor in
layer additions to form an external shell layer on each of the
plurality of nanocrystal cores. In some embodiments, the resulting
population of FRET capable non-blinking nanoparticles has an
.alpha..sub.on value which is less than about 1.4. In other
embodiments, the resulting population of FRET capable non-blinking
nanoparticles has an on-time fraction of least about 0.8. In some
embodiments, the resulting population of FRET capable non-blinking
nanoparticles has diameters which are less than about 15 nm. In
some embodiments, the resulting population of FRET capable
non-blinking nanoparticles has a FRET efficiency of at least 20%.
In some embodiments, the resulting population of FRET capable
non-blinking nanoparticles has a quantum yield of at least about
40%.
[0440] In some embodiments, the functionalized
organophosphorous-based hydrophilic ligands are multi-functional
surface ligands which include a phosphonate/phosphinate nanocrystal
binding center, a linker, and a functional group, which imparts
functionality on the nanocrystal. As used herein the term
"functional group" may refer to a group which affects reactivity,
solubility, or both reactivity and solubility when present on a
multi-functional surface ligand. Embodiments can include a wide
variety of functional groups which can impart various types of
functionality on the nanocrystal including hydrophilicity,
water-solubility, or dispersibility and/or reactivity, and the
functionality may generally not include only hydrophobicity or only
solubility in organic solvents without increasing reactivity. For
example, a functional group which is generally hydrophobic but
which increases reactivity such as an alkene or alkyne and certain
esters and ethers can be encompassed by embodiments, whereas alkyl
groups, which do not generally impart reactivity but increase
hydrophobicity may be excluded.
[0441] In certain embodiments, the FRET capable and non-blinking
nanoparticles produced by the disclosed methods may be coated with
ligands which impart water solubility and/or reactivity on the
nanoparticle obviating the need for ligand replacement. Without
wishing to be bound by theory, eliminating ligand replacement may
provide more consistent thermodynamic properties, which may lead to
reduction in variability of coating and less loss of quantum yield,
among other improvements in the properties of nanoparticles
produced by the methods embodied herein. Eliminating ligand
replacement may also allow for the production of nanoparticles
having a wide variety of functional groups associated with the
coating. In particular, while ligand replacement is generally
limited to production of nanoparticles having amine and/or
carboxylic acid functional groups, in various embodiments, the
skilled artisan may choose among numerous functional groups when
preparing the multi-functional ligands and may, therefore, generate
nanoparticles which provide improved water-solubility or
water-dispersity and/or support improved crosslinking and/or
improved reactivity with cargo molecules. See PCT Application
Serial No. PCT/US09/059,117 which is expressly incorporated herein
by reference as if set forth in full.
[0442] In another aspect, provided herein is a method of making a
nanoparticle or population thereof comprising a core and a layered
gradient shell, wherein the shell comprises an multi-component
(e.g., alloy, etc.) shell material of the form
M.sup.1.sub.xM.sup.2.sub.yX, where x and y represent the ratio of
M.sup.1 and M.sup.2 in the shell material. The method comprising:
(a) providing a mixture comprising a core, at least one
coordinating solvent; (b) heating said mixture to a temperature
suitable for formation of the shell layer; and (c) adding a first
inner shell precursor comprising M.sup.1.sub.x and M.sup.2.sub.y
alternately with a second inner shell precursor comprising X in
layer additions, wherein the ratio of y to x gradually increases in
sequential layer additions, such that the shell layers becomes
successively enriched in M.sup.2, to form a layered gradient shell
which is a desired number of monolayers thick. If the coordinating
solvent is not an amine, at least one amine can be included in step
(a).
[0443] In one embodiment, the method described above provides a
nanoparticle having a layered gradient shell, wherein the core
comprises CdSe and the shell comprises sequential layers of
Cd.sub.xZn.sub.yS, where the ratio of y to x increases gradually
from the innermost shell layer to the outermost shell layer, to
provide a layered gradient shell with a finely graded potential. In
some such embodiments, the outermost shell layer is essentially
pure ZnS. In some embodiments, the percent of Zn in the gradient
shell varies from less than about 10% at the innermost shell layer
to greater than about 80% at the outermost shell layer.
[0444] Typically, the heating steps in the disclosed methods are
conducted at a temperature within the range of about
150-350.degree. C., more preferably within the range of about
200-300.degree. C. In some embodiments, the temperature suitable
for formation of at least one inner shell layer is about
215.degree. C. In some embodiments, the temperature suitable for
formation of at least one outer shell layer is about 245.degree. C.
It is understood that the above ranges are merely exemplary and are
not intended to be limiting in any manner as the actual temperature
ranges may vary, dependent upon the relative stability of the
precursors, ligands, and solvents. Higher or lower temperatures may
be appropriate for a particular reaction. The determination of
suitable time and temperature conditions for providing
nanoparticles is within the level of skill in the art using routine
experimentation.
[0445] It can be advantageous to conduct the nanoparticle-forming
reactions described herein with the exclusion of oxygen and
moisture. In some embodiments the reactions are conducted in an
inert atmosphere, such as in a dry box. The solvents and reagents
are also typically rigorously purified to remove moisture and
oxygen and other impurities, and are generally handled and
transferred using methods and apparatus designed to minimize
exposure to moisture and/or oxygen. In addition, the mixing and
heating steps can be conducted in a vessel which is evacuated and
filled and/or flushed with an inert gas such as nitrogen. The
filling can be periodic or the filling can occur, followed by
continuous flushing for a set period of time.
[0446] In some embodiments, the at least one coordinating solvent
comprises a trialkylphosphine, a trialkylphosphine oxide, a
phosphonic acid, or a mixture of these. Sometimes, the at least one
coordinating solvent comprises TOP, TOPO, TDPA, OPA or a mixture of
these. The solvent for these reactions often comprises a primary or
secondary amine, for example, decylamine, hexadecylamine, or
dioctylamine. In some embodiments, the amine is decylamine. In some
embodiments, the first inner shell precursor is Cd(OAc).sub.2 and
the second inner shell precursor is bis(trimethylsilyl)sulfide
(TMS.sub.2S). Sometimes, the first and second inner shell
precursors are added as a solution in TOP. In some embodiments, the
first outer shell precursor is Et.sub.2Zn and the second inner
shell precursor is TMS.sub.2S. Sometimes, the first and second
outer shell precursors are added as a solution in TOP.
[0447] In certain embodiments, the disclosed nanoparticles may be
prepared using the method described herein to build a layered
CdS--ZnS shell on a CdSe quantum size core. The shells for these
materials can have varying numbers of layers of CdS and ZnS.
Prototypical materials containing a CdSe core and approximately 4
monolayers CdS and 3.5 monolayers of ZnS (the final 0.5 monolayer
is essentially pure Zn), or a CdSe core and 9 monolayers CdS and
3.5 monolayers of ZnS were prepared as described in the
examples.
[0448] In some embodiments, for either the inner or outer layer, or
both, less than a full layer of the appropriate first shell
precursor can be added alternately with less than a full layer of
the appropriate second shell precursor, so the total amount of the
first and second shell precursor required is added in two or more
portions. Sometimes, the portion is about 0.25 monolayers of shell
material, so that the 4 portions of 0.25 monolayer of first shell
precursor are added alternately with 4 portions of 0.25 monolayer
of second shell precursor; sometimes the portion is about 0.5
monolayers of shell material, and sometimes about 0.75 monolayers
of shell material.
[0449] Examples of compounds useful as the first precursor can
include, but are not limited to: organometallic compounds such as
alkyl metal species, salts such as metal halides, metal acetates,
metal carboxylates, metal phosphonates, metal phosphinates, metal
oxides, or other salts. In some embodiments, the first precursor
provides a neutral species in solution. For example, alkyl metal
species such as diethylzinc (Et.sub.2Zn) or dimethyl cadmium are
typically considered to be a source of neutral zinc
atoms)(Zn.sup.0) in solution. In other embodiments, the first
precursor provides an ionic species (i.e., a metal cation) in
solution. For example, zinc chloride (ZnCl.sub.2) and other zinc
halides, zinc acetate (Zn(OAc).sub.2) and zinc carboxylates are
typically considered to be sources of Zn.sup.2+ cations in
solution.
[0450] By way of example only, suitable first precursors providing
neutral metal species include dialkyl metal sources, such as
dimethyl cadmium (Me.sub.2Cd), diethyl zinc (Et.sub.2Zn), and the
like. Suitable first precursors providing metal cations in solution
include, e.g., cadmium salts, such as cadmium acetate
(Cd(OAc).sub.2), cadmium nitrate (Cd(NO.sub.3).sub.2), cadmium
oxide (CdO), and other cadmium salts; and zinc salts such as zinc
chloride (ZnCl.sub.2), zinc acetate (Zn(OAc).sub.2), zinc oleate
(Zn(oleate).sub.2), zinc chloro(oleate), zinc undecylenate, zinc
salicylate, and other zinc salts. In some embodiments, the first
precursor is salt of Cd or Zn. In some embodiments, it is a halide,
acetate, carboxylate, or oxide salt of Cd or Zn. In other
embodiments, the first precursor is a salt of the form
M(O.sub.2CR)X, wherein M is Cd or Zn; X is a halide or O.sub.2CR;
and R is a C4-C24 alkyl group which is optionally unsaturated.
Other suitable forms of Groups 2, 12, 13 and 14 elements useful as
first precursors are known in the art.
[0451] Precursors useful as the "second" precursor in the disclosed
methods include compounds containing elements from Group 16 of the
Periodic Table of the Elements (e.g., S, Se, Te, and the like),
compounds containing elements from Group 15 of the Periodic Table
of the Elements (N, P, As, Sb, and the like), and compounds
containing elements from Group 14 of the Periodic Table of the
Elements (Ge, Si, and the like). Many forms of the precursors can
be used in the disclosed methods. It will be understood that in
some embodiments, the second precursor will provide a neutral
species in solution, while in other embodiments the second
precursor will provide an ionic species in solution.
[0452] When the first precursor comprises a metal cation, the
second precursor can provide an uncharged (i.e., neutral) non-metal
atom in solution. In frequent embodiments, when the first precursor
comprises a metal cation, the second precursor contributes a
neutral chalcogen atom, most commonly S.sup.0, Se.sup.0 or
Te.sup.0.
[0453] Suitable second precursors for providing a neutral chalcogen
atom include, for example, elemental sulfur (often as a solution in
an amine, e.g., decylamine, oleylamine, or dioctylamine, or an
alkene, such as octadecene), and tri-alkylphosphine adducts of S,
Se and Te. Such trialkylphosphine adducts are sometimes described
herein as R3P=X, wherein X is S, Se or Te, and each R is
independently H, or a C1-C24 hydrocarbon group which can be
straight-chain, branched, cyclic, or a combination of these, and
which can be unsaturated. Exemplary second precursors of this type
include tri-n (butylphosphine)selenide (TBP=Se),
tri-n-(octylphosphine)selenide (TOP=Se), and the corresponding
sulfur and tellurium reagents, TBP=S, TOP=S, TBP=Te and TOP=Te.
These reagents are frequently formed by combining a desired
element, such as Se, S, or Te with an appropriate coordinating
solvent, e.g., TOP or TBP. Precursors which provide anionic species
under the reaction conditions are typically used with a first
precursor which provides a neutral metal atom, such as alkylmetal
compounds and others described above or known in the art.
[0454] In some embodiments, the second precursor provides a
negatively charged non-metal ion in solution (e.g., S-2, Se-2 or
Te-2). Examples of suitable second precursors providing an ionic
species include silyl compounds such as bis(trimethylsilyl)selenide
((TMS).sub.2Se), bis(trimethylsilyl)sulfide ((TMS).sub.2S) and
bis(trimethylsilyl)telluride ((TMS).sub.2Te). Also included are
hydrogenated compounds such as H2Se, H2S, H2Te; and metal salts
such as NaHSe, NaSH or NaHTe. In this situation, an oxidant can be
used to oxidize a neutral metal species to a cationic species which
can react with the anionic precursor in a `matched` reaction, or an
oxidant can be used increase the oxidation state of the anionic
precursor to provide a neutral species which can undergo a
`matched` reaction with a neutral metal species.
[0455] Other exemplary organic precursors are described in U.S.
Pat. Nos. 6,207,229 and 6,322,901 to Bawendi et al., and synthesis
methods using weak acids as precursor materials are disclosed by Qu
et al., (2001), Nano Lett., 1(6):333-337, the disclosures of each
of which are incorporated herein by reference in their
entirety.
[0456] Both the first and the second precursors can be combined
with an appropriate solvent to form a solution for use in the
disclosed methods. The solvent or solvent mixture used to form a
first precursor solution may be the same or different from that
used to form a second precursor solution. Typical coordinating
solvents include alkyl phosphines, alkyl phosphine oxides, alkyl
phosphonic acids, alkyl phosphinic acids, or carboxylic acid
containing solvents, or mixtures of these.
[0457] Suitable reaction solvents include, by way of illustration
and not limitation, hydrocarbons, amines, alkyl phosphines, alkyl
phosphine oxides, carboxylic acids, ethers, furans, phosphoacids,
pyridines and mixtures thereof. The solvent may actually comprise a
mixture of solvents, often referred to in the art as a "solvent
system". In some embodiments, the solvent comprises at least one
coordinating solvent. In some embodiments, the solvent system
comprises a secondary amine and a trialkyl phosphine (e.g., TBP or
TOP) or a trialkylphosphine oxide (e.g., TOPO). If the coordinating
solvent is not an amine, an amine can be included.
[0458] A coordinating solvent might be a mixture of an essentially
non-coordinating solvent such as an alkane and a ligand as defined
below.
[0459] Suitable hydrocarbons include alkanes, alkenes and aromatic
hydrocarbons from 10 to about 30 carbon atoms; examples include
octadecene and squalane. The hydrocarbon may comprise a mixture of
alkane, alkene and aromatic moieties, such as alkylbenzenes (e.g.,
mesitylene).
[0460] Suitable amines include, but are not limited to,
monoalkylamines, dialkylamines, and trialkylamines, for example
dioctylamine, oleylamine, decylamine, dodecylamine,
hexyldecylamine, and so forth. Alkyl groups for these amines
typically contain about 6-24 carbon atoms per alkyl, and can
include an unsaturated carbon-carbon bond, and each amine typically
has a total number of carbon atoms in all of its alkyl groups
combined of about 10-30 carbon atoms.
[0461] Exemplary alkyl phosphines include, but are not limited to,
the trialkyl phosphines, tri-n-butylphosphine (TBP),
tri-n-octylphosphine (TOP), and so forth. Alkyl groups for these
phosphines contain about 6-24 carbon atoms per alkyl, and can
contain an unsaturated carbon-carbon bond, and each phosphine has a
total number of carbon atoms in all of its alkyl groups combined of
about 10-30 carbon atoms.
[0462] Suitable alkyl phosphine oxides include, but are not limited
to, the trialkyl phosphine oxide, tri-n-octylphosphine oxide
(TOPO), and so forth. Alkyl groups for these phosphine oxides
contain about 6-24 carbon atoms per alkyl, and can contain an
unsaturated carbon-carbon bond, and each phosphine oxide has a
total number of carbon atoms in all of its alkyl groups combined of
about 10-30 carbon atoms.
[0463] Exemplary fatty acids include, but are not limited to,
stearic, oleic, palmitic, myristic and lauric acids, as well as
other carboxylic acids of the formula R--COOH, wherein R is a
C6-C24 hydrocarbon group and can contain an unsaturated
carbon-carbon bond. It will be appreciated that the rate of
nanocrystal growth generally increases as the length of the fatty
acid chain decreases.
[0464] Exemplary ethers and furans include, but are not limited to,
tetrahydrofuran and its methylated forms, glymes, and so forth.
[0465] Suitable phosphonic and phosphinic acids include, but are
not limited to hexylphosphonic acid (HPA), tetradecylphosphonic
acid (TDPA), and octylphosphinic acid (OPA), and are frequently
used in combination with an alkyl phosphine oxide such as TOPO.
Suitable phosphonic and phosphinic acids are of the formula
RPO.sub.3H.sub.2 or R.sub.2PO.sub.2H, wherein each R is
independently a C6-C24 hydrocarbon group and can contain an
unsaturated carbon-carbon bond.
[0466] Exemplary pyridines include, but are not limited to,
pyridine, alkylated pyridines, nicotinic acid, and so forth.
[0467] Suitable alkenes include, e.g., octadecene and other C4-C24
hydrocarbons which are unsaturated.
[0468] Nanoparticle core or shell precursors can be represented as
a M-source and an X-donor. The M-source can be an M-containing
salt, such as a halide, carboxylate, phosphonate, carbonate,
hydroxide, or diketonate, or a mixed salt thereof (e.g., a halo
carboxylate salt, such as Cd(halo)(oleate)), of a metal, M, in
which M can be, e.g., Cd, Zn, Mg, Hg, Al, Ga, In, or Tl. In the
X-donor, X can be, e.g., O, S, Se, Te, N, P, As, or Sb. The mixture
can include an amine, such as a primary amine (e.g., a C8-C20 alkyl
amine). The X donor can include, for example, a phosphine
chalcogenide, a bis(trialkylsilyl)chalcogenide, a dioxygen species,
an ammonium salt, or a tris(trialkylsilyl)phosphine, or the
like.
[0469] The M-source and the X donor can be combined by contacting a
metal, M, or an M-containing salt, and a reducing agent to form an
M-containing precursor. The reducing agent can include an alkyl
phosphine, a 1,2-diol or an aldehyde, such as a C.sub.6-C.sub.20
alkyl diol or a C.sub.6-C.sub.20 aldehyde.
[0470] Suitable M-containing salts include, for example, cadmium
acetylacetonate, cadmium iodide, cadmium bromide, cadmium chloride,
cadmium hydroxide, cadmium carbonate, cadmium acetate, cadmium
oxide, zinc acetylacetonate, zinc iodide, zinc bromide, zinc
chloride, zinc hydroxide, zinc carbonate, zinc acetate, zinc oxide,
magnesium acetylacetonate, magnesium iodide, magnesium bromide,
magnesium chloride, magnesium hydroxide, magnesium carbonate,
magnesium acetate, magnesium oxide, mercury acetylacetonate,
mercury iodide, mercury bromide, mercury chloride, mercury
hydroxide, mercury carbonate, mercury acetate, aluminum
acetylacetonate, aluminum iodide, aluminum bromide, aluminum
chloride, aluminum hydroxide, aluminum carbonate, aluminum acetate,
gallium acetylacetonate, gallium iodide, gallium bromide, gallium
chloride, gallium hydroxide, gallium carbonate, gallium acetate,
indium acetylacetonate, indium iodide, indium bromide, indium
chloride, indium hydroxide, indium carbonate, indium acetate,
thallium acetylacetonate, thallium iodide, thallium bromide,
thallium chloride, thallium hydroxide, thallium carbonate, or
thallium acetate. Suitable M-containing salts also include, for
example, carboxylate salts, such as oleate, stearate, myristate,
and palmitate salts, mixed halo carboxylate salts, such as
M(halo)(oleate) salts, as well as phosphonate salts.
[0471] Alkyl is a branched or unbranched saturated hydrocarbon
group of 1 to 100 carbon atoms, preferably 1 to 30 carbon atoms,
such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,
t-butyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl
and the like, as well as cycloalkyl groups such as cyclopentyl,
cyclohexyl and the like. Optionally, an alkyl can contain 1 to 6
linkages selected from the group consisting of --O--, --S--, -M-
and --NR-- where R is hydrogen, or C1-C8 alkyl or lower
alkenyl.
[0472] The X donor is a compound capable of reacting with the
M-containing salt to form a material with the general formula MX.
The X donor is generally a chalcogenide donor or a phosphine donor,
such as a phosphine chalcogenide, a bis(silyl) chalcogenide,
dioxygen, an ammonium salt, or a tris(trialkylsilyl) phosphine.
Suitable X donors include dioxygen, elemental sulfur,
bis(trimethylsilyl) selenide ((TMS).sub.2Se), trialkyl phosphine
selenides such as (tri-n-octylphosphine) selenide (TOPSe) or
(tri-n-butylphosphine) selenide (TBPSe), trialkyl phosphine
tellurides such as (tri-n-octylphosphine) telluride (TOPTe) or
hexapropylphosphorustriamide telluride (HPPTTe),
bis(trimethylsilyl)telluride ((TMS).sub.2Te), sulfur,
bis(trimethylsilyl)sulfide ((TMS).sub.2S), a trialkyl phosphine
sulfide such as (tri-n-octylphosphine) sulfide (TOPS),
tris(dimethylamino) arsine, an ammonium salt such as an ammonium
halide (e.g., NH.sub.4Cl), tris(trimethylsilyl) phosphide
((TMS).sub.3P), tris(trimethylsilyl) arsenide ((TMS).sub.3As), or
tris(trimethylsilyl) antimonide ((TMS).sub.3Sb). In certain
embodiments, the M donor and the X donor can be moieties within the
same molecule.
Ligand Exchange Processes for Coating Nanoparticles
[0473] Provided herein are ligand exchange processes that permit
efficient conversion of a conventional hydrophobic nanoparticle or
population thereof into a water-dispersible and functionalized
nanoparticle or population of nanoparticles. It also permits
preparation of small nanoparticles which are highly stable and
bright enough to be useful in biochemical and biological assays.
The resulting nanoparticles can also be linked to a target molecule
or cell or enzyme (e.g., polymerase) of interest.
[0474] Typically, the nanoparticle used for this process is a
core/shell nanocrystal which is coated with a hydrophobic ligand
such as tetradecylphosphonic acid (TDPA), trioctylphosphine oxide
(TOPO), trioctyl phosphine (TOP), octylphosphonic acid (OPA), and
the like, or a mixture of such ligands; these hydrophobic ligands
typically have at least one long-chain alkyl group, i.e. an alkyl
group having at least 8 carbons, or for the phosphine/phosphine
oxide ligands, this hydrophobic character may be provided by two or
three alkyl chains on a single ligand molecule having a total of at
least 10 carbon atoms. Therefore, in some embodiments, the surface
of the core/shell nanocrystal or population thereof can be coated
with varying quantities of TDPA hydrophobic ligands prior to
replacement with hydrophilic ligand(s). For example, TDPA can
represent at least about 10%, at least about 20%, at least about
30%, at least about 40%, at least about 50%, at least about 80%, at
least about 95%, at least about 98%, at least about 99% or more of
the total surface ligands coating the core/shell nanoparticles.
Moreover, certain hydrophobic ligands show an unexpected and
apparent ease of replacement with the hydrophilic ligand. For
example, nanoparticles with OPA on the surface have been observed
to transfer into aqueous buffer more readily and more completely
than the same type of core-shell with TDPA on the surface.
Therefore, in some embodiments, the surface of the core/shell
nanocrystal or populations thereof can be coated with varying
quantities of OPA hydrophobic ligands prior to replacement with
hydrophilic ligand(s). For example, OPA can represent at least
about 10%, at least about 20%, at least about 30%, at least about
40%, at least about 50%, at least about 80%, at least about 95%, at
least about 98%, at least about 99% or more of the total surface
ligands coating the core/shell nanocrystal.
[0475] In one aspect, provided herein is a "one-step" ligand
exchange process to apply various types of ligands to the surface
of a nanoparticle, by substituting a desired hydrophilic ligand for
a conventional hydrophobic ligand like TOPO, TOP, TDPA, OPA, and
the like. The process steps, comprising: providing a nanocrystal
coated with a surface layer comprising a hydrophobic ligand, and
dissolved or dispersed in a non-aqueous solvent, contacting the
nanocrystal dispersion with a phase transfer agent and an aqueous
solution comprising a hydrophilic ligand, to form a biphasic
mixture having an aqueous phase and a non-aqueous phase and
maintaining the mixture under conditions that cause the nanocrystal
to migrate from the non-aqueous solvent into the aqueous phase. See
PCT Application Serial No. PCT/US09/053,018 which is expressly
incorporated herein by reference as if set forth in full.
[0476] The `one-step` ligand exchange process described herein
utilizes phase transfer catalysts which are particularly effective,
and provide faster exchange reactions. Butanol has been utilized as
a phase transfer catalyst for this type of exchange reaction;
however, the reaction takes several days typically, and requires
heating to about 70.degree. C. The time for this reaction exposes
the nanoparticles to these reaction conditions for a long period of
time, which may contribute to some reduction in its ultimate
stability. The embodiments disclosed herein provide more efficient
conditions which achieve ligand exchange more rapidly, thus better
protecting the nanoparticles. As a result of accelerating the
exchange reaction and allowing use of milder conditions, these
phase transfer catalysts produce higher quality nanoparticles.
[0477] The phase transfer agent for this process can be a crown
ether, a PEG, a trialkylsulfonium, a tetralkylphosphonium, and an
alkylammonium salt, or a mixture of these. In some embodiments, the
phase transfer agent is 18-crown-6,15-crown-5, or 12-crown-4. In
some embodiments, the phase transfer agent is a PEG, which can have
a molecular weight from about 500 to about 5000. In some
embodiments, the phase transfer agent is a trialkylsulfonium,
tetralkylphosphonium, or alkylammonium (including
monoalkylammonium, dialkylammonium, trialkylammonium and
tetralkylammonium) salt.
[0478] Tetralkylammonium salts are sometimes preferred as phase
transfer agents. Examples of suitable tetralkylammonium salts
include triethylbenzyl ammonium, tetrabutylammonium,
tetraoctylammonium, and other such quaternary salts. Other
tetralkylammonium salts, where each alkyl group is a C1-C12 alkyl
or arylalkyl group, can also be used. Typically, counting all of
the carbons on the alkyl groups of a trialkylsulfonium,
tetralkylphosphonium, and alkylammonium salt, the phase transfer
agent will contain a total of at least 2 carbons, at least 10
carbons and preferably at least 12 carbon atoms. Each of the
trialkylsulfonium, tetralkylphosphonium, and alkylammonium salts
has a counterion associated with it; suitable counterions include
halides, preferably chloride or fluoride; sulfate, nitrate,
perchlorate, and sulfonates such as mesylate, tosylate, or
triflate; mixtures of such counterions can also be used. The
counterion can also be a buffer or base, such as borate, hydroxide
or carbonate; thus, for example, tetrabutylammonium hydroxide can
be used to provide the phase transfer catalyst and a base. Specific
phase transfer salts for use in these methods include
tetrabutylammonium chloride (or bromide) and tetraoctylammonium
bromide (or chloride).
[0479] Suitable hydrophilic ligands are organic molecules which
provide at least one binding group to associate tightly with the
surface of a nanocrystal. The hydrophilic ligand typically is an
organic moiety having a molecular weight between about 100 and
1500, and contains enough polar functional groups to be water
soluble. Some examples of suitable hydrophilic ligands include
small peptide having 2-10 amino acid residues (preferably including
at least one histidine or cysteine residue), mono- or polydentate
thiol containing compounds.
[0480] Following ligand exchange, the surface layer can optionally
be crosslinked.
[0481] In another aspect, provided herein is a "two-step" ligand
exchange process to apply various types of ligands to the surface
of a nanoparticle, by substituting a desired hydrophilic ligand for
a conventional hydrophobic ligand like TOPO, TOP, TDPA, OPA, and
the like. The process involves the removal of phosphonate or
phosphinate ligands from the surface of a nanoparticle or
nanocrystal by treatment with sulfonate reagents, particularly
silylsulfonate derivatives of weak bases or other poorly
coordinating groups.
[0482] The process steps, comprising: providing a nanocrystal whose
surface comprises a phosphonate ligand, contacting the nanocrystal
with a sulfonate reagent in an organic solvent, contacting the
sulfonate ligand coated nanocrystal with a functionalized organic
molecule (i.e., hydrophilic ligand) comprising at least one
nanocrystal surface attachment group, contacting the nanocrystal
dispersion with an aqueous solution to form a biphasic mixture
having an aqueous phase and a non-aqueous phase, and maintaining
the biphasic mixture under conditions which cause the nanocrystal
to migrate from the non-aqueous phase into the aqueous phase. See
PCT Application Serial No. PCT/US09/59456 which is expressly
incorporated herein by reference as if set forth in full.
[0483] The result of this removal of phosphonate ligands is
replacement of the phosphonates with the weakly coordinating
groups. One example is the use of silyl sulfonates, such as
trimethylsilyl triflate, to form a sulfonate-coated nanoparticle.
Triflate is a conventional/common name for a
trifluoromethanesulfonyloxy group, CF.sub.3SO.sub.2O--.
[0484] The same type of replacement process can also occur on
nanoparticles having phosphinic acid ligands of the formula
R.sub.2P(.dbd.O)--OH or on nanoparticles having carboxylic acid
ligands of the formula RC(.dbd.O)--OH, which could be incorporated
on the surface of a nanocrystal by known methods; R can be a
C.sub.1-C.sub.24 hydrocarbon group in these phosphinates, and the
two R groups can be the same or different. Thus, it is understood
that when phosphonate-containing nanocrystals are described herein,
phosphinate-containing nanocrystals can be used instead, with
similar results.
[0485] This process provides a mild and selective method for
removing phosphonate, phosphinate, and carboxylate ligands from the
surface of a nanocrystal. As a result, it provides a way for a user
to remove these groups and replace them, without removing other
ligands which are not displaced or affected by the
silylsulfonate.
[0486] The sulfonate ligands can comprise an alkyl or aryl moiety
linked to --SO.sub.3X, where X can represent whatever the sulfonate
group is attached to. For example, where the sulfonate ligand is a
sulfonate anion (i.e., triflate), X would represent a nanocrystal,
or the surface of a nanocrystal. Some of the sulfonate embodiments
disclosed herein can also be described with reference to feature
`A` of Formula I, as set forth below.
##STR00002##
[0487] wherein R.sup.1, R.sup.2, R.sup.3 and A are each,
independently, C1-C10 alkyl or C5-C10 aryl; and each alkyl and aryl
is optionally substituted.
[0488] The alkyl groups for Formula I compounds are independently
selected, and can be straight chain, branched, cyclic, or
combinations of these, and optionally can include a C1-C4 alkoxy
group as a substituent. Typically, the alkyl groups are lower
alkyls, e.g., C1-C4 alkyl groups which are linear or branched.
Methyl is one suitable example.
[0489] The aryl group for the compounds of Formula I can be phenyl,
naphthyl or a heteroaryl having up to 10 ring members, and can be
monocyclic or bicyclic, and optionally contain up to two
heteroatoms selected from N, O and S as ring members in each ring.
(It will be understood by those skilled in the art that the
5-membered aryl is a heteroaryl ring.) Phenyl is a preferred aryl
group; and an aryl group is typically only present if the other
organic groups on the silicon other than the sulfonate are lower
alkyls, and preferably they are each Me.
[0490] Examples of silylsulfonate ligands can include, but are not
limited to: (trimethylsilyl)triflate, (triethylsilyl)triflate,
(t-butyldimethylsilyl)triflate, (phenyldimethylsily)triflate,
trimethylsilyl fluoromethanesulfonate, trimethylsilyl
methanesulfonate, trimethylsilyl nitrophenylsulfonate,
trimethylsilyl trifluoroethylsulfonate, trimethylsilyl
phenylsulfonate, trimethylsilyl toluenesulfonate, diisopropylsilyl
bis(trifluoromethanesulfonate), tertbutyldimethylsilyl
trifluoromethanesulfonate, triisopropylsilyl
trifluoromethanesulfonate and trimethylsilyl chlorosulfonate.
[0491] Examples of other sulfonate ligands can include, but are not
limited to: trifluoromethanesulfonate (triflate),
fluoromethanesulfonate, methanesulfonate (mesylate),
nitrophenylsulfonate (nosylate), trifluorethylsulfonate,
phenylsulfonate (besylate) and toluenesulfonate (tosylate).
[0492] Some suitable examples of the hydrophilic ligand are
disclosed, for example, in Naasani, U.S. Pat. Nos. 6,955,855;
7,198,847; 7,205,048; 7,214,428; and 7,368,086. Suitable
hydrophilic ligands also include imidazole containing compounds
such as peptides, particularly dipeptides, having at least one
histidine residue, and peptides, particularly dipeptides, having at
least one cysteine residue. Specific ligands of interest for this
purpose can include carnosine (which contains beta-alanine and
histidine); His-Leu; Gly-His; His-Lys; His-Glu; His-Ala; His-His;
His-Cys; Cys-His; His-Ile; His-Val; and other dipeptides where H is
or Cys is paired with any of the common alpha-amino acids; and
tripeptides, such as Gly-His-Gly, His-Gly-His, and the like. The
chiral centers in these amino acids can be the natural
L-configuration, or they can be of the D-configuration or a mixture
of L and D. Thus a dipeptide having two chiral centers such as
His-Leu can be of the L,L-configuration, or it can be L,D- or D,L;
or it can be a mixture of diastereomers.
[0493] Furthermore, suitable hydrophilic ligands can also include
mono- or polydentate thiol containing compounds, for example:
monodentate thiols such as mercaptoacetic acid, bidentate thiols
such as dihydrolipoic acid (DHLA), tridentate thiols such as
compounds of Formula II-VII as shown below, and the like.
##STR00003##
[0494] In compounds of Formula II-VI, R.sup.1, R.sup.2, R.sup.3 can
independently be H, halo, hydroxyl, (--(C.dbd.O)--C.sub.1-C.sub.22,
--(C.dbd.O)CF.sub.3,) alkanoyl, C.sub.1-C.sub.22 alkyl,
C.sub.1-C.sub.22 heteroalkyl, ((CO)OC.sub.1-C.sub.22)
alkylcarbonato, alkylthio (C.sub.1-C.sub.22) or
(--(CO)NH(C.sub.1-C.sub.20) or --(CO)N(C.sub.1-C.sub.20).sub.2)
alkylcarbamoyl. In some embodiments, R.sup.1, R.sup.2, and R.sup.3
are different. In other embodiments, R.sup.1, R.sup.2, and R.sup.3
are the same.
[0495] In compounds of Formula II-VI, R.sup.4, and R.sup.5 can
independently be H, C.sub.1-C.sub.20 alkyl, C.sub.6-C.sub.18 aryl,
C.sub.1-C.sub.22 heteroalkyl or C.sub.1-C.sub.22 heteroaryl. In
some embodiments, R.sup.4 and R.sup.5 are different. In other
embodiments, R.sup.4 and R.sup.5 are the same.
[0496] In compounds of Formula II-VI, R.sup.6 can be H or a
polyethylene glycol based moiety of Formula VIII:
##STR00004##
[0497] In certain embodiments of Formula VII, R.sup.7 can be
--NH.sub.2, --N.sub.3, --NHBoc, --NHFmoc, --NHCbz, --COOH,
--COOt-Bu, --COOMe, iodoaryl, hydroxyl, alkyne, boronic acid,
allylic alcohol carbonate, --NHBiotin, --(CO)NHNHBoc,
--(CO)NHNHFmoc or --OMe. In some embodiments, n can be an integer
from 1 to 100.
[0498] In still further embodiments, the tridentate thiol ligands
can be a compound of Formula IX, X, XI, XII, XIII, XIV, XV, XVI,
XVII, XVIII, XIX, XX, XXI, XXII, XXIII or XXIV:
##STR00005## ##STR00006## ##STR00007##
Functionalized TDPA Ligands on Nanoparticles
[0499] Provided herein are methods for preparing water-soluble
semi-conducting, insulating, or metallic nanoparticles including
the steps of admixing one or more nanocrystal precursors and one or
more multi-functional surface ligands with a solvent to form a
solution and heating the solution to a suitable temperature, and in
certain embodiments, methods may include the steps of admixing
nanocrystal cores, one or more nanocrystal precursors, and one or
more multi-functional surface ligands with a solvent to form a
solution and heating the solution to a suitable temperature. In
such embodiments, the one or more multi-functional surface ligands
may at least include a nanocrystal binding center, a linker, and a
functional group, which imparts functionality on the nanocrystal.
As used herein the term "functional group" may refer to a group
which affects reactivity, solubility, or both reactivity and
solubility when present on a multi-functional surface ligand.
Embodiments can include a wide variety of functional groups which
can impart various types of functionality on the nanocrystal
including hydrophilicity, water-solubility, or dispersibility
and/or reactivity, and the functionality may generally not include
only hydrophobicity or only solubility in organic solvents without
increasing reactivity. For example, a functional group which is
generally hydrophobic but which increases reactivity such as an
alkene or alkyne and certain esters and ethers can be encompassed
by embodiments, whereas alkyl groups, which do not generally impart
reactivity but increase hydrophobicity may be excluded.
[0500] In certain embodiments, the nanoparticles produced by the
methods of such embodiments may be coated with ligands which impart
water solubility and/or reactivity on the nanoparticle obviating
the need for ligand replacement. Without wishing to be bound by
theory, eliminating ligand replacement may provide more consistent
thermodynamic properties, which may lead to reduction in
variability of coating and less loss of quantum yield, among other
improvements in the properties of nanoparticles produced by the
methods embodied herein. Eliminating ligand replacement may also
allow for the production of nanoparticles having a wide variety of
functional groups associated with the coating. In particular, while
ligand replacement is generally limited to production of
nanoparticles having amine and/or carboxylic acid functional
groups, in various embodiments, the skilled artisan may choose
among numerous functional groups when preparing the
multi-functional ligands and may, therefore, generate nanoparticles
which provide improved water-solubility or water-dispersity and/or
support improved crosslinking and/or improved reactivity with cargo
molecules. See for example PCT Application Serial No.
PCT/US09/59117 filed Sep. 30, 2009 which are expressly incorporated
herein by reference as if set forth in full.
Solid Surfaces
[0501] The methods, compositions, systems and kits disclosed herein
can involve the use of surfaces (e.g., solid surfaces) which can be
attached covalently or non-covalently with the nanoparticles and/or
the biomolecules (polymerases, nucleotides, target nucleic acid
molecules, primers, and/or oligonucleotides) described herein. The
attachment can be reversible or irreversible. The immobilized
biomolecules include the: polymerases, nucleotides, target nucleic
acid molecules, primer molecules and/or oligonucleotides which are
components in the nucleotide binding and/or nucleotide
incorporation reactions. The immobilized nanoparticles and/or
biomolecules may be attached to the surface in a manner that they
are accessible to components of the nucleotide incorporation
reaction and/or in a manner which does not interfere with
nucleotide binding or nucleotide incorporation. The immobilized
nanoparticles and/or biomolecules may be attached to the surface in
a manner which renders them resistant to removal or degradation
during the incorporation reactions, including procedures which
involve washing, flowing, temperatures or pH changes, and reagent
changes. In another aspect, the immobilized nanoparticles and/or
biomolecules may be reversibly attached to the surface.
[0502] The surface may be a solid surface, and includes planar
surfaces, as well as concave, convex, or any combination thereof.
The surface may comprise texture (e.g., etched, cavitated or
bumps). The surface includes the inner walls of a capillary, a
channel, a well, groove, channel, reservoir, bead, particle,
sphere, filter, gel or a nanoscale device. The surface can be
optically transparent, minimally reflective, minimally absorptive,
or exhibit low fluorescence. The surface may be non-porous. The
surface may be made from materials such as glass, borosilicate
glass, silica, quartz, fused quartz, mica, polyacrylamide, plastic
polystyrene, polycarbonate, polymethacrylate (PMA), polymethyl
methacrylate (PMMA), polydimethylsiloxane (PDMS), silicon,
germanium, graphite, ceramics, silicon, semiconductor, high
refractive index dielectrics, crystals, gels, polymers, or films
(e.g., films of gold, silver, aluminum, or diamond). The surface
can include a solid substrate having a metal film or metal
coat.
[0503] The immobilized nanoparticles and/or biomolecules may be
arranged in a random or ordered array on a surface. The ordered
array includes rectilinear and hexagonal patterns. The distance and
organization of the immobilized molecules may permit distinction of
the signals generated by the different immobilized molecules. The
surface can be coated with an adhesive and/or resist layer which
can be applied to the surface to create the patterned array and can
be applied to the surface in any order. The adhesive layer can
bind/link the nanoparticle or biomolecules (e.g., polymerases,
nucleotides, target nucleic acid molecules, primers, and/or
oligonucleotides). The resist layer does not bind/link, or exhibits
decreased binding/linking, to the nanoparticle or biomolecules
(e.g., polymerases, nucleotides, target nucleic acid molecules,
primers, and/or oligonucleotides).
[0504] The immobilized nucleic acid molecules (e.g., target and/or
primer molecules) may be attached to the surface at their 5' ends
or 3' ends, along their length, or along their length with a 5' or
3' portion exposed. The immobilized proteins (e.g., polymerases)
can be attached to the surface in a manner which orients them to
mediate their activities (nucleotide binding or nucleotide
incorporation).
[0505] The surface can be coated to facilitate attachment of
nucleic acid molecules (target and/or primers). For example, a
glass surface can be coated with a polyelectrolyte multilayer (PEM)
via light-directed attachment (U.S. Pat. Nos. 5,599,695, 5,831,070,
and 5,959,837) or via chemical attachment. The PEM chemical
attachment can occur by sequential addition of polycations and
polyanions (Decher, et al., 1992 Thin Solid Films 210:831-835). In
one embodiment, the glass surface can be coated with a
polyelectrolyte multilayer which terminated with polyanions or
polycations. The polyelectrolyte multilayer can be coated with
biotin and an avidin-like compound. Biotinylated molecules (nucleic
acid molecules or polymerases or nanoparticles) can be attached to
the PEM/biotin/avidin coated surface (Quake, U.S. Pat. Nos.
6,818,395; 6,911,345; and 7,501,245).
Nanoscale Devices
[0506] The surface can be the surface of a nanoscale device. The
components of the nucleotide binding or nucleotide incorporation
reaction (e.g., nanoparticles, polymerase, nucleotides, target
nucleic acid molecules, primers and/or oligonucleotides) can be
associated with or immobilized onto the nanoscale device.
[0507] The nanoscale device can have microscopic features (e.g., at
the micro meter, nano meter size level, or pico meter level) which
permit manipulation or analysis of biological molecules at a
nanoscale level.
[0508] The nanoscale device can include open or enclosed (i.e.,
sealed) structures (e.g., nanostructures) including: channels,
slits, pores, wells, pillars, loops, arrays, pumps valves. The
nanostructures can have length, width, and height dimensions. The
nanostructures can be linear or branched, or can have inlet and/or
outlet ports. The branched nanostructures (e.g., branched channels)
can form a T or Y junction, or other shape and geometries.
[0509] The nanostructure dimensions can be between about 10-25 nm,
or about 25-50 nm, or about 50-100 nm, or about 100-200 nm, or
about 200-500 nm, or about 500-700 nm, or about 700-900 nm, or
about 900-1000 nm. The nanostructures can have a trench width equal
to or less than about 150 nanometers. The nanostructures can be
wells which are 50-10,000 nm in diameter. The nanostructures can
have a trench depth equal to or less than about 200 nanometers
(e.g., 50-100 nm thickness).
[0510] The nanoscale device can comprise one or a plurality of
nanostructures, typically more than 5, 10, 50, 100, 500, 1000,
10,000 and 100,000 nanostructures for binding, holding, streaming,
flowing, washing, flushing, or stretching samples. The samples can
include the nanoparticles, polymerase, nucleotides, target nucleic
acid molecules, primers and/or oligonucleotides. The fluid which
runs through the nanoscale device can be liquid, gas or slurry.
Nanoscale devices are also known as nanofluidic devices.
[0511] Nanoscale devices and/or their component nanostructures may
be fabricated from any suitable substrate including: silicon,
carbon, glass, polymer (e.g., poly-dimethylsiloxane), metals, boron
nitrides, nickel, platinum, copper, tungsten, titanium, aluminum,
chromium, gold, synthetic vesicles, carbon nanotubes, or any
combination thereof.
[0512] The nanoscale devices and/or nanostructures may be
fabricated using any suitable method, including: lithography;
photolithography; diffraction gradient lithography (DGL);
nanoimprint lithography (NIL); interference lithography;
self-assembled copolymer pattern transfer; spin coating; electron
beam lithography; focused ion beam milling; plasma-enhanced
chemical vapor deposition; electron beam evaporation; sputter
deposition; bulk or surface micromachining; replication techniques
such as embossing, printing, casting and injection molding; etching
including nuclear track or chemical etching, reactive ion-etching,
wet-etching; sacrificial layer etching; wafer bonding; channel
sealing; and combinations thereof.
[0513] The nanoscale device can be used to react, confine,
elongate, mix, sort, separate, flow, deliver, flush, wash, or
enrich the nanoparticles or biomolecules, or the intermediates or
products of nucleotide incorporation. For example, the target
nucleic acid molecule (e.g., nucleic acid molecules, or chromosomal
or genomic DNA) can be elongated using pulsed field
electrophoresis, or in a nanofluidic device via flow stretching
(with or without tethering) or confinement elongation. Elongated
nucleic acid molecules can be used to: measure the contour length
of a nucleic acid molecule, locate landmark restriction sites along
the length of the molecule, or detect sequencing reactions along
the molecule (Schwartz, U.S. Pat. Nos. 6,221,592, 6,294,136 and
U.S. Published App. Nos. 2006/0275806 and 2007/0161028). In one
aspect, the nanostructure can be one or more nanochannels, which
are capable of transporting a macromolecule (e.g., nucleic acid
molecule) across its entire length in elongated form. In another
aspect, the nanostructure can detect an elongated macromolecule, or
detect sequencing of a single nucleic acid molecule.
[0514] The nanochannels can be enclosed by surmounting them with a
sealing material using suitable methods. See, for example, U.S.
Publication No. 2004/0197843. The nanoscale device can comprise a
sample reservoir capable of releasing a fluid, and a waste
reservoir capable of receiving a fluid, wherein both reservoirs are
in fluid communication with the nanofluidic area. The nanoscale
device may comprise a microfluidic area located adjacent to the
nanofluidic area, and a gradient interface between the microfluidic
and nanofluidic area which reduces the local entropic barrier to
nanochannel entry. See, for example, U.S. Pat. No. 7,217,562.
[0515] The nanoscale device comprising a nanochannel array can be
used to isolate individual nucleic acid molecules prior to
sequencing, wherein the sample population of nucleic acid molecules
is elongated and displayed in a spatially addressable format.
Isolation of the nucleic acid molecules to be sequenced may be
achieved using any suitable nanoscale device which comprises
nanostructures or nanofluidic constrictions of a size suited to
achieve isolation and separation of the test nucleic acid molecule
from other sample components in a manner which will support direct
sequencing of the test molecule in situ. For example, a nucleic
acid molecule, such as a chromosome, is isolated from a sample
mixture using a nanofluidic device which is capable of receiving a
sample comprising mixed population of nucleic acid molecules and
elongating and displaying them in an ordered format without the
need for prior treatment or chemical attachment to a support.
[0516] The nanoscale device supports analysis of intact chromosomes
without the need for fragmentation or immobilization of sequencing
components. The nanoscale device comprises at least one
nanostructure, typically a nanochannel, which is designed to admit
only a single polymeric molecule and elongate it as it flows
through the nanostructure. Suitable nanoscale devices have been
described, for example, in U.S. Pat. No. 6,635,163 (nanofluidic
entropic trapping and sieving devices). Suitable nanoscale devices
comprise microfluidic and nanofluidic areas separated by a gradient
interface which reduces the local entropic barrier to nanochannel
entry thereby reducing clogging of the device at the
microfluidic-nanofluidic interface. See, for example, Cao, U.S.
Pat. No. 7,217,562 and U.S. Pub. No. 2007/0020772.
[0517] The nanoscale device can include an array of nanochannels.
Introduction of a sample comprising a mixed population of nucleic
acid molecules into the nanoscale device results in the isolation
and elongation of a single nucleic acid molecule within each
nanostructure, so that an entire population of nucleic acid
molecules is displayed in an elongated and spatially addressable
format. After the nucleic acid molecules enter and flow through
their respective nanochannel, they are contacted with one or more
components of a nucleotide incorporation reaction mixture, and the
progress of the incorporation reaction is monitored using suitable
detection methods. The ordered and spatially addressable
arrangement of the population allows signals to be detected and
monitored along the length of each nucleic acid molecule. Separate
sequencing reactions occur within each nanochannel. The spatially
addressable nature of the arrayed population permits discrimination
of signals generated by separate priming events, and permitting
simultaneous detection and analysis of multiple priming events at
multiple points in the array. The emission data can be gathered and
analyzed to determine the time-sequence of incorporation events for
each individual nucleic acid (DNA) in the nanochannel array.
Nanoscale devices can permit the simultaneous observation of
macromolecules in multiple channels, thereby increasing the amount
of sequence information obtainable from a single experiment and
decreasing the cost of sequencing of an entire genome. See, for
example, U.S. Pub. No. 2004/0197843, also U.S. Ser. Nos.
61/077,090, filed on Jun. 30, 2008, and 61/089,497, filed on Aug.
15, 2008, and 61/090,346, filed on Aug. 20, 2008.
[0518] In one embodiment, the nanoscale device can include a flow
cell which includes a two-sided multi-channel flow cell comprising
multiple independently-addressable sample channels and removable
loading blocks for sample loading (Lawson, U.S. published patent
application No. 2008/0219888).
[0519] In another embodiment, the nanoscale device can include a
light source for directing light to the nucleotide incorporation
reaction, a detector (e.g., photon detector), a camera, and/or
various plumbing components such as microvalves, micropumps,
connecting channels, and microreservoirs for controlled flow (in
and/or out) of the reagents of the nucleotide incorporation
reactions. The reagents can be pulled through the inlet or outlet
ports via capillary action, or by vacuum (Lawson, U.S. published
patent application No. 2008/0219890; and Harris, et al., 2008
Science 320:106-109, and Supplemental Materials and Methods from
the supporting online material), or moved via a pressure-driven
fluidics system. The reagents can be pulled through the inlet or
outlet ports using a passive vacuum source (Ulmer, U.S. Pat. No.
7,276,720).
[0520] In another embodiment, the nucleotide incorporation methods
can be practiced in a nanoscale device such as a patterned metal
masked array which includes a metal layer disposed on a glass
support, where the metal layer is perforated with holes ranging in
size from 50-10,000 nm. The holes can be any shape including round,
rectilinear, triangular, slit, and the like. The metal layer can
have a thickness of about 50-100 nm. The metal layer can be gold,
chrome, silver, aluminum, titanium, nickel, platinum, copper,
tungsten, titanium-tungsten, carbon, carbon nanotubes,
nanoparticles, or polymers. The surface can be spin-coated with an
imaging resist using e-beam or photo resist procedures. The metal
can be global-coated using evaporation or sputtering procedures.
The exposure step can be achieved using e-beam or photomask
lithography. See for example, U.S. Ser. No. 61/245,248, filed Sep.
23, 2009.
[0521] The nanoparticles and biomolecules (e.g., polymerases,
nucleotides, target nucleic acid molecules, primers, and/or
oligonucleotides) can be isolated, modified, sorted, collected,
distributed, linked and/or immobilized using suitable procedures
and devices.
[0522] The nanoparticles and biomolecules (e.g., polymerases,
nucleotides, target nucleic acid molecules, primers, and/or
oligonucleotides) can be used presently for any procedure described
herein, or can be stored or preserved for later use by employing
suitable procedures.
Modified Surfaces
[0523] The surface can be chemically or enzymatically modified to
have one or more reactive groups, including amines, aldehyde,
hydroxyl, sulfate or carboxylate groups, which can be used to
attach the surface to the nanoparticles, polymerases, nucleotides,
target nucleic acid molecules, primers, and/or
oligonucleotides.
Attaching Nucleic Acid Molecules to the Surface
[0524] Nucleic acid molecules can be attached to a surface. The
target nucleic acid molecules, primers, and/or oligonucleotides can
be modified at their 5' or 3' end, or internally, to carry a
reactive group which can bind to a reactive group on the surface.
Typically, the surface is treated or untreated to provide reactive
groups such as silanol, carboxyl, amino, epoxide, and methacryl
groups. The nucleic acid molecules can be treated or untreated to
provide reactive groups including: amino, hydroxyl, thiol, and
disulfide. The nucleic acid molecules can include non-natural
nucleotides having reactive group which will attach to a surface
reactive group. For example, the non-natural nucleotides include
peptide nucleic acids, locked nucleic acids, oligonucleotide
N3'.fwdarw.P5' phosphoramidates, and
oligo-2'-O-alkylribonucleotides.
[0525] In one aspect, nucleic acid molecules modified with one or
more amino groups at the 5' or 3' end, or internally, can be
attached to modified surfaces.
[0526] In another aspect, the nucleic acid molecules can be
attached at their 5' ends with one or more amino groups, including:
a simple amino group; a short or long tethering arm having one or
more terminal amino groups; or amino-modified thymidine or
cytosine. The tethering arms can be linear or branched, have
various lengths, charged or uncharged, hydrophobic, flexible,
cleavable, or have one or multiple terminal amino groups. The
number of plural valent atoms in a tethering arm may be, for
example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30 or a larger
number up to 40 or more.
[0527] In another aspect, the 3' end of nucleic acid molecules can
be modified to carry an amino group. Typically, the amino group is
initially protected by a fluorenylmethylcarbamoyl (Fmoc) group. To
expose the amino group, the protecting group can be removed and
acylated with an appropriate succinimidyl ester, such as an
N-hydroxy succinimidyl ester (NHS ester).
[0528] In another aspect, the nucleic acid molecules can carry
internal amino groups for binding to the solid surface. For
example, 2' amino modified nucleic acid molecules can be produce by
methoxyoxalamido (MOX) or succinyl (SUC) chemistry to produce
nucleotides having amino linkers attached at the 2' C of the sugar
moiety.
[0529] In another aspect, the surface can be modified to bind the
amino modified nucleic acid molecules. For example, 5'
amino-modified nucleic acid molecules can be attached to surfaces
modified with silane, such as epoxy silane derivatives (J. B.
Lamture, et al., 1994 Nucleic Acids Res. 22:2121-2125; W. G.
Beattie et al., 1995 Mol. Biotechnol. 4:213-225) or isothiocyanate
(Z. Guo, et al., 1994 Nucleic Acids Res. 22:5456-5465). Acylating
reagents can be used to modify the surface for attaching the
amino-modified nucleic acid molecules. The acylating reagents
include: isothiocyanates, succinimidyl ester, and sulfonyl
chloride. The amino-modified nucleic acid molecules can attach to
surface amino groups which have been converted to amino reactive
phenylisothiocyanate groups by treating the surface with
p-phenylene 1,4 diisothiocyanate (PDC). In other methods, the
surface amino groups can be reacted with homobifunctional
crosslinking agents, such as disuccinimidylcaronate (DCS),
disuccinimidyloxalate (DSO), phenylenediisothiocyanate (PDITC) or
dimethylsuberimidate (DMS) for attachment to the amino-modified
nucleic acid molecules. In another example, metal and metal oxide
surfaces can be modified with an alkoxysilane, such as
3-aminopropyltriethoxysilane (APTES) or
glycidoxypropyltrimethoxysilane (GOPMS).
[0530] In another aspect, succinylated nucleic acid molecules can
be attached to aminophenyl- or aminopropyl-modified surfaces (B.
Joos et al., 1997 Anal. Biochem. 247: 96-101).
[0531] In yet another aspect, a thiol group can be placed at the 5'
or 3' end of the nucleic acid molecules. The thiol group can form
reversible or irreversible disulfide bonds with the surface. The
thiol attached to the 5' or 3' end of the nucleic acid molecule can
be a phosphoramidate. The phosphoramidate can be attached to the 5'
end using S-trityl-6-mercaptohexyl derivatives.
[0532] In another aspect, the thiol-modified nucleic acid molecules
can be attached to a surface using heterobifunctional reagents
(e.g. cross linkers). For example, the surface can be treated with
an alkylating agent such as iodoacetamide or maleimide for linking
with thiol modified nucleic acid molecules. In another example,
silane-treated surfaces (e.g., glass) can be attached with
thiol-modified nucleic acid molecules using succinimidyl
4-(malemidophenyl)butyrate (SMPB).
[0533] In another aspect, the nucleic acid molecule can be modified
to carry disulfide groups can be attached to thiol-modified
surfaces (Y. H. Rogers et al., 1999 Anal. Biochem. 266:23-30).
[0534] Still other aspects include methods which employ modifying
reagents such as: carbodiimides (e.g., dicyclohexylcarbodiimide,
DCC), carbonyldiimidazoles (e.g., carbonyldiimidazole, CDI.sub.Z),
and potassium periodate. The nucleic acid molecules can have
protective photoprotective caps (Fodor, U.S. Pat. No. 5,510,270)
capped with a photoremovable protective group. DMT-protected
nucleic acid molecules can be immobilized to the surface via a
carboxyl bond to the 3' hydroxyl of the nucleoside moiety (Pease,
U.S. Pat. No. 5,599,695; Pease et al., 1994 Proc. Natl. Acad. Sci.
USA 91(11):5022-5026). The nucleic acid molecules can be
functionalized at their 5' ends with activated 1-O-mimethoxytrityl
hexyl disulfide
1'4-[(2-cyanoethyl)-N,N-diisopropyl)]phosphoramidate (Rogers et
al., 1999 Anal. Biochem. 266:23). Exemplary methods of attaching
nucleic acid molecules to suitable substrates are disclosed, for
example, in Schwartz, U.S. Pat. Nos. 6,221,592, 6,294,136 and U.S.
Published App. Nos. 2006/0275806 and 2007/0161028 (Schwartz et
al.). Linking agents, can be symmetrical bifunctional reagents,
such as bis succinimide (e.g., bis-N-hydroxy succinimide) and
maleimide (bis-N-hydroxy maleimide) esters, or toluene diisocyanate
can be used. Heterobifunctional cross-linkers include: m-maleimido
benzoyl-N-hydroxy succinimidyl ester (MBS);
succinimidyl-4-(p-maleimido phenyl)-Butyrate (SMPB); and
succinimidyl-4-(N-Maleimidomethyl)Cyclohexane-1-Carboxylate (SMCC)
(L. A. Chrisey et al., 1996 Nucleic Acids Res. 24:3031-3039). In
one example, a glass surface can be layered with a gold (e.g.,
about 2 nm layer) which is reacted with mercaptohexanoic acid. The
mercaptohexanoic acid can be placed in a patterned array. The
mercaptohexanoic acid can be reacted with PEG. The PEG can be
reacted to bind nucleic acid molecules such as the target nucleic
acid molecules.
[0535] In another aspect, the target nucleic acid molecule can be
linked to an amine-functionalized solid surface. In one embodiment,
the amine-functionalized solid surface can be a spot surrounded by
PEG molecules, where the target molecule preferentially binds the
amine-functionalized spots (see Fry, et al., U.S. Ser. No.
61/245,248, filed on Sep. 23, 2009).
Capture Probes:
[0536] The surfaces, nanoparticles, polymerases, nucleotides,
target nucleic acid molecules, primers, and/or oligonucleotides can
be attached to each other in any combination via capture nucleic
acid probes.
[0537] For example, the surface may comprise capture nucleic acid
probes which form complexes with single or double stranded nucleic
acid molecules. In one embodiment, the capture probes anneal with
target nucleic acid molecules. The capture probes include
oligonucleotide clamps (U.S. Pat. No. 5,473,060). The parameters
for selecting the length and sequence of the capture probes are
well known (Wetmur 1991 Critical Reviews in Biochemistry and
Molecular Biology, 26: 227-259; Britten and Davidson, chapter 1 in:
Nucleic Acid Hybridization: A Practical Approach, Hames et al,
editors, IRL Press, Oxford, 1985). The length and sequence of the
capture probes may be selected for sufficiently stability during
low and/or high stringency wash steps. The length of the capture
probes ranges from about 6 to 50 nucleotides, or from about 10 to
24 nucleotides, or longer.
Attaching Proteins to the Solid Surface
[0538] In one aspect, the surface can be modified to attach the
protein molecules (e.g., polymerases) via covalent or non-covalent
linkage. The polymerases may be attached to the surface via
covalent cross-linking bridges, including disulfide, glycol, azo,
sulfone, ester, or amide bridges. Some exemplary methods for
attaching polymerases to a surface are disclosed in U.S. Pat. Nos.
7,056,661, 6,982,146, 7,270,951, 6,960,437, 6,255,083, 7,229,799
and published application U.S. No. 2005/0042633.
[0539] The polymerases can be modified at their amino- or
carboxyl-terminal ends, or internally, to carry a reactive group
which can bind to a reactive group on the surface.
[0540] The polymerases can be attached to the modified surfaces
using standard chemistries including: amination, carboxylation or
hydroxylation. The attachment agents can be cyanogen bromide,
succinimide, aldehydes, tosyl chloride, photo-crosslinkable agents,
epoxides, carbodiimides or glutaraldehyde (in: Protein
immobilization: Fundamentals and Applications, Richard F. Taylor,
ed. (M. Dekker, New York, 1991). The surface can be treated or
untreated to provide reactive groups such as silanol, carboxyl,
amino, epoxide, and methacryl groups. The protein molecules can be
treated or untreated to provide reactive groups including: amino,
hydroxyl, thiol, and disulfide. The surface can be coated with an
electron-sensitive compound such as polymethyl methacrylate-like
material (PMMA).
[0541] The polymerases can be attached to a surface which is
untreated or modified via physical or chemical interaction. See
Nakanishi for a review of protein immobilization methods (K.
Nakanishi, 2008 Current Proteomics 5:161-175).
[0542] The polymerases can be adsorbed onto a surface. The
adsorption can occur via ion exchange, charge-charge interaction,
or hydrogen bond interactions. The adsorption can occur on to
untreated surfaces, including polystyrene, polyvinylidene fluoride
(PVDF), glass coated with poly-lysine (H. Ge 2000 Nucl. Acids Res.
28: e3; B. B. Haab, et al., 2001Genome Biol. 2: R4-13; Zhu and
Snyder 2003 Curr. Opin. Chem. Biol. 7: 55-63), or onto surfaces
having hydrophobic properties (Y. Sanghak, et al., 2006 Cum Appl.
Phys. 6: 267-70).
[0543] The polymerases can be attached to the surface using a
hydrogel (P. Arenkov, et al., 2000 Anal. Biochem. 278: 123-31; S.
Kiyonaka, et al., 2004 Nat. Mater. 3: 58-64).
[0544] The polymerases can be linked to an affinity His-tag (e.g.,
6.times.His-tag (SEQ ID NO: 63)) which interacts with Ni.sup.2+,
Co.sup.2+, or Cu.sup.2+ surfaces (T. Nakaji-Hirabayashi, et al.,
2007 Biomaterials 28: 3517-29; R. Vallina-Garcia, et al., 2007
Biosens. Bioelectron. 23: 210-7; T. Cha, et al., 2004 Proteomics 4:
1965-76; T. Cha, et al., 2005 Proteomics 5: 416-9). For example,
the polymerases can be a fusion protein which includes the His-tag
sequence. The glass surface can be functionalized with a chelate
group by treating with nitrotriacetic acid (NTA) or imidoacetic
acid (IDA) and reacted with Ni.sup.2+ or Cu.sup.2+,
respectively.
[0545] The polymerases can be attached to the surface via
chemisorption between a thiol (e.g., SH group of cysteines) on the
polymerase and a gold surface (S. V. Rao, et al., 1998 Mikrochim
Acta 128: 127-43).
[0546] The polymerases can be attached to the surface via a
Schiff's base linkage reaction. For example, a glass surface can be
silanized with silane, polysilane, trimethoxysilane, or
aminosilane. The silanized glass surface can interact with amino
groups (e.g., lysine) on the polymerase (MacBeath and Schreiber
2000 Science 289: 1760-1763; H. Zhu, et al., 2000 Nat. Genet. 26:
283-289). Metal and metal oxide surfaces can be modified with an
alkoxysilane, such as 3-aminopropyltriethoxysilane (APTES) or
glycidoxypropyltrimethoxysilane (GOPMS).
[0547] The polymerases can be immobilized via protein coil-coil
interaction between a heterodimeric Leu zipper pair (J. R. Moll, et
al., 2001 Protein Sci. 10: 649-55; K. Zhang, et al., 2005 J. Am.
Chem. Soc. 127: 10136-7). For example, the surface can be
functionalized to bind one of the zipper proteins, and the
polymerases can be linked with the other zipper protein. The
polymerases can be fusion proteins which include a zipper protein
sequence. The glass surface can be coated with a bifunctional
silane coupling reagent comprising aldehyde (e.g.,
octyltrichlorosilane (OTC)) and functionalized with a hydrophobic
elastin mimetic domain (ELF) as a hydrophobic surface anchor which
serves to bind a leucine zipper sequence. The anchored zipper
sequence can interact with a partner leucine zipper sequence linked
to the polymerases.
[0548] The polymerases can be immobilized via an acyl transfer
reaction. For example, transglutaminase (TGase) can catalyze an
acyl transfer reaction between a primary amino group and a
carboxyamide group (J. Tominaga, et al., 2004 Enz. Microb. Technol.
35: 613-618). In one embodiment, carboxyamide groups from a
casein-coated surface can react with the primary amine groups
(e.g., lysine as a peptide tag or part of the polymerase) on the
polymerases. In another embodiment, the amine groups on the surface
can react with carboxyamide groups (e.g., glutamine-tag or
glutamine groups on the polymerases).
[0549] The polymerases can be immobilized via interaction between
an affinity peptide sequence (e.g., motif) and its cognate peptide
binding partner. For example, the affinity motif could bind a
protein kinase. In one embodiment, the affinity motif comprises the
"minimal" motif, R-X-X-S*/T*(T. R. Soderling 1996 Biochim Biophys.
Acta 1297: 131-138), including peptide motifs RRATSNVFA (SEQ ID
NO:17), RKASGPPV (SEQ ID NO:18), or LRRASLG (SEQ ID NO:19), which
bind a calmodulin-dependent protein kinase.
[0550] Oriented poly-His tagged protein molecules can be
immobilized on to a glass surface modified with PEG and reacted
with a chelate group such as iminodiacetic acid (IDA) or
nitrolotriacetic acid (NTA), and metal ions such as Ni.sup.2+ or
Cu.sup.2+ (T. Cha, et al., 2004 Proteomics 4:1965-1976).
[0551] EDAC chemistry can be use to link a carboxylated silica
surface to an avidin. The avidin can bind to a biotinylated protein
(e.g., polymerase). The avidin-silica surface can bind one or more
biotinylated protein molecules, or bind more than one type of
biotinylated protein (e.g., binds biotinylated polymerase).
[0552] In one aspect, a peptide linker can be used to attach the
protein molecules (e.g., polymerases) to the nanoparticle or to the
solid surface. The peptide linkers can be part of a fusion protein
comprising the amino acid sequences of the polymerases. The fusion
protein can include the peptide linker positioned at the N- or
C-terminal end or in the interior of the fusion protein. In another
embodiment, the peptide linkers can be separate linkers which are
attached to the protein and the solid surface or nanoparticle.
[0553] For example, the peptide linker can be a flexible linker
comprising the amino acid sequence GGGGSGGGGSAAAGSAA (SEQ ID
NO:20). In another example, the peptide linker can be a rigid
linker comprising the amino acid sequence GAAAKGAAAKGSAA (SEQ ID
NO:21). In another example, the peptide linker can be a poly-lysine
linker, comprising between about 4-15 lysine residues (e.g., 12
lysine residues). BS3 coupling (bis(sulfo-succinimidyl)suburate)
can be used to attach the poly-lysine linkers to PEG-amine groups
on the solid surfaces or on nanoparticles. In yet another example,
the peptide linker can be a poly-cysteine linker comprising between
about 4-15 cysteine residues (e.g., 12 cysteine residues). SMCC
coupling
(succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate) can
be used to attach the poly-cysteine linkers to PEG-amine groups on
solid surfaces or on nanoparticles. In yet another example, the
peptide linker can be a transglutaminase tag comprising the amino
acid sequence PKPQQF (SEQ ID NO:22) or PKPQQFM (SEQ ID NO:23). The
transglutaminase tag can provide site specific attachment of the
protein (polymerase) to the solid surface or nanoparticle.
Transglutaminase enzyme can catalyze an acyl transfer reaction
between the .gamma.-carboxyamide group of an acceptor glutamine
residue and a primary amine donor on the solid surface or
nanoparticles. In yet another example, the peptide linker can be a
protein kinases (PKA) tag comprising the amino acid sequence LRRASL
(SEQ ID NO: 62). The PKA tag can provide site specific attachment
of the protein (polymerase) to the solid surface or nanoparticle.
SPDP(N-succinimidyl 3-(2-pyridyldithio) propionate) and iodoacetic
acid are heterobifunctional cross-linking agents which can react
with amines and sulfhydryl groups to link proteins to the solid
surfaces or nanoparticles.
[0554] In yet another embodiment, the peptide linker can include a
poly-histidine tag: MNHLVHHHHHHIEGRHMELGTLEGS (SEQ ID NO:14), or
MSHHHHHHSMSGLNDIFEAQKIEWHEGAPGARGS (SEQ ID NO:15), or
MHHHHHHLLGGGGSGGGGSAAAGSAAR (SEQ ID NO:16).
[0555] In one embodiment, the solid surface can be modified to
provide avidin (or avidin-like) binding groups. In one embodiment,
the surface material is glass. In another embodiment, the glass
surface is reacted with silane or its derivative. In another
embodiment, the glass surface is reacted with PEG, biotin, and
avidin (or avidin-like protein) to provide avidin (or avidin-like)
binding sites. In yet another embodiment, the glass surface is
reacted with PEG and avidin (or avidin-like protein) to provide
avidin (or avidin-like) binding site. The binding sites on the
glass slide can attach to the nanoparticles, proteins (e.g.,
polymerases, or any fusion proteins thereof), target nucleic acid
molecules, primers, or oligonucleotides.
[0556] In another embodiment, the polymerase (or polymerase fusion
protein) is linked to the surface. In another embodiment, the solid
surface can be modified for binding to a His-tagged protein. In
another embodiment, the polymerase can be a biotinylated protein
bound to a surface which is coated with avidin or avidin-like
protein. In another embodiment, the polymerase can be a
poly-His-tagged protein bound to a nickel-conjugated surface. In
another embodiment, the polymerase (or polymerase fusion protein)
can be linked to a nanoparticle. In another embodiment, polymerase
and nanoparticle can be separately linked to the surface. The
immobilized polymerase can bind the target nucleic acid molecule,
which may or may not be base-paired with the polymerization
initiation. The immobilized polymerase can bind the nucleotide
and/or can incorporate the nucleotide onto the polymerization
initiation site.
Reducing Non-Specific Binding
[0557] In one aspect, the surfaces, nanoparticles, polymerases,
nucleotides, target nucleic acid molecules, primers, and/or
oligonucleotides can be modified to reduce non-specific binding by
dyes or nucleotides. For example, the surface can be coated with
sugar molecules (e.g., mono or disaccharides as described in
Jogikalmath, U.S. 2008/0213910), silane (Menchen, U.S. Ser. No.
11/943,851), and/or PEG to reduce non-specific binding with dyes
and/or nucleotides. Silane includes:
N-(3-aminopropyl)-3-mercapto-benzamide;
3-aminopropyl-trimethoxysilane; 3-mercaptopropyl-trimethoxysilane;
3-(trimethoxysilyl)propyl-maleimide; and
3-(trimethoxysilyl)propyl-hydrazide. In another example, the
nanoparticles can be reacted with bovine serum albumin (BSA) to
reduce non-specific binding to polymerases.
Linking Methods
[0558] In some embodiments, the surfaces, reporter moieties
(including, e.g., energy transfer moieties, nanoparticles and
organic dyes), polymerases, nucleotides and nucleic acid molecules
(including, e.g., targets, primers and/or oligonucleotides) can be
linked to each other, in any combination and in any order, using
well known linking chemistries. Such linkage can optionally include
a covalent bond and/or a non-covalent bond selected from the group
consisting of an ionic bond, a hydrogen bond, an affinity bond, a
dipole-dipole bond, a van der Waals bond, and a hydrophobic
bond.
[0559] In some embodiments, the linking procedure used to link the
biomolecules, reporter moieties and/or surfaces of the present
disclosure comprises a chemical reaction that includes formation of
one or more covalent bonds between a first and second moiety,
resulting in the linkage of the first moiety to the second moiety.
In some embodiments, the chemical reaction occurs between a first
group of the moiety and a second group of the second moiety. Such
chemical reaction can include, for example, reaction of activated
esters, acyl azides, acyl halides, acyl nitriles, or carboxylic
acids with amines or anilines to form carboxamide bonds. Reaction
of acrylamides, alkyl halides, alkyl sulfonates, aziridines,
haloacetamides, or maleimides with thiols to form thioether bonds.
Reaction of acyl halides, acyl nitriles, anhydrides, or carboxylic
acids with alcohols or phenols to form an ester bond. Reaction of
an aldehyde with an amine or aniline to form an imine bond.
Reaction of an aldehyde or ketone with a hydrazine to form a
hydrazone bond. Reaction of an aldehyde or ketone with a
hydroxylamine to form an oxime bond. Reaction of an alkyl halide
with an amine or aniline to form an alkyl amine bond. Reaction of
alkyl halides, alkyl sulfonates, diazoalkanes, or epoxides with
carboxylic acids to form an ester bond. Reaction of an alkyl
halides or alkyl sulfonates with an alcohol or phenol to form an
ether bond. Reaction of an anhydride with an amine or aniline to
form a carboxamide or imide bond. Reaction of an aryl halide with a
thiol to form a thiophenol bond. Reaction of an aryl halide with an
amine to form an aryl amine bond. Reaction of a boronate with a
glycol to form a boronate ester bond. Reaction of a carboxylic acid
with a hydrazine to form a hydrazide bond. Reaction of a
carbodiimide with a carboxylic acid to form an N-acylurea or
anhydride bond. Reaction of an epoxide with a thiol to form a
thioether bond. Reaction of a haloplatinate with an amino or
heterocyclic group to form a platinum complex. Reaction of a
halotriazine with an amine or aniline to form an aminotriazine
bond. Reaction of a halotriazines with an alcohol or phenol to form
a triazinyl ether bond. Reaction of an imido ester with an amine or
aniline to form an amidine bond. Reaction of an isocyanate with an
amine or aniline to form a urea. Reaction of an isocyanate with an
alcohol or phenol to form a urethane bond. Reaction of an
isothiocyanate with an amine or aniline to form a thiourea bond.
Reaction of a phosphoramidate with an alcohol to form a phosphite
ester bond. Reaction of a silyl halide with an alcohol to form a
silyl ether bond. Reaction of a sulfonate ester with an amine or
aniline to form an alkyl amine bond. Reaction of a sulfonyl halide
with an amine or aniline to form a sulfonamide bond. Reaction of a
thioester with thiol group of a cysteine followed by rearrangement
to form an amide bond. Reaction of an azide with an alkyne to form
a 1,2,3-traizole.
[0560] In some embodiments, water-insoluble substances can be
chemically modified in an aprotic solvent such as
dimethylformamide, dimethylsulfoxide, acetone, ethyl acetate,
toluene, or chloroform. Similar modification of water-soluble
substances can be accomplished using reactive compounds to make
them more readily soluble in organic solvents.
Linkage to Surface
[0561] In some embodiments the biomolecules and/or reporter
moieties of the present disclosure are linked to a surface.
Optionally, such linkage can result in reversible or non-reversible
immobilization of the nanoparticles, polymerases, nucleotides,
nucleic acid molecules, primers, and/or oligonucleotides onto the
surface. Non-limiting examples of such linkage can include: nucleic
acid hybridization, protein aptamer-target binding, non-specific
adsorption, and solvent evaporation. In some embodiments, the
biomolecule that is linked to a surface is a polymerase (such as,
for example, a polymerase fusion protein). The polymerase can be
attached to a surface via a linker comprising an anchor or
tethering moiety. The anchor or tethering moiety can be flexible or
rigid. The anchor or tether can orient the polymerase, or
polymerase fusion protein, in a manner that does not interfere with
the nucleotide binding and/or polymerase activity.
Conjugation Methods--Biomolecules
[0562] Linkage of biomolecules to reporter moieties, surfaces
and/or to each other can be accomplished by any suitable method
(for example, Brinkley et al., 1992 Bioconjugate Chem. 3: 2). In
some embodiments, a biomolecule can comprise a single type of
reactive site (as is typical for polysaccharides), or it can
comprise multiple types of reactive sites, e.g., amines, thiols,
alcohols, phenols, may be available (as is typical for proteins).
Conjugation selectivity can be obtained by selecting an appropriate
reactive moiety. For example, modification of thiols with a
thiol-selective reagent such as a haloacetamide or maleimide, or
modification of amines with an amine-reactive reagent such as
1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide (variously known as
EDC or EDAC), an activated ester, acyl azide, isothiocyanate or
3,5-dichloro-2,4,6-triazine. Partial selectivity can also be
obtained by careful control of the reaction conditions.
[0563] In some embodiments, the biomolecule can be linked to the
reporter moiety through a bond selected from group consisting of: a
covalent bond, a hydrogen bond, a hydrophilic bond, a hydrophobic
bond, an electrostatic bond, a Van der Waals bond, and an affinity
bond.
[0564] In some embodiments, the biomolecule comprises a peptide and
the bond is a covalent bond formed between an amine group of a
lysine residue of the biomolecule and an amine-reactive moiety,
wherein the amine reactive moiety is linked to the reporter moiety.
In some embodiments, the biomolecule comprises a peptide and the
bond is a covalent bond formed between a carboxy group of an amino
acid residue of the biomolecule and a maleimide moiety, wherein the
maleimide moiety is linked to the reporter moiety.
[0565] In some embodiments, the biomolecule can be linked to a
reporter moiety, such as, for example a nanoparticle. Optionally,
the nanoparticle further comprises at least one carboxyl group on
its surface, and the one or more biomolecules or fragments at least
one primary amine group, and the cross-linking agent EDC is
employed to form a covalent amide bond between the at least one
nanoparticle and the one or more biomolecules or fragments.
[0566] In some embodiments, the biomolecule can be attached to a
reporter moiety (including, e.g., a FRET donor or acceptor moiety)
using any suitable chemical linking procedure, including chemical
linking procedures that are known in the art. In some embodiments,
the at least one biomolecule or biologically active fragment can be
operably linked to the nanoparticle via chemical linking
procedures. Many linking procedures are well known in the art,
including: maleimide, iodoacetyl, or pyridyl disulfide chemistry
which targets thiol groups on polypeptides; or succinimidyl esters
(NHS), sulfonyl chlorides, iso(thio)cyanates, or carbonyl azide
chemistry which targets primary amines in a polypeptide, and
dichlorotriazine-based linking procedures. Additional exemplary
linking procedures are described in more detail herein.
[0567] In some embodiments, the appropriate reactive compounds can
be dissolved in a nonhydroxylic solvent (usually DMSO or DMF) in an
amount sufficient to give a suitable degree of conjugation when
added to a solution of the protein to be conjugated. These methods
have been used to prepare protein conjugates from antibodies,
antibody fragments, avidins, lectins, enzymes, proteins A and G,
cellular proteins, albumins, histones, growth factors, hormones,
and other proteins. The resulting protein (e.g., polymerase)
attached to the energy transfer or reporter moiety can be used
directly or enriched, e.g., chromatographically enriched to
separate the desired linked compound from the undesired unlinked
compound. Several linking procedures are described in U.S. patents
and U.S. Pat. No. 5,188,934. Other suitable linking procedures are
also known in the art.
[0568] When conjugating biomolecules to nanoparticles, the
residual, unreacted compound or a compound hydrolysis product can
be removed by dialysis, chromatography or precipitation. The
presence of residual, unconjugated moieties can be detected by
methods such as thin layer chromatography which elutes the
unconjugated forms away from its conjugate. In some embodiments,
the reagents are kept concentrated to obtain adequate rates of
conjugation.
Modification to Facilitate Linkage
[0569] In some embodiments, the surfaces, reporter moieties
(including, e.g., dyes and/or nanoparticles) and/or biomolecules
(including, e.g., polymerases, nucleotides and nucleic acid
molecules) disclosed herein can be modified to facilitate their
linkage to each other. Such modification can optionally include
chemical or enzymatic modification. The modification can be
practiced in any combination and in any order. In some embodiments,
the modification can mediate covalent or non-covalent linkage of
the surfaces, reporter moieties and/or biomolecules with each
other.
[0570] In some embodiments, the biomolecule can be attached, fused
or otherwise associated with a moiety that facilitates purification
and/or isolation of the biomolecule. For example, the moiety can be
an enzymatic recognition site, an epitope or an affinity tag that
facilitates purification of the biomolecule.
[0571] In some embodiments, the polymerase can include an amino
acid analog which provides a reactive group for linking to the
nanoparticle, target, substrate and/or surface. For example, the
amino acid analog can be produced using a cell (e.g., bacterial
cell) which is genetically engineered to have a 21 amino acid
genetic code which is capable of inserting the amino acid analog
into the encoded polymerase (or fusion protein). The inserted amino
acid analog can be used in a linking chemistry procedure to attach
the polymerase (or fusion protein) to the energy transfer donor
moiety, biomolecule or the surface.
His Tag Modification
[0572] In some embodiments, the biomolecule is a protein and is
modified with a His tag. In some embodiments, the His tag may be
fused directly with the protein; alternatively, a linker comprising
various lengths of amino acid residues can be placed between the
protein and the His tag. The linker can be flexible or rigid.
[0573] Optionally, the presence of the His tag can facilitate
purification of the protein. For example, His tagged protein can be
purified from a raw bacterial lysate by contacting the lysate with
any suitable affinity medium comprising bound metal ions to which
the histidine residues of the His-tag can bind, typically via
chelation. The bound metal ions can comprise, e.g., zinc, nickel or
cobalt, to which the His tag can bind with micromolar affinity.
Suitable affinity media include Ni Sepharose, NTA-agarose,
HisPur.RTM. resin (Thermo Scientific, Pierce Protein Products,
Rockford, Ill.), or Talon.RTM. resin (Clontech, Mountain View,
Calif.). The affinity matrix can then be washed with suitable
buffers, e.g., phosphate buffers, to remove proteins that do not
specifically interact with the cobalt or nickel ion. Washing
efficiency can be improved by the addition of 20 mM imidazole. The
biomolecule can optionally be eluted from the proteins are usually
eluted with 150-300 mM imidazole). The purity and amount of
purified biomolecule can then be assessed using suitable methods,
e.g., SDS-PAGE and Western blotting.
[0574] Optionally, the His tag can be fused to a suitable amino
acid sequence that facilitates removal of the His-tag using a
suitable endopeptidase. Alternatively, the His tag may be removed
using a suitable exopeptidase, for example the Qiagen TAGZyme
exopeptidase.
[0575] In some embodiments, the His tag can facilitate linkage of
the biomolecule to a metal surface, for example, a surface
comprising Zn.sup.2+, Ni.sup.2+, Co.sup.2+, or Cu.sup.2+ ions.
Optionally, the His-tag can facilitate linkage of the biomolecule
to the surface of a nanoparticle comprising one or more metal ions,
typically via chelation interactions, as described in more detail
herein.
Linkers
[0576] Suitable linkers can be used to link the biomolecules
(including, e.g., the polymerases, nucleotides and nucleic acid
molecules), the labels (including, e.g., nanoparticles, organic
dyes, energy transfer moieties and/or other reporter moieties)
and/or the surfaces of the present disclosure to each other, in any
combination. The linkers can be attached (to the surfaces,
nanoparticles, polymerases, nucleotides, target nucleic acid
molecules, primers, oligonucleotides, reporter moieties, and/or
energy transfer moieties) via covalent bonding, non-covalent
bonding, ionic bonding, hydrophobic interactions or any combination
thereof. The type and length of the linker can be selected to
optimize tethering, proximity, flexibility, rigidity, or
orientation. The attachment can be reversible or
non-reversible.
[0577] Suitable linkers include without limitation homobifunctional
linkers and heterobifunctional linkers. For example,
heterobifunctional linkers contain one end having a first reactive
functionality to specifically link to a first molecule, and an
opposite end having a second reactive functionality to specifically
link to a second molecule. Depending on such factors as the
molecules to be linked and the conditions in which the method of
strand synthesis is performed, the linker can vary in length and
composition for optimizing properties such as stability, length,
FRET efficiency, resistance to certain chemicals and/or temperature
parameters, and be of sufficient stereo-selectivity or size to link
a nanoparticle to the biomolecule such that the resultant conjugate
is useful reporting biomolecular activity such as approach,
bonding, fusion or catalysis of a particular chemical reaction.
Linkers can be employed using standard chemical techniques and
include but not limited to, amine linkers for attaching reporter
moieties to nucleotides (see, for example, U.S. Pat. No.
5,151,507); a linker containing a primary or secondary amine for
linking a reporter moiety to a nucleotide; and a rigid hydrocarbon
arm added to a nucleotide base (see, for example, Science
282:1020-21, 1998).
[0578] In some embodiments, the linker comprises a polyethylene
glycol (PEG) or PEG derivative. See, e.g., U.S. Provisional
Applications 61/086,750; 61/102,709; 61/102,683; and 61/102,666.
Such PEG moieties can be functionalized at one or both ends. In
some embodiments, functionalization at both ends with the same
reactive moiety can be employed to create a homobifunctional PEG
derivative. Some examples of homobifunctional PEG derivatives
include without limitation COOH-PEG-COOH; NH2--PEG-NH2; and
MAL-PEG-MAL (where MAL denotes a maleimide group).
[0579] The linker moiety can optionally include: a covalent or
non-covalent bond; amino acid tag; chemical compound (e.g.,
polyethylene glycol); protein-protein binding pair (e.g.,
biotin-avidin); affinity coupling; capture probes; or any
combination of these.
[0580] Optionally, the linker can be selected such that they do not
significantly interfere with the function or activity of the
biomolecules, reporter moieties and/or surfaces that it links to
each other. For example, when the biomolecule is a polymerase, the
linker can be selected such that it does not significantly
interfere with nucleotide binding to the polymerase, or with
cleavage of the phosphodiester bonds, or with nucleotide
incorporation, or with release of the polyphosphate product, or
with translocation of the polymerase or with energy transfer, or
with emission of a detectable signal.
[0581] In some embodiments, the linker can comprise a single
covalent bond or a series of covalent bonds. Optionally, the linker
can be linear, branched, bifunctional, trifunctional,
homofunctional, or heterofunctional. The linker can be cleavable.
The linkers can be rigid or flexible. The linker can be capable of
energy transfer. The linker can be a chemical chain or a chemical
compound. The linker can be resistant to heat, salts, acids, bases,
light and chemicals. The linker can include a short or long spacer,
a hydrophilic spacer, or an extended spacer.
[0582] In another embodiment, the rigid linker can be used to
improve a FRET signal. Examples of rigid linkers include benzyl
linkers, proline or poly-proline linkers (S. Flemer, et al., 2008
Journal Org. Chem. 73:7593-7602), bis-azide linkers (M. P. L.
Werts, et al., 2003 Macromolecules 36:7004-7013), and rigid linkers
synthesized by modifying the so-called "click" chemistry scheme
which is described by Megiatto and Schuster 2008 Journal of the Am.
Chem. Soc. 130:12872-12873. In yet another embodiment, the linker
can be an energy transfer linker synthesized using methods
described in U.S. published patent application No. 2006/0057565,
which is incorporated in its entirety. In yet another embodiment,
the spacer linking moiety can be a cationic arginine spacer or an
imidazolium spacer molecule.
[0583] In some embodiments, the linker moiety comprises about 1-40
plural valent atoms or more selected from the group consisting of
C, N, O, S and P. The number of plural valent atoms in a linker may
be, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, or
40, or more. A linker may be linear or non-linear; some linkers
have pendant side chains or pendant functional groups (or both).
Examples of such pendant moieties are hydrophilicity modifiers, for
example solubilizing groups like, e.g., sulfo (--SO.sub.3H-- or
--SO.sup.3--). In some embodiments, a linker is composed of any
combination of single, double, triple or aromatic carbon-carbon
bonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds,
carbon-oxygen bonds and carbon-sulfur bonds. Exemplary linking
members include a moiety which includes --C(O)NH--, --C(O)O,
--NH--, --S--, --O--, and the like. Linkers may by way of example
consist of a combination of moieties selected from alkyl, alkylene,
aryl, --C(O)NH--, --C(O)O--, --NH--, --S--, --O--, --C(O)--,
--S(O).sub.4-- where n is 0, 1, 2, 3, 4, 5, or 6-membered
monocyclic rings and optional pendant functional groups, for
example sulfo, hydroxy and carboxy.
[0584] In some embodiments, the linker can result from "click"
chemistries schemes (see, e.g., Gheorghe, et al., 2008 Organic
Letters 10:4171-4174) which can be used to attach any combination
of biomolecules, reporter moieties and surfaces as disclosed herein
to each other
[0585] In one aspect, the linker can attach two or more energy
transfer or reporter moieties to each other (the same type or
different types of moieties). In another aspect, a trifunctional
linker (e.g., Graham, U.S. published patent application No.
2006/0003383) can be linked to two fluorescent dye moieties (the
same type or different types) to amplify the fluorescent signal
upon nucleotide binding or nucleotide incorporation. For example, a
trifunctional linker can be linked to two energy transfer acceptor
moieties, or to an energy transfer acceptor and a reporter moiety.
In another example, multiple trifunctional linkers can be linked to
each other, which can be linked to multiple fluorescent dyes for
dendritic amplification of the fluorescent signal (e.g., Graham,
U.S. published patent application No. 2007/0009980).
[0586] In some embodiments, the linker can be a cleavable linker
such as, for example, a photocleavable linker, a chemically
cleavable linker or a self-cleaving linker.
[0587] In some embodiments, the linker is a self-cleaving linker.
Optionally, such linker can be a trimethyl lock or a quinone
methide linker, which can each optionally link to two energy
transfer acceptor and/or reporter moieties and the nucleotide.
[0588] In some embodiments, the linkers can be cleavable where
cleavage is mediated by a chemical reaction, enzymatic activity,
heat, acid, base, or light. For example, photo-cleavable linkers
include nitrobenzyl derivatives, phenacyl groups, and benzoin
esters. Many cleavable groups are known in the art and are
commercially available. See, for example, J. W. Walker, et al.,
1997 Bioorg. Med. Chem. Lett. 7:1243-1248; R. S. Givens, et al.,
1997 Journal of the American Chemical Society 119:8369-8370; R. S.
Givens, et al., 1997 Journal of the American Chemical Society
119:2453-2463; Jung et al., 1983 Biochem. Biophys. Acta, 761:
152-162; Joshi et al., 1990 J. Biol. Chem., 265: 14518-14525;
Zarling et al., 1980 J. Immunol., 124: 913-920; Bouizar et al.,
1986 Eur. J. Biochem., 155: 141-147; Park et al., 1986 J. Biol.
Chem., 261: 205-210; and Browning et al., 1989 J. Immunol., 143:
1859-1867; see also U.S. Pat. No. 7,033,764. A broad range of
cleavable, bifunctional (both homo- and hetero-bifunctional) spacer
arms with varying lengths are commercially available.
[0589] A rigid linker can be used. In some embodiments, use of a
rigid linker can be useful in improving a FRET signal. Examples of
rigid linkers include benzyl linkers, proline or poly-proline
linkers (S. Flemer, et al., 2008 Journal Org. Chem. 73:7593-7602),
bis-azide linkers (M. P. L. Werts, et al., 2003 Macromolecules
36:7004-7013), and rigid linkers synthesized by modifying the
so-called "click" chemistry scheme that is described by Megiatto
and Schuster 2008 Journal of the Am. Chem. Soc.
130:12872-12873.
[0590] In yet another embodiment, the linker can be an energy
transfer linker synthesized using methods described in U.S.
Published Patent Application No. 2006/0057565.
[0591] In yet another embodiment, the linker can comprise a spacer,
for example a cationic arginine spacer or an imidazolium spacer
molecule.
[0592] In some embodiments, the linker can be a fragmentable
linker, including non-lamellar "detergent-like" micelles or
lamellar vesicle-like micelles such as small unilamellar vesicles
or liposomes ("SUVs"), small multilamellar vesicles or liposomes
(SMVs"), large unilamellar vesicles or liposomes ("LUVs") and/or
large multilamellar vesicles or liposomes ("LMVs") (see U.S.
application Ser. No. 11/147,827) and see U.S. Application Nos.
60/577,995, and 12/188,165.
[0593] In some embodiments, the linker can include multiple amino
acid residues (e.g., arginine) which serve as an intervening linker
between the terminal phosphate group and the reporter moiety. For
example, the linker can be can four arginine residues which connect
a dye moiety to a nucleotide comprising one or more phosphate
groups, wherein the linker links the dye moiety to the terminal
phosphate group of the nucleotide.
[0594] In some embodiments, linkers can be used to attach energy
transfer or reporter moieties to nucleotides using any suitable
linking procedure, including: amine linkers for attaching reporter
moieties to nucleotides (see, for example, Hobbs, U.S. Pat. No.
5,151,507); a linker comprising a primary or secondary amine for
operably linking a reporter moiety to a nucleotide; and a rigid
hydrocarbon arm added to a nucleotide base (see, for example, R. F.
Service, 1998 Science 282(5391):1020-21). Some exemplary linking
procedures for attaching energy transfer or reporters moieties to
base molecules are provided in European Patent Application
87310256.0; International Application PCT/US90/05565; Marshall,
1975 Histochemical Journal 7:299-303; and Barone et al., 2001
Nucleosides, Nucleotides, and Nucleic Acids, 20(4-7): 1141-1145.
Other examples include linkers for attaching energy transfer or
reporter moieties to oligonucleotides synthesized using
phosphoramidate to incorporate amino-modified dT (see Mathies, U.S.
Pat. No. 5,707,804).
PEG Linkers
[0595] In one aspect, a linker comprising a polymer of ethylene
oxide can be used to attach the surfaces, reporter moieties
(including, e.g., dyes and nanoparticles), polymerases, nucleotides
and/or nucleic acid molecules of the present disclosure to each
other in any combination. Non-limiting examples of such polymers of
ethylene oxide include polyethylene glycol (PEG), including short
to very long PEG, branched PEG, amino-PEG-acids, PEG-amines,
PEG-hydrazines, PEG-guanidines, PEG-azides, biotin-PEG, PEG-thiols,
and PEG-maleinimides. For example, PEG includes: PEG-1000,
PEG-2000, PEG-12-OMe, PEG-8-OH, PEG-12-COOH, and PEG-12-NH.sub.2.
In some embodiments, the PEG molecule may be linear or branched. In
some embodiments, it can have a molecular weight greater than or
approximately equal to 1000, 2000, 3000, 4000, 5000 or greater.
[0596] In some embodiments, functionalization with different
reactive moieties can be used create a heterobifunctional PEG
derivative comprising different reactive groups at each end. Such
heterobifunctional PEGs can be useful in linking two entities,
where a hydrophilic, flexible and biocompatible spacer is needed.
Some examples of heterobifunctional PEG derivatives include without
limitation Hydroxyl PEG Carboxyl (HO-PEG-COOH): Thiol PEG Carboxyl
(HS-PEG-COOH); Hydroxyl PEG Amine (HO-PEG-NH2); t-Boc Amine PEG
Amine (TBOC-PEG-NH2); Amine PEG Carboxyl (NH2--PEG-COOH); t-Boc
Amine PEG NHS Ester (TBOC-PEG-NHS); FMOC Amine PEG NHS Ester
(FMOC-PEG-NHS): Acrylate PEG NHS Ester (ACLT-PEG-NHS); Maleimide
PEG Carboxyl (MAL-PEG-COOH); Maleimide PEG Amine (MAL-PEG-NH2),
including the TFA Salt thereof; Maleimide PEG NHS Ester
(MAL-PEG-NHS); Biotin PEG NHS Ester (BIOTIN-PEG-NHS); Biotin
Polyethylene Glycol Maleimide (BIOTIN-PEG-MAL); OPSS PEG NHS Ester
(OPSS-PEG-NHS).
[0597] Optionally, the PEG derivative can be a multi-arm PEG
derivative. In some embodiments, the multi-arm PEG derivative can
be a PEG derivative having a core structure comprising
pentaerythritol (including, for example, 4arm PEG Amine
(4ARM-PEG-NH2); 4arm PEG Carboxyl (4ARM-PEG-COOH); 4arm PEG
Maleimide (4ARM-PEG-MAL); 4arm PEG Succinimidyl Succinate
(4ARM-PEG-SS); 4arm PEG Succinimidyl Glutarate (4ARM-PEG-SG)); a
PEG derivative having a core structure comprising hexaglycerin
(including, for example, 8arm PEG Amine (8ARM-PEG-NH2); 8arm PEG
Carboxyl (8ARM-PEG-COOH); 8arm PEG Succinimidyl Succinate
(8ARM-PEG-SS); 8arm PEG Amine (8ARM-PEG-SG); PEG derivative having
a core structure comprising tripentaerythritol (including, for
example, 8arm PEG Amine (8ARM(TP)-PEG-NH2); 8arm PEG Carboxyl
(8ARM(TP)-PEG-COOH); 8arm PEG Succinimidyl Succinate
(8ARM(TP)-PEG-SS); 8arm PEG Amine (8ARM(TP)-PEG-SG)). Optionally,
end groups for heterobifunctional PEGs can include maleimide, vinyl
sulfones, pyridyl disulfide, amine, carboxylic acids and NHS
esters. The activated PEG derivatives can then be used to attach
the PEG to the desired biomolecule and/or nanoparticle. Optionally,
one or both ends of the PEG derivative can be attached to the
N-terminal amino group or the C-terminal carboxylic acid of a
protein-comprising biomolecule.
Signal Detection
[0598] The methods, compositions, systems and kits disclosed herein
can involve the use of a detection system for optical or spectral
detection of a signal, or a change in a signal, generated (emitted)
by the energy transfer moiety(ies) or reporter moiety(ies) in the
nucleotide binding or nucleotide incorporation reactions.
[0599] The systems and methods can detect and/or measure a signal,
or a change or an amount of change of an optical or spectral
characteristic of a signal (e.g., fluorescence or quenching) from a
reporter moiety, such as an energy transfer donor and/or acceptor
moiety. The change in the signal can include changes in the:
intensity of the signal; duration of the signal; wavelength of the
signal; amplitude of the signal; duration between the signals;
and/or rate of the change in intensity, duration, wavelength or
amplitude. The change in the signal can include a change in the
ratio of the change of the energy transfer donor relative to change
of the energy transfer acceptor signals.
[0600] The detection system comprises: excitation illumination,
optical transmission elements, detectors, and/or computers.
[0601] In one aspect, detecting radiation emitted by an excited
energy transfer or reporter moiety during nucleotide binding
comprises: the nucleotide, which can be labeled with a FRET
acceptor, binds the polymerase which can be labeled with a FRET
donor, bringing the FRET acceptor/donor pair in proximity to each
other, and the FRET donor can be excited resulting in energy
transfer to the FRET acceptor which emits a signal which is
detectable by the detection system.
[0602] The detection system comprises excitation illumination which
can excite the energy transfer or reporter moieties which produce a
detectable signal. The excitation illumination can be
electromagnetic energy, such as radio waves, infrared, visible
light, ultraviolet light, X-rays or gamma rays. The source of the
electromagnetic radiation can be a laser, which possesses
properties of mono-chromaticity, directionality, coherence,
polarization, and/or intensity. The laser can produce a continuous
output beam (e.g., continuous wave laser) or produce pulses of
light (e.g., Q-switching or mode-locking). The laser can be used in
a one-photon or multi-photon excitation mode. The laser can produce
a focused laser beam. The wavelength of the excitation
electromagnetic radiation can be between about 325-850 nm, or
between about 325-752 nm, or between about 330-752 nm, or between
about 405-752 nm. The laser can be generated by a mercury, xenon,
halogen, or other lamps.
[0603] The wavelength and/or power of the excitation illumination
can be selected to avoid interfering with or damaging the
polymerase enzymatic activities. The excitation illumination can be
focused on a stationary position or moved to a different field of
view (FOV). The excitation illumination can be directed at a
nucleotide incorporation reaction which is: in a liquid volume
(e.g., aqueous or oil); on a surface; in or on a nanodevice; in a
waveguide; or in an evanescent illumination system (e.g., total
internal reflection illumination). The excitation illumination can
pass through a transparent or partially transparent surface which
is conjugated (covalently or non-covalently) with the components of
the nucleotide incorporation reaction.
[0604] The energy transfer moiety (e.g., a FRET donor) can be
excited by the excitation illumination at a particular wavelength,
and transmit the excitation energy to an acceptor moiety which is
excited and emits a signal at a longer wavelength. The energy
transfer moiety or reporter moiety can undergo multi-photon
excitation with a longer wavelength, typically using a pulsed
laser.
[0605] The detection system comprises suitable optical transmission
elements which are capable of transmitting light from one location
to another with the desired refractive indices and geometries. The
optical transmission elements transmit the excitation illumination
and/or the emitted energy in an unaltered or altered form. The
optical transmission elements include: lens, optical fibers,
polarization filters (e.g., dichroic filters), diffraction gratings
(e.g., etched diffraction grating), arrayed waveguide gratings
(AWG), optical switches, mirrors, dichroic mirrors, dichroic beam
splitter, lenses (e.g., microlens and nanolens), collimators,
filters, prisms, optical attenuators, wavelength filters (low-pass,
band-pass, or high-pass), wave-plates, and delay lines, or any
combination thereof.
[0606] The detection system comprises suitable detectors which are
capable of detecting and/or distinguishing the excitation
illumination and/or the emitted energy. A wide variety of detectors
are available in the art, including: single or multiple channel
detectors, high-efficiency photon detection systems, optical
readers, charge couple devices (CCD), photodiodes (e.g. avalanche
photo diodes (APD)), APD arrays, cameras, electron-multiplying
charge-coupled device (EMCCD), intensified charge coupled device
(ICCD), photomultiplier tubes (PMT), multi-anode PMT, complementary
metal oxide semiconductor (CMOS) chip(s), and a confocal microscope
equipped with any of the foregoing detectors. The location of the
nucleotide incorporation reaction can be aligned, with respect to
the excitation illumination and/or detectors, to facilitate proper
optical transmission.
[0607] Suitable detection methods can be used for detecting and/or
distinguishing the excitation illumination (or change in excitation
illumination) and/or the emitted energy (or change in emitted
energy), including: confocal laser scanning microscopy, Total
Internal Reflection (TIR), Total Internal Reflection Fluorescence
(TIRF), near-field scanning microscopy, far-field confocal
microscopy, wide-field epi-illumination, light scattering, dark
field microscopy, photoconversion, wide field fluorescence, single
and/or multi-photon excitation, spectral wavelength discrimination,
evanescent wave illumination, scanning two-photon, scanning wide
field two-photon, Nipkow spinning disc, multi-foci multi-photon, or
any combinations thereof.
[0608] The signals emitted from different energy transfer moieties
can be resolved using suitable discrimination methods which are
based on: fluorescence resonance energy transfer measurements;
photoconversion; fluorescent lifetime measurements; polarization;
fluorescent lifetime determination; correlation/anti-correlation
analysis; Raman; intensity; ratiometric; time-resolved methods;
anisotropy; near-field or far field microscopy; fluorescence
recovery after photobleaching (FRAP); spectral wavelength
discrimination; measurement and separation of fluorescence
lifetimes; fluorophore identification; background suppression,
parallel multi-color imaging, or any combination thereof. See, for
example, J. R. Lakowitz 2006, in: "Principles of Fluorescence
Spectroscopy", Third Edition. If the different nucleotides are
labeled with different energy transfer or reporter moieties, then
resolving the emitted signals can be used to distinguish between
the different nucleotides which bind the polymerase and/or which
are incorporated by the polymerase.
[0609] In one embodiment, a system and method for detecting
radiation emitted by an excited energy transfer or reporter moiety
comprises: an illumination source (e.g., a laser) which produces
the excitation energy (e.g., one or multi-photon excitation
radiation) which is directed, via a dichroic beam splitter, through
a lens, and through a transparent surface or onto a surface, where
the nucleotide binding reaction or the nucleotide incorporation
reaction is attached to the surface or is in a solution. The
excitation illumination excites the energy transfer or reporter
moiety (e.g., fluorescent dye and/or nanoparticle) resulting in
emitted radiation (or a change in radiation) which passes back
through the dichroic beam splitter and is directed to the detector
(or an array of detectors) which is capable of identifying and/or
resolving the type of emission. Information about the detected
emitted signals is directed to the computer where the information
is registered and/or stored. The computer can process the
registered and/or stored information to determine the identity of
the nucleotide which bound the polymerase or the identity of the
incorporated nucleotide.
[0610] In one aspect, the system and method for detecting radiation
emitted by an excited energy transfer or reporter moiety includes a
multifluorescence imaging system. For example, the different
nucleotides may each be linked to different FRET acceptor moieties.
The FRET acceptor moieties can be selected to have minimal overlap
between the absorption and emission spectra, and the absorption and
emission maxima. The multifluorescence imaging system can
simultaneously (or substantially simultaneously) detect signals
from the FRET acceptor moieties, and resolve the signals. Such
multifluorescent imaging can be accomplished using suitable
filters, including: band pass filters, image splitting prisms, band
cutoff filters, wavelength dispersion prisms, dichroic mirrors, or
diffraction gratings, or any combination thereof.
[0611] In another aspect, the multifluorescence imaging system is
capable of detecting the signals emitted by the different energy
transfer and reporter moieties attached to the different
nucleotides. Such a system can include special filter combinations
for each excitation line and/or each emission band. In one
embodiment, the detection system includes tunable excitation and/or
tunable emission fluorescence imaging. For tunable excitation,
light from a light source can pass through a tuning section and
condenser prior to irradiating the sample. For tunable emissions,
emissions from the sample can be imaged onto a detector after
passing through imaging optics and a tuning section. The tuning
sections can be controlled to improve performance of the
system.
[0612] In yet another aspect, the detection system comprises an
optical train which directs signals emitted from an organized array
onto different locations of an array-based detector to detect
multiple optical signals from multiple locations. The optical
trains typically include optical gratings and/or wedge prisms to
simultaneously direct and separate signals having differing
spectral characteristics from different addressable locations in an
array to different locations on an array-based detector, e.g., a
CCD.
[0613] In another aspect, the detection methods include detecting
photon bursts from the labeled nucleotides during incorporation.
The photon bursts can be the fluorescent signals emitted by the
energy transfer moiety which is linked to the nucleotide. The
photon bursts can be a FRET event. The methods can additionally
include analyzing the time trace of the photon bursts. The methods
can be practiced using time-resolved fluorescence correlation
spectroscopy.
[0614] Nucleotide incorporation reactions using nucleotides labeled
at the terminal phosphate with a fluorescent dye have been
previously demonstrated (Sood, U.S. published patent application
No. 2004/0152119; and Kumar, U.S. Pat. No. 7,393,640). Furthermore,
fluorescence detection of single molecule nucleotide incorporation
reactions has been routinely obtained (Kao, U.S. Pat. No.
6,399,335; and Fuller, U.S. Pat. No. 7,264,934).
[0615] The nucleotide labeling strategy can be used as a basis for
selecting any suitable detection system for detecting and/or
resolving signals emitted by the nucleotide binding reaction or the
nucleotide incorporation reaction. Exemplary labeling and detection
strategies include but are not limited to optical train and TIRF
detection methods such as those disclosed in U.S. Pat. No.
6,423,551; and U.S. Pub. Nos. 2006/0176479, 2007/0109536,
2007/0111350, and 2007/0250274.
Sequence Analysis of Detected Signals
[0616] Following detection of the sample emissions, the raw
emission data can be analyzed to identify events involving
nucleotide polymerization. In some embodiments, the emissions can
be analyzed in single molecule format to identify nucleotide
polymerization.
[0617] In one aspect, a labeled enzyme conjugate is a labeled
polymerase conjugate, and a time series of nucleotide
incorporations by the labeled polymerase conjugate is detected and
analyzed to deduce the ordered sequence of nucleotides (identifying
the nucleotide bases) in the single nucleic acid substrate that is
being replicated by the polymerase.
[0618] In one exemplary embodiment, the labeled polymerase
conjugate comprises an energy transfer moiety that undergoes FRET
with the energy transfer moiety of an incoming labeled nucleotide
that is polymerized by the polymerase of the conjugate. Nucleic
acid sequence analysis is performed by first analyzing the raw
emission data to computationally determine the occurrence of a FRET
event. In some embodiments, FRET events i.e., a detectable change
in a signal produced from a donor or acceptor resulting from a
change in the distance between the donor and acceptor, can be
identified using a Hidden Markov Model (HMM)-based or equivalent
generalized likelihood ratio test that determines the location of
an intensity change point based on individual photon arrival times;
this test can then be applied recursively to an entire single
molecule intensity trajectory, thus finding each change points. The
true number of states accessible to the system is then computed.
See, e.g., Watkins et al., "Detection of Intensity Change Points in
Time-Resolved Single-Molecule Measurements" J. Phys. Chem. B.,
109(1):617-628 (2005). An exemplary FRET detection method using
this technique is described herein in Example 14.
[0619] In one aspect, a system can collect and analyze chemical
and/or physical event data occurring at one or a plurality of
locations within a viewing volume or field of an imaging apparatus.
In some embodiments, the system comprises a sample subsystem for
containing a sample to be detected and analyzed, where the sample
includes at least one moiety (e.g., enzyme, substrate, reporter
moiety, etc) having detectable property that undergoes a change
before, during or after one or a sequence of chemical and/or
physical events involving the moiety. The system can also includes
a detection apparatus having a viewing field that permits the
detection of changes in the detectable property of the moiety
within the viewing field. The system also includes a data
processing subsystem connected to the imaging apparatus for
collecting, storing and analyzing data corresponding to the
chemical and/or physical events occurring at definable locations in
the viewing field involving one or more moieties within the viewing
field of the imaging subsystem. The data processing subsystem
converts the data into classifications of events according the
event type determined by a set of parameters defining or
characterizing each event type. See, e.g., U.S. Published Patent
Application No. 2007/0250274, Volkov et al. which is incorporated
herein as if set forth in full.
[0620] In one aspect, FRET events can be identified by
computationally determining the occurrence of an anti-correlated
FRET event (typically involving a correlated decrease in donor
signal and increase in acceptor signal). In one exemplary
embodiment, FRET events corresponding to interactions between a
donor fluorophore associated with a first moiety, e.g., a
polymerase and an acceptor fluorophore associated with a second
moiety, e.g., a nucleotide can be analyzed by first collecting or
receiving data from a viewing volume of an imaging apparatus such
as an CCD or iCCD detection system. In some embodiments, the data
can be in a single data channel or a plurality of data channels,
each data channel representing a different frequency range of
emitted fluorescent light, e.g., one channel can include
fluorescent light data emitted by a donor, a donor channel, while
other channels include fluorescent light data emitted by an
acceptor, an acceptor channel, or by another donor, a second donor
channel. In certain embodiments, a channel will exit for each
different fluorophore being detected simultaneously. In some
embodiments, the acceptors are selected so that they can be
separately identified based on detectable attributes of their
signals e.g., intensity, frequency shifts, signal duration,
attenuation, etc. After data collection, the separate data channels
are spatially correlated within the viewing volume so that active
fluorophores can be spatially and temporally related, called
calibration or registration. The goal of calibration is to
determine the pixel coordinates in each quadrant that correspond to
a single position on the slide or a single location within the
viewing field--to make sure that the data in each channel is
spatially coincident over the viewing field and through time of
detection. After reading the configuration file and the open log
file, calibrations, if any, are loaded from the command line. After
loading the calibration information, a corresponding directory is
read as specified in the command line with all subdirectories, for
each one. This read step includes: (1) scanning for calibration
stacks, and if there are some not matched by the available
calibrations, generate new calibrations out of them; (2) scanning
for stacks; if there are some, assume this directory is a slide;
and (3) scanning the directory path for a date and slide name
comprising reaction conditions such as donor identity, acceptor
identity, buffers, etc. See, for example, U.S. Published Patent
Application No. 2007/0250274, Volkov et al.
[0621] Once FRET events have been identified, they can be analyzed
to determine the order and sequence of nucleotide
incorporations.
Analysis of Fluorescence Data To Extrapolate Sequence
Information
[0622] To convert the observed fluorescence emissions detected
during the sequencing reaction into nucleotide sequence
information, the raw data comprising a movie of observed emissions
was first processed by using a Hidden Markov Model (HMM)-based
algorithm or equivalent to detect and identify FRET events. The
subsequent detected FRET events were filtered and filtered
sequences were aligned. Each of these two steps, FRET event
detection and sequence analysis, are described in more detail
below.
Detection of FRET Events
[0623] The analysis underlying FRET event detection is designed to
process spatially correlated movie(s) comprising sequence
fluorescence emission data, and extract time-series of interest
from those data. A movie typically contains one or more channels
where each channel represents the same spatial location at
different wavelengths. The analysis chain begins with the
submission of one or more movies to the analysis machine via a
comprehensive user interface. The user interface requires the user
to input various parameters that describe the movie(s) (e.g.
channel regions, dye emission properties, etc.). Once this data is
submitted the movie(s) are then processed by the image analysis
software where a sliding window of N frames propagates through the
movie calculating a temporal local average of the frames within the
window. At each position of the window in the movie, the local
average image is then further processed and enhanced using well
known image processing algorithms and a record of the maximum
projection of all the local average images is recorded to produce a
global image of the movie. This global image is the input into a
spot identification algorithm which produces a set of spots
identified by a unique spot id, its x and y location and its
corresponding channel, for the sake convenience referred to as a
spot-tuple. Each set of spots for a given channel is then
registered to the set of spots in every other channel. In this way
a set of spot tuples is constructed. If a detected spot in one
channel does not have a corresponding detected spot in another
channel, then the position of the undetected spot using the
transformation between the two channels and the location of the
detected spot is inferred. Once a complete set of spot tuples is
constructed the movie is iterated over and at each frame the
amplitude of each spot is calculated and appended to the
appropriate time-series.
[0624] The collection of time-series from a spot tuple consists of
time-series from donor and corresponding acceptor channels. This
collection is called a Vector Time-Series (VTS). The FRET detection
process starts with a data segmentation step using a Markov Chain
Monte-Carlo (MCMC) algorithm. Each segment of VTS is modeled by a
multivariate Gaussian model, with each of the channel modeled by a
mean and a standard deviation. This model establishes a baseline
for each channel, from which quantities such as "Donor Down" and
"Acceptor Up" can be calculated. A Hidden Markov Model (HMM) or
equivalent algorithm is used to model the observed data. The
underlying states consist of a null state, a blink state and a
number of FRET states (one for each acceptor channel). Each state
has its emission probability, which reflects the state's
corresponding physical concept. FRET states are characterized by
significant "donor down" and "acceptor up" signals. Blink state is
characterized by significant "donor down" with no "acceptor up".
Null state is characterized by no "donor down" and no "acceptor
up". Given the observed VTS signal, the emission matrix, and a
state transition probability matrix, the most probable state path
can be computed using the Viterbi algorithm. This state path
assigns each of the frames to a state. Temporally neighboring FRET
frames are grouped into FRET events. For each of the detected FRET
events, a list of event features are calculated, including event
duration, signal average, signal to noise ratio, FRET efficiency,
probability of event, color calling and other features. This list
of events and corresponding features are stored in a file.
[0625] The final stage of the automated analysis generates a report
summarizing the results in the form of a web page containing
summary image, statistics of the spots and FRET detection, together
with line intensity plots and base call plots.
[0626] Using the above process, the movie data obtained from the
sequencing reactions was analyzed to detect and identify FRET
events according to the process described above. The FRET events
were then processed to identify sequences as described below.
Sequence Analysis
[0627] The string of FRET events from the same spot-tuple are then
aligned to a reference sequence. Each color call in the string is
associated with a nucleotide, creating a DNA sequence. That DNA
sequence and a reference sequence are fed into a Smith-Waterman
alignment or equivalent algorithm to determine where the read comes
from in the template sequence and the similarity between the
sequences.
Kits
[0628] Provided herein are kits for conducting the nucleotide
binding reactions and/or the nucleotide incorporation reactions
described herein. The kits can include, in one or more containers,
the components of nucleotide binding and/or nucleotide
incorporation disclosed herein, including: the solid surfaces,
energy transfer moieties, reporter moieties, nanoparticles,
polymerases, nucleotides, target nucleic acid molecules (e.g., a
control test target molecules), primers, and/or
oligonucleotides.
[0629] In the kits, the solid surfaces, energy transfer moieties,
reporter moieties, nanoparticles, polymerases, nucleotides, target
nucleic acid molecules, primers, and/or oligonucleotides can be
attached to each other in any combination, and/or be unattached.
The kits can include positive and/or negative control samples.
[0630] Additional components can be included in the kit, such as
buffers and reagents. For example, the buffers can include Tris,
Tricine, HEPES, or MOPS, or chelating agents such as EDTA or EGTA.
In another example, the reagents can include monovalent ions, such
as KCl, K-acetate, NH.sub.4-acetate, K-glutamate, NH.sub.4Cl, or
ammonium sulfate. In yet another example, the reagents can include
divalent ions, such as Ca.sup.2+, CaCl.sub.2, Mg.sup.2+,
MgCl.sub.2, Mg-acetate, Mn.sup.2+, MnCl.sub.2, and the like. The
kits can include the components in pre-measured unit amounts. The
kits can include instructions for performing the nucleotide binding
reactions and/or the nucleotide incorporation reactions. Where the
kit is intended for diagnostic applications, the kits may further
include a label indicating regulatory approval for the diagnostic
application.
EXAMPLES
[0631] All of the compositions and methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. In some cases, the compositions
and methods of this invention have been described in terms of
embodiments, however these embodiments are in no way intended to
limit the scope of the claims, and it will be apparent to those of
skill in the art that variations may be applied to the compositions
and/or methods and in the steps or in the sequence of steps of the
methods described herein without departing from the concept, spirit
and scope of the invention. More specifically, it will be apparent
that certain components which are both chemically and
physiologically related may be substituted for the components
described herein while the same or similar results would be
achieved. All such similar substitutes and modifications apparent
to those skilled in the art are deemed to be within the spirit,
scope and concept of the invention as defined by the appended
claims.
Example 1
Synthesis of Nucleotide Tetraphosphate Molecules Labeled with Alexa
Dyes
[0632] The synthesis scheme of amino-dN tetraphosphate is
illustrated in scheme 1 using amino-dG4P as an illustrative
example. Amino-attached dA4P, dC4P and dT4P were synthesized by the
same method.
1.) Synthesis of compound 2
[0633] Compound 1 (678 mg, 2 mmol) was suspended in trimethyl
phosphate (5 mL) and cooled to 0.degree. C. POCl.sub.3 (280 .mu.L)
was added to the stirred mixture under argon. The mixture was
warmed up and stirred at room temperature overnight. The reaction
was quenched by adding slowly 4 mL of TEAB buffer (1 M) at
0.degree. C. Triethylamine was added to adjust to pH 7. The solvent
was evaporated and the residue was purified by column
chromatography on silica gel, eluting with 10% H.sub.2O/CH.sub.3CN.
After evaporation of the solvent, the solid was dissolved in water.
The pH of the solution was adjusted to pH 7 with TEAB buffer (1 M),
followed by coevaporation with methanol. Yield: 400 mg of compound
2.
##STR00008##
2.) Synthesis of Compound 3
[0634] The sodium salt of dGTP (20 mg) was converted into its
triethylammonim salt by passing a trethylammonium resin and dried
in high vacuum. Compound 2 (42 mg) was dissolved in 2 mL of dry
DMF. arbonyldiimidazole (CDI) (65 mg) was added and the solution
was stirred for 4 hours at room temperature, followed by the
addition of anhydrous methanol (18 .mu.L) and stirred for a further
hour. The dried dGTP triethylammonium salt was dissolved in dry DMF
(2 mL), and to this solution was added the prepared
phosphoimidazolate solution of 2 under argon. The mixture was
stirred under argon overnight. Triethylamine (1 mL) was added and
stirred for 4 hours. The solvent was evaporated, washed with
CHCl.sub.3, dissolved in water and purified by sephadex A-25 DEAE
ion exchange chromatography, eluting with a linear gradient of 0.05
M to 0.6 M TEAB buffer. After coevaporation with methanol and
lyophilization, ca. 5 mg of compound 3 was obtained. The reaction
was checked by TLC
(Dioxane/IPA/H.sub.2O/NH.sub.4OH=40/20/40/36).
3.) Synthesis of Amino-Attached dA4P (4), dC4P (5) and dT4P (6)
##STR00009##
[0635] These compounds were synthesized by the same method as
described for amino-dG4P (3).
4.) Labeling Amino-dGP4 with Alexa Dyes
##STR00010##
[0636] A solution of amino-dG4P (3) (0.5 mg) in DMF-water (2:1, 300
.mu.L) was mixed with 50 .mu.L of saturated sodium bicarbonate
solution. To this solution was added the Alexa dye SE (2 mg). The
solution was stirred at room temperature until the completion of
the reaction (ca. 1 hour). The product was purified by column
chromatography on sephadex LH-20, eluting with water. The desired
fraction was concentrated to ca. 300 .mu.L and stored at
-20.degree. C.
[0637] The Alexa dye SE used includes AF633 SE, AF647 SE, AF660 SE,
AF680 SE, AF700 SE and AF750 SE.
5.) Labeling Amino-dAP4, Amino-dC4P and Amino-dT4P with Alexa Fluor
Dyes
[0638] These amino-dN tetraphosphates were labeled with Alexa dyes
by the same method as described in procedure 4.
Example 2
Preparing PEG and Biotin-Streptavidin Coated Surfaces
Low-Density Streptavidin Coating
[0639] Low density streptavidin layers were coated on the surface
of glass coverslips using a flowcell. PEG/PEG-biotin coated glass
cover slips (MicroSurfaces, Inc., Minneapolis, Minn.) were
assembled into 8-lane reaction chambers with laser-cut 3M
double-sided adhesive and custom fabricated plastic superstructures
with inlet/outlet ports for fluid addition. The surface was wetted
by flowing 1 milliliter of TBSB solution which contains
Tris-buffered saline (50 mM Tris, pH 7.5, 150 mM NaCl) and 0.5%
bovine serum albumin (Sigma, catalog #A8577). 250 microliters of 1%
BSA/TBS (50 mM Tris, pH 7.5, 1% BSA) was flowed across the chip and
allowed to incubate at room temperature for 5 minutes. The surface
was coated with streptavidin by flowing 100 microliters of 60
.mu.pM streptavidin, (Zymed, Cat # 43-4302) diluted in TBSB, and
incubating for 30 minutes at room temperature. The lanes were
washed with 1 milliliter of TBSB and passivated for a second time
with 250 microliters of 1% BSA/TB S-biotinylated DNA, in the form
of a self-annealing 5'-overhanging hairpin molecule, was diluted to
10-100 .mu.pM in 1% BSA/TBS and 100 microliters was flowed into the
reaction chamber and incubated 30 minutes at room temperature. The
lanes were washed with 1 milliliter of TBSB. The density of the DNA
bound to the low density PEG-biotin-streptavidin coated glass
surface was imaged using total internal reflection microscopy
(TIRF) and a 633 nm laser.
High-Density Streptavidin Coating
[0640] High density streptavidin layers were coated on the surface
of glass coverslips using a flowcell. PEG/PEG-biotin coated cover
slips (MicroSurfaces, Inc., Minneapolis, Minn.) were assembled into
8-lane reaction chambers with laser-cut 3M double-sided adhesive
and custom fabricated plastic superstructures with inlet/outlet
ports for fluid addition. The surface was wetted by flowing 1
milliliter of TBSB solution which contains Tris-buffered saline (50
mM Tris, pH 7.5, 150 mM NaCl) containing 0.5% bovine serum albumin
(Sigma, catalog #A8577). 250 microliters of 1% BSA/TBS (50 mM Tris,
pH 7.5, 1% BSA) was flowed across the chip and allowed to incubate
at room temperature for 5 minutes. The surface was coated with
streptavidin by flowing 100 microliters of 200 .mu.g/ml
streptavidin, (Zymed, Cat # 43-4302) diluted in TBSB, and
incubating for 10 minutes at room temperature. The lanes were
washed with 1 milliliter of TBSB and passivated for a second time
with 250 microliters of 1% BSA/TBS. Biotinylated DNA, in the form
of a self-annealing 5'-overhanging hairpin molecule, was diluted to
10-100 .mu.pM in 1% BSA/TBS and 100 microliters was flowed into the
reaction chamber and incubated 30 minutes at room temperature. The
lanes were washed with 1 milliliter of TBSB. The density of the DNA
bound to the high density PEG-biotin-streptavidin coated glass
surface was imaged using total internal reflection microscopy
(TIRF) and a 633 nm laser.
Example 3
Linking Chemistries for Attaching Nanoparticles with
Polymerases
Preparing Phosphorothiolated Phi29 Polymerases
[0641] Phi29 polymerase protein, comprising the protein kinase A
recognition sequence LRRASLG (SEQ ID NO:19) at the N-terminus (SEQ
ID NO:7), was incubated with kinase and ATP-.gamma.S to form a
phosphorothioate functional group on the serine residue of the
recognition sequence.
Modifying the Nanoparticles with Adipic Dihydrazide
[0642] C8 Nanoparticles having outer shells which are pre-modified
with methoxy-terminated PEG were obtained from Molecular Probes.
These nanoparticles have residual carboxylate functional groups.
300 .mu.l of 4.1 .mu.M the nanoparticles were buffer exchanged into
100 mM MES, 300 mM NaCl, pH 5.5 using ultrafiltration (VivaSpin
100K MWCO spin filters). The reaction was started by adding: 260
.mu.l of 4.08 .mu.M buffer exchanged nanoparticles, 10.6 .mu.l of
20 mM adipic dihydrazide (dissolved in water) and 13.5 .mu.l of 10
mM EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
hydrochloride dissolved in water). 25 minutes after the start of
the reaction another 13.5 .mu.l aliquot of 10 mM EDC was added to
the reaction mix. After two hours incubation at room temperature,
the reaction mix was concentrated by ultrafiltration (VivaSpin 100K
MWCO) then washed three times with 200 .mu.l of 100 mM MES, 300 mM
NaCl, pH 7.5 using the same ultrafiltration unit. The nanoparticles
have hydrazide functional groups.
Reacting the Nanoparticles with Iodoacetic Acid
[0643] The nanoparticles (having hydrazide reactive groups) were
modified with iodoacetic acid. The following reagents were added:
185 .mu.l of 3.98 .mu.M hydrazide-modified nanoparticles, 14.7
.mu.l of 10 mM iodoacetic acid (sodium salt, dissolved in water)
and 10 .mu.l of 10 mM EDC (dissolved in water). 25 minutes after
the start of the reaction another 10 .mu.l aliquot of 10 mM EDC was
added to the reaction mix. The reaction mix was allowed to incubate
at room temperature, in the dark for three hours. After incubation,
the reaction mix was concentrated by ultrafiltration and washed
5.times.200 .mu.l with 100 mM MES, 300 mM NaCl, pH 5.5 also using
ultrafiltration. The nanoparticles have iodoacetyl functional
groups.
Attaching Iodoacetyl Nanoparticles with Phi29 Polymerases
[0644] The phosphorothioated phi29 polymerase was buffer exchanged
into 100 mM MES, 300 mM NaCl, pH 5.5 using a NAPS column (GE
Healthcare). For the conjugation reaction, 392 .mu.l of 13.2 .mu.M
phosphorothioated phi29 polymerase was added to 95 .mu.l of 2.73
.mu.M iodoacetyl nanoparticles. The reaction mix was allowed to
incubate overnight at room temperature in the dark. The reaction
mix was concentrated to approximately 30 .mu.l then purified over a
SUPERDEX 200 (GE Healthcare) 8 mm.times.5.5 cm column (2 mL
disposable column from Thermo Scientific) using 100 mM TRIS, 300 mM
NaCl, pH 7.5 as the elution buffer. Three fractions were collected
and assayed for concentration, extension activity and template
binding.
Materials:
[0645] Hairpin oligonucleotide 221 sequence:
TABLE-US-00002 (SEQ ID NO: 24)
5'-TTTTTTTGCAGGTGACAGGTTTTTCCTGTCACCXGC-3'
where X=fluorescein dT. Hairpin oligonucleotide ALEXA
FLUOR-647-labeled 199 sequence:
TABLE-US-00003 (SEQ ID NO: 25)
5'-TTATCTTTGTGGGTGACAGGTTTTTCCTGTCACCX-3'
where X=ALEXA FLUOR 647-dC 1.times. extension buffer: 50 mM Tris
(pH 8), 50 mM NaCl, and 10 mM MgCl.sub.2.
Activity Assay
[0646] A 150 nM master mix solution of a labeled hairpin
oligonucleotide 221 was prepared by diluting the appropriate
quantity of a 50 .mu.M stock solution with extension buffer (50 mM
TRIS, pH 8, 50 mM NaCl, 10 mM MgCl.sub.2). 450 .mu.l of a master
mix was prepared for each sample being tested.
[0647] The conjugate being tested was diluted in 450 .mu.l of the
master mix such that the final concentration of the conjugate is in
the range of 10 nM to 50 nM. The positive control samples of free
PKA.PHI.29 were similarly diluted. The sample solution was
deposited in four microtiter plate wells, at 100 .mu.l/well.
[0648] The microtiter plate was placed in a plate reader (Molecular
Devices, SpectraMax M5) and set up to monitor the fluorescence as
function of time (excitation 490 nm, emission 535 nm, cutoff filter
515 nm). Just prior to starting the plate reader, 2 .mu.l of 1 mM
dATP was added to each of two microtiter wells to start the
extension reaction. The other two microtiter wells with sample
represent no extension controls. The plate was read for an hour or
until the samples reached saturation. The results indicate that
phi29 polymerase, attached to nanoparticles, can incorporate
nucleotides.
Binding Assay
[0649] Each sample to be tested was diluted to 20 nM in 650 .mu.l
of extension buffer. 50 .mu.l of the sample was pipetted into each
well of the top row of a microtiter plate.
[0650] A 2 .mu.M solution of an ALEXA FLUOR-labeled hairpin
oligonucleotide JX338 was prepared by dissolving the appropriate
amount of stock oligonucleotide in extension buffer. 140 .mu.l of
each sample to be tested was prepared. The hairpin primer/template
solution was pipetted into the first well of the second row in the
microtiter plate. Into the remaining 11 wells of the second row of
the microtiter plate, 70 .mu.l of extension buffer was pipetted. 70
.mu.l of the hairpin primer/template was removed from the first
well of the second row and mixed with the extension buffer in the
second well. 70 .mu.l from the second well was removed and mixed
with the extension buffer in the third well. The serial dilution
was prepared up to the last well in row two.
[0651] 50 .mu.l of the primer/template was transferred from each
well of row two into 50 .mu.l of the sample in each well of row
one.
[0652] The microtiter plate was placed on the plate reader which
was set to measure fluorescence at 605 nm and 670 nm with
excitation at 450 nm. The results showed an increase in FRET
acceptor signal with an increase in the amount of the labeled
oligonucleotide-199, or a decrease in FRET donor signal.
Example 4
Preparing Nanoparticles Attached with His-Tagged Polymerases
Materials:
[0653] Hairpin ALEXA FLUOR 647 labeled-oligonucleotide 192
sequence:
TABLE-US-00004 (SEQ ID NO: 26)
5'-TTTTTTTGCCCCCAGGGTGACAGGTTTTTCCTGTCACCC-3'
where the 192 oligo is labeled at the 3' end with ALEXA FLUOR 647.
Hairpin ALEXA FLUOR 647 labeled-oligonucleotide 199 sequence:
TABLE-US-00005 (SEQ ID NO: 25)
5'-TTATCTTTGTGGGTGACAGGTTTTTCCTGTCACCX-3'
where X=ALEXA FLUOR 647-dC. Hairpin fluorescein
labeled-oligonucleotide 221 sequence:
TABLE-US-00006 (SEQ ID NO: 24)
5'-TTTTTTTGCAGGTGACAGGTTTTTCCTGTCACCXGC-3'
where X=fluorescein dT. Hairpin ALEXA FLUOR 647
labeled-oligonucleotide 229 sequence:
TABLE-US-00007 (SEQ ID NO: 27)
5'-TTTTTGCGGGTGACAGGTTTTTCCTGTCACCC-3'
where the 229 oligo is labeled at the 3' end with ALEXA FLUOR
647.
[0654] 1.times. extension buffer: 50 mM Tris (pH 7.5), 50 mM NaCl,
10 mM MgCl.sub.2, and 0.5 mM MnCl.sub.2.
Preparing Nanoparticles Attached with Phi29 Polymerase
[0655] 300 .mu.L, of a stock solution of His-tagged phi29
polymerase (SEQ ID NO:8) (56 .mu.M) which is exonuclease minus
(flexible linker: GGGGSGGGGSAAAGSAA, SEQ ID NO:20) (stock solution
in: 10 mM Tris (pH 7.5) buffer with 100 mM NaCl, 1 mM DTT, 0.5%
Tween-20, 0.1 mM EDTA and 50% v/v glycerol) was buffer exchanged
into 100 mM Tris (pH 7.5) buffer with 300 mM NaCl using an NAP-5
column.
[0656] C8 Nanoparticles (160 .mu.L, 4.9 .mu.M in 50 mM borate
buffer pH 8.0) was concentrated to approximately 30 .mu.L by
ultrafiltration (VivaSpin, at 100K MWCO0, and mixed with the buffer
exchanged phi29 polymerase (440 .mu.L, 26.9 .mu.M in 100 mM Tris
(pH 7.5) buffer with 300 mM NaCl n a 1:15 molar ratio (nanoparticle
to polymerase). The resulting solution was incubated overnight at
4.degree. C., concentrated to .about.30 .mu.L, by ultra-filtration
with a 100K MWCO VivaSpin centrifugal concentrator, further
purified on SUPERDEX 200 column using 100 mM Tris (pH 7.5) buffer
with 300 mM NaCl as the eluent.
[0657] The conjugated nanoparticle-phi29 was assayed to determine
nucleotide incorporation activity and DNA binding by detecting FRET
signals. The incorporation reaction contained: 1.times. extension
buffer, 10 nM nanoparticle-phi29 conjugates (or non-conjugated
phi29 as a control), 150 nM oligonucleotide 221, and 20 .mu.M
dATP.
[0658] The results indicate that phi29 polymerase, attached to
nanoparticles, can incorporate nucleotides.
[0659] The binding reactions contained: 1.times. extension buffer,
C8 nanoparticles-phi29 conjugates (or phi29 non-conjugated),
oligonucleotide 199, and dATP. The binding reactions were serially
diluted. The results showed an increase in FRET acceptor signal
with an increase in the amount of the labeled oligonucleotide-199,
or a decrease in FRET donor signal.
Preparing GST-Nanoparticles Attached with Phi 29 Polymerase
[0660] C8 Nanoparticles (50 .mu.L, 3.5 .mu.M in 50 mM borate buffer
pH 8.0) was diluted with 100 .mu.L of 100 mM Tris buffer pH 7.5
with 300 mM NaCl and concentrated to .about.20 .mu.L, by
ultrafiltration (VivaSpin, 100K MWCO). The concentrated
nanoparticle solution was mixed with His-tagged-GST (184 .mu.L, 19
.mu.M in 50 mM Tris pH7.5 with 200 mM NaCl) in a 1:20 molar ratio
(nanoparticle to His-tagged-GST). The resulting solution was
incubated at room temperature for 5 hours. Phi29 polymerase (SEQ ID
NO:8) (60 .mu.L, 14.5 .mu.M in 100 mM Tris (pH 7.5) buffer with 300
mM NaCl) was added to the nanoparticles in a 5:1 molar ratio (phi29
to nanoparticle). The resulting solution was incubated overnight at
4.degree. C., concentrated to .about.30 .mu.L by ultra-filtration
with 100K MWCO VivaSpin centrifugal concentrator, purified on a
SUPERDEX 200 column using 100 mM Tris (pH 7.5) buffer with 300 mM
NaCl as the eluent.
[0661] The conjugated GST-nanoparticle-phi29 were assayed to
determine template extension activity and DNA binding by detecting
FRET signals. The incorporation reaction contained: 1.times.
extension buffer, 10 nM nanoparticle-phi29 conjugates (or
non-conjugated phi29 as a control), 150 nM oligonucleotide 221, and
20 .mu.M dATP.
[0662] The results indicated that phi29 polymerase, attached to
GST-treated nanoparticles, can incorporate nucleotides.
[0663] The binding reactions contained: 1.times. extension buffer,
C8 nanoparticles-phi29 conjugates (or phi29 non-conjugated),
oligonucleotide 199, and dATP. The binding reactions were serially
diluted. The results showed an increase in FRET acceptor signal
with an increase in the amount of the labeled oligonucleotide-199,
or a decrease in FRET donor signal.
Preparing UDG-ugi-Nanoparticles Attached with Phi29 Polymerase
[0664] His-tagged UDG protein (uracil DNA glycosylase) (500 .mu.L,
27 mM in 30 mM Tris buffer (pH 7.5) with 200 mM NaCl) was mixed ugi
(uracil-DNA glycosylase inhibitor) (50 .mu.L, 347 .mu.M in 30 mM
Tris buffer (pH 7.5) with 200 mM NaCl) in 1:1.2 molar ratio
(His-tagged-UDG to ugi protein), and incubated at 4.degree. C.
overnight.
[0665] C8 Nanoparticles (140 .mu.L, 4.9 .mu.M in 50 mM borate
buffer pH 8.0) was diluted by 200 .mu.L of 100 mM Tris buffer (pH
7.5) with 300 mM NaCl and concentrated to .about.30 .mu.L by
ultrafiltration (VivaSpin, 100K MWCO). The concentrated
nanoparticle solution was mixed with the His-tagged-UDG-ugi protein
conjugate (550 .mu.L, 24.7 .mu.M in 30 mM Tris buffer (pH 7.5) with
200 mM NaCl) in a 1:20 molar ratio (nanoparticle to
His-tagged-UDG-ugi) to prepare the UDG-ugi-nanoparticles. The
resulting solution was incubated at room temperature for 5
hours.
[0666] The phi29 polymerase (SEQ ID NO:8) was added (220 .mu.L,
15.4 .mu.M in 100 mM Tris (pH 7.5) buffer with 300 mM NaCl) in a
1:5 molar ratio (UDG-ugi-nanoparticle to phi29). The resulting
solution was incubated overnight at 4.degree. C., concentrated to
.about.30 .mu.L by ultra-filtration with 100K MWCO VivaSpin
centrifugal concentrator, and purified on a SUPERDEX 200 column
using 100 mM Tris (pH 7.5) buffer with 300 mM NaCl as the
eluent.
[0667] The conjugated UDG-ugi-nanoparticle-phi29 was assayed to
determine template extension activity and DNA binding by detecting
FRET signals. The incorporation reaction contained: 1.times.
extension buffer, 10 nM nanoparticle-phi29 conjugates (or
non-conjugated phi29 as a control), 150 nM oligonucleotide 199, and
20 .mu.M dATP.
[0668] The results showed that phi29 polymerase, attached to
UDG/ugi-treated nanoparticles, can incorporate nucleotides.
[0669] The binding reactions contained: 1.times. extension buffer,
C8 nanoparticles-phi29 conjugates (or phi29 non-conjugated),
oligonucleotide 199, and dATP. The binding reactions were serially
diluted. The results showed an increase in FRET acceptor signal
with an increase in the amount of the labeled oligonucleotide-199,
or a decrease in FRET donor signal.
Preparing BSA-Nanoparticles Attached with Phi29 Polymerase
[0670] Bovine serum albumin (BSA) (20 mg, catalog #B4287, Sigma)
was dissolved in 2 mL deionized water. The BSA solution (200 .mu.L,
10 mg/mL in H.sub.2O was mixed with DTT (8 .mu.L, 1M), and
incubated at room temperate overnight. The resulting solution was
purified on an NAP-5 column using deionized water as the
eluent.
[0671] A1 Nanoparticles (100 .mu.L, 1.0 .mu.M in 50 mM Tris buffer
(pH 8)) was diluted by 100 .mu.L of 100 mM Tris buffer (pH 7.5)
with 300 mM NaCl and concentrated to .about.30 .mu.L by
ultrafiltration (VivaSpin, 100K MWCO).
[0672] The concentrated nanoparticle solution was mixed with DTT
(1.0 .mu.L, 100 mM), and with the above-described BSA solution (27
.mu.L, 75.8 .mu.M in deionized water) in a 1:20 molar ratio
(nanoparticle to BSA). The resulting solution was incubated at room
temperature overnight, concentrated to .about.30 .mu.L by
ultra-filtration 100K MWCO VivaSpin centrifugal concentrator.
[0673] The concentrated nanoparticle-BSA solution was mixed with
the phi29 polymerase (SEQ ID NO:8) (48 .mu.L, 20.8 .mu.M in 100 mM
Tris (pH 7.5) buffer with 300 mM NaCl) in a 1:10 molar ration
(BSA-nanoparticles to phi29). The resulting solution was incubated
overnight at 4.degree. C., concentrated to .about.30 .mu.L by
ultra-filtration with 100K MWCO VivaSpin centrifugal concentrator,
and purified on a SUPERDEX 200 column using 100 mM Tris (pH 7.5)
buffer with 300 mM NaCl as the eluent.
[0674] The conjugated BSA-nanoparticle-phi29 was assayed to
determine template extension activity and DNA binding by detecting
FRET signals. The incorporation reaction contained: 1.times.
extension buffer, 10 nM nanoparticle-phi29 conjugates (or
non-conjugated phi29 as a control), 150 nM oligonucleotide 229, and
20 .mu.M dATP.
[0675] The results showed that phi29 polymerase, attached to
BSA-treated nanoparticles, can incorporate nucleotides.
[0676] The binding reactions contained: 1.times. extension buffer,
C8 nanoparticles-phi29 conjugates (or phi29 non-conjugated),
oligonucleotide 229, and dATP. The binding reactions were serially
diluted. The results showed an increase in FRET acceptor signal
with an increase in the amount of the labeled oligonucleotide-229,
or a decrease in FRET donor signal.
Example 5
Nucleotide Polymerization Using Polymerases Attached to
Nanoparticles
Materials
[0677] Nanoparticle shapes: A1 are spherical, and A2 and A4 are
rod-shaped. The spherical nanoparticles are about 8 nm in diameter,
and the rod-shaped ones are about 5.times.12 nm
(width.times.length). These nanoparticles have ligand coatings
which include: L-carnosine; dipeptides (e.g., His-Leu and Gly-His);
4-aminobenzophenone; citric acid; glycine;
tris(hydroxymethyl)phosphine; and amino-dPEG24-acid.
[0678] The nanoparticles were reacted with HRP, BSA, biotin, and
conjugated with one of three different phi29 polymerases: HP1,
HP1-Q380A or HP1-S388G.
[0679] HP1 is a 6.times.His-tagged phi29 polypeptide (`6.times.His`
disclosed as SEQ ID NO: 63) which is exonuclease-minus (SEQ ID
NO:9). HP1-Q380A is a 6.times.His-tagged phi29 mutant polypeptide
(`6.times.His ` disclosed as SEQ ID NO: 63) which is
exonuclease-minus (SEQ ID NO:10). HP1-S388G is a 6.times.His-tagged
phi29 mutant polypeptide (`6.times.His` disclosed as SEQ ID NO: 63)
which is exonuclease-minus (SEQ ID NO:11).
Attaching Nanoparticles Attached with Polymerases
[0680] Horseradish peroxidase (HRP; Invitrogen; Cat#01-2001)
reduction reaction: 3 mg of HRP was reacted with 150 mg of
Cleland's REDUCTACRYL Reagent (VWR; Cat# 80056-208) in 600 .mu.l of
50 mM sodium borate buffer, pH 8.2 for 45 minutes at room
temperature. The reaction was filtered through a Micro Bio-Spin
Empty Column (Bio-Rad; Cat#732-6204). 360 .mu.mol of spherical (A1)
or rod-shaped (A2 or A4) nanoparticles (1 eq.) were added in 50
.mu.l of 50 mM sodium borate buffer, pH 8.2 containing 5 .mu.L of
10% BSA (Invitrogen; Cat#P2489) for 1 hour at room temperature. The
reaction mixture was concentrated using a VivaSpin 500 100 KDa MWCO
ultrafiltration unit (VWR; Cat#14005-008) and washed (5 times) with
50 mM sodium borate buffer (pH 8.2). 3 mg of LC-sulfo-NHS-Biotin
(Molecular Biosciences; Cat#00598) was added in 300 .mu.l of 50 mM
sodium borate buffer, pH 8.2 for 30 mM at room temperature. The
reaction was filtered and washed again as above (5 times), diluted
with 100 .mu.l of sodium borate buffer containing 300 mM NaCl
(final concentration in a final reaction volume). Phi29 polymerase
(HP1 or HP1-Q380A (15 eq.) was added and incubated at 4.degree. C.
overnight. Reaction mixtures were purified using a SUPERDEX column
(VWR; Cat# 95017-068) eluting with a borate buffer containing 300
mM NaCl and concentrated to 1-2 .mu.M of conjugation products using
VivaSpin 500 100 KDa MWCO filters and centrifugation at
6,000.times.G.
Assay: Confirming Nanoparticles Are Conjugated with Polymerases
[0681] Assays were performed to confirm that the phi29 polymerases
were attached to the nanoparticles. The assay included 250 nM of
ALEXA FLUOR 647 labeled oligonucleotide:
TABLE-US-00008 (SEQ ID NO: 64)
(5'-TTATCTTTGTGGGTGACAGGTTTTTCCTGTCACC-3'-ALEXA FLUOR 647)
[0682] and 40 nM of the nanoparticle-polymerase conjugates in 50 mM
Tris, 50 mM NaCl and 10 mM MgCl.sub.2. The reaction was excited at
450 nm and emission (e.g., FRET) was detected as a ratio of
intensities at 605/670 (nanoparticle emission/ALEXA FLUOR 647
emission). Control nanoparticles were reacted with HRP, BSA,
biotin, and ALEXA FLUOR 647, but no phi29 polymerase.
[0683] The results showed that the control nanoparticles exhibit a
higher intensity peak compared to the nanoparticles conjugated with
phi29 polymerase and dye-labeled oligonucleotides at the same
concentration, and the signal intensity peaks at 670 nm. This
demonstrates that the nanoparticles are bound with the phi29
polymerase and with the ALEXA FLUOR 647-labeled
oligonucleotide.
Assay: Nucleotide Incorporation
[0684] Assays were performed to determine if the polymerases, which
are attached to the nanoparticles, could incorporate nucleotides.
The assay included 150 nM of a hairpin oligonucleotide,
fluorescein-labeled oligo-221:
[0685] (5'-TTTTTTTGCAGGTGACAGGTTTTTCCTGTCACC(fluorescein-T)GC-3')
(SEQ ID NO:28), and 40 nM of the nanoparticle-polymerase
conjugates, 20 .mu.M dATP in 50 mM Tris, 50 mM NaCl, and 10 mM
MgCl.sub.2 buffer. The reaction was excited at 490 nm and emission
was detected at 525 nm Control nanoparticles were reacted with HRP,
BSA, biotin, and ALEXA FLUOR 647, and no phi29 polymerase. The
results showed that the control nanoparticles exhibit baseline
intensity fluorescence levels compared to nanoparticles bound with
phi29 polymerase and dye-labeled oligonucleotides. These results
demonstrate that phi29 enzyme conjugated with a nanoparticle
retains its nucleotide incorporation activity.
Assay: Nucleotide Incorporation and DNA Extension
[0686] Assays were performed to determine if the polymerases, which
are attached to the nanoparticles, could polymerize nucleotides.
The assay included 50 mM Tris (pH 7.0), 2 mM MnCl.sub.2, 62.5-70 mM
NaCl (from the various nanoparticle-polymerase conjugate stocks),
0.5% BSA, 1 .mu.M each dNTP, 50 nM duplex (primer Top:
5'-GGTACTAAGCGGCCGCATG-3' (SEQ ID NO:29) with template C6gOV:
5'-TAAAGCCCCCCCATGCGGCCGCTTAGTACC-3' (SEQ ID NO:30) or template
T6gOV: 5'-TAAAGTTTTTTCATGCGGCCGCTTAGTACC-3') (SEQ ID NO:31), and
100 nM of HP1 phi29 polymerase (no nanoparticles), 100 nM
A1/HRP-HP1 (A1 spherical nanoparticles conjugated with phi29
polymerase), or 100 nM A4/HRP-HP1 (A4 rod-shaped nanoparticles
conjugated with phi29 polymerase). The reaction was initiated with
the addition of dNTPs (1 .mu.M) including dA4P labeled at the
terminal phosphate group with one ALEXA FLUOR 647, dG4P labeled at
the terminal phosphate group with ALEXA FLUOR 680, and dCTP labeled
at the nucleo-base with Cy5 dye (GE Healthcare Biosciences; catalog
#PA55021). The reaction was quenched with EDTA and analyzed by
electrophoresis in a 20% 7M urea denaturing gel followed by
fluorescence imaging. The results showed extension products from
phi29 polymerase in all three forms (unbound; bound to spherical
nanoparticles (A1); and bound to rod nanoparticles (A4)). The
results also showed extension products produced by phi29
polymerase, bound to nanoparticles, and incorporating fluorescent
dye labeled deoxynucleotide tetraphosphate molecules (dA4P and
dG4P).
Assay: Nucleotide Incorporation and DNA Extension
[0687] Assays were performed to determine if the polymerases, which
are attached to the nanoparticles, could polymerize nucleotides.
The assay included 50 mM Tris (pH 7.0), 2 mM MnCl.sub.2, 42.5-167.5
mM NaCl (from various nanoparticle-polymerase conjugate stocks),
0.5% BSA, 1 .mu.M each dNTP, 100 nM duplex (primer Top:
5'-GGTACTAAGCGGCCGCATG-3' (SEQ ID NO:29) with template C6gOV:
5'-TAAAGCCCCCCCATGCGGCCGCTTAGTACC-3' (SEQ ID NO:30) or template
A6A: 5'-GGTACTAAGCGGCCGCATGAAAAAAA-3') (SEQ ID NO:32),
and 200 nM of HP1 phi29 polymerase (no nanoparticles) or 200 nM of
A2/HRP-HP1 (rod-shaped nanoparticles conjugated with phi29
polymerase). The reaction was initiated with the addition of 1
.mu.M of dNTPs, including dCTP labeled at the nucleo-base with Cy5
dye (GE Healthcare Biosciences; catalog #PA55021) in combination
with dG4P labeled at the terminal phosphate group with ALEXA FLUOR
680 or with dGTP. For the A6A template, the reaction was conducted
in the presence of dU4P labeled at the terminal phosphate group
with ALEXA FLUOR 680 and labeled at the nucleo-base with ALEXA
FLUOR 647. The reactions were quenched with EDTA and analyzed by
gel electrophoresis in a 20% 7M urea denaturing gel followed by
fluorescence imaging. The results showed extension products from
phi29 polymerase in four forms: (1) unbound HP1 polymerase, (2) HP1
polymerase bound to A2 rod-shaped nanoparticles (A2-HP1), (3) HP1
polymerase mutant Q380A bound to A2 rod-shaped nanoparticles
A2-HP1-Q380A), and (4) HP1 polymerase mutant S388G bound to A2
rod-shaped nanoparticles (A2-S388G-Phi29). The results also showed
extension products produced by phi29 polymerase bound to
nanoparticles and incorporating deoxynucleotide tetraphosphate
molecules (dG4P) and fluorescent-dye labeled deoxynucleotide
tetraphosphate molecules (dG4P-Alexa 680).
Detecting FRET Signals in a Single Molecule Assay
[0688] Chambered glass cover slips were prepared to facilitate
injection and multiple experiments data collection from several
chambers using a single slide. The PEG-neutravidin glass coverslips
were functionalized as described by Taekjip Ha (2002 Nature
419:638-641) but using neutravidin instead of streptavidin.
Duplexes of primer/template strands were prepared by reacting 1
.mu.M of the template and 1 .mu.M of the primer strands in 1.times.
Duplexing buffer (50 mM Tris (pH 7.2), 10 mM NaCl).
TABLE-US-00009 Reaction 1: Primer: (SEQ ID NO: 33)
5'-TGATAGAACCTCCGTGT-3' Template: (SEQ ID NO: 34)
5'-GGAACACGGAGGTTCTATCATCGTCATCGTCATCGTCATCG-3'; Reactions 2 and 3:
Primer: (SEQ ID NO: 35) 5'-GGTACTAAGCGGCCGCATG-3' Template: (SEQ ID
NO: 36) 5'-TTTTACCCATGCGGCCGCTTAGTACC-3'; Reaction 4: Primer: (SEQ
ID NO: 37) 5'-GGTACTAAGCGGCCGC-dd-3' Template: (SEQ ID NO: 38)
5'-TTTTACCCATGCGGCCGCTTAGTACC-3'.
[0689] 10 nM of the nanoparticles (which were conjugated with phi29
polymerase mutant Q380A) were reacted with 300 nM of the DNA
primer/template duplex on ice for 30 minutes in 1.times.
pre-complexing buffer (50 mM Tris (pH 7.2), 100 mM NaCl) in a total
volume of 100 .mu.L. This reaction forms the binary complex of
nanoparticle/polymerase bound with template/primer.
[0690] The binary complex was diluted to a nanoparticle/polymerase
(100 .mu.M) and template/primer duplex (3000 .mu.M) to a ratio of
1:30. 100 .mu.L of the diluted binary complex was injected into a
chamber and was allowed to immobilize on the PEG-neutravidin
surface for 5 minutes. An extension mix was injected and the
reaction was allowed to occur for 2 minutes, followed by a 200
.mu.L of EDTA and an oxygen scavenging system containing buffer
wash. The extension mix consisted of 50 mM Tris (pH 7.2), 2 mM
MnCl.sub.2, 100 mM NaCl, 0.5% BSA and natural dNTPs (dGTP) or
dye-labeled dNTPs (dG4P labeled at the terminal phosphate group
with ALEXA FLUOR 680 and Cy5 base-labeled dUTP) at 1 .mu.M each.
The oxygen scavenging system consisted of 50 nM
protocatechuate-3,4-dioxygenase, 2.5 mM protocatechuic acid and 1
mM TROLOX (6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic Acid;
Hoffmann-LaRoche).
[0691] Four separate reactions were performed: Reaction #1 included
Cy5 base-labeled dUTP (GE Healthcare Biosciences; catalog
#PA55022). Reaction #2 included dGTPs and Cy5 base-labeled dUTP (GE
Healthcare Biosciences; catalog #PA55022). Reaction #3 included
dG4P labeled at the terminal phosphate group with ALEXA FLUOR 680
and Cy5 base-labeled dUTP (GE Healthcare Biosciences; catalog
#PA55022). Reaction #4 included dG4P labeled at the terminal
phosphate group with ALEXA FLUOR 680 and Cy5 base-labeled dUTP (GE
Healthcare Biosciences; catalog #PA55022), and the primer having a
ddG at the 3' end (negative control).
[0692] The data were collected on the single molecule detection
system, which included an ANDOR back-illuminated EMCCD camera
(iXonEM), and an inverted Olympus microscope (IX71), with a
100.times.TIRE objective. The samples were excited using a 405 nm
laser (Coherent; Cat#1069413) at 460 .mu.W, and the data was
collected at 100 ms integration time for 2000 frames and 3 to 5
consecutive streams were collected by moving to new fields of views
(FOVs). The signals were separated using dichroics (535 nm, 667 nm)
before forming an image on the camera.
[0693] FRETAN software (Volkov et al., U.S. Ser. No. 11/671,956)
was used to obtain donor and acceptor FRET traces. Custom-designed
MATLAB scripts were used to extract the data and obtain percent
FRET or percent activity data. Only acceptor donor type signals and
acceptors with S/N greater than 2 were counted for the percent
activity numbers.
Example 6
Preparation of Core-shell Nanoparticle CdSe/4CdS-3.5ZnS
Core Synthesis
[0694] Cores are prepared using standard methods, such as those
described in U.S. Pat. No. 6,815,064, the only change being that
the growth is halted at 535 nm emission. These cores were
precipitated and cleaned in the standard methods and resuspended
into hexane for use in the shell reaction.
Shell Synthesis:
[0695] A 1:1 (w:v) mixture of tri-n-octylphosphine oxide (TOPO) and
tri-n-octylphosphine (TOP) was introduced into a flask.
Tetradecylphosphonic acid (TDPA) was added to the flask in an
amount suitable to fully passivate the final material, as can be
calculated from the reaction scale and the expected final
nanoparticle size. The contents of the flask were heated to
125.degree. C. under vacuum and then the flask was refilled with
N.sub.2 and cooled.
[0696] Inside the glovebox, a solution of a suitable cadmium
precursor (such as dimethylcadmium or cadmium acetate) in TOP was
prepared in a quantity sufficient to produce a desired thickness of
shell, as can be calculated by one of ordinary skill in the art.
When a zinc shell was also desired, a solution of a suitable zinc
precursor (such as diethylzinc or zinc stearate) was prepared in
TOP in a quantity sufficient to produce the desired shell
thickness. Separately, a solution of trimethylsilylsulfide
[(TMS).sub.2S] in TOP was prepared in a quantity sufficient to
produce the desired shell thickness. Each of these solutions was
taken up in separate syringes and removed from the glove box.
[0697] Of the previously prepared core/hexane solution, 17 mL (at
an optical density of 21.5 at the band edge) was added to the
reaction flask and the hexane was removed by vacuum; the flask was
then refilled with N.sub.2. The flask was heated to the desired
synthesis temperature, typically about 200 to about 250.degree. C.
During this heat-up, 17 mL of decylamine was added.
[0698] The cadmium and sulfur precursor solutions were then added
alternately in layer additions, which were based upon the starting
size of the underlying cores. So this means that as each layer of
shell material was added, a new "core" size was determined by
taking the previous "core" size and adding to it the thickness of
just-added shell material. This leads to a slightly larger volume
of the following shell material needing to be added for each
subsequent layer of shell material.
[0699] After a desired thickness of CdS shell material was added,
the cadmium precursor solution was replaced with the zinc precursor
solution Zinc and sulfur solutions were then added alternately in
layer additions until a desired thickness of ZnS was added. A final
layer of the zinc solution was added at the end, the reaction flask
was cooled, and the product was isolated by conventional
precipitation methods.
Example 7
Exchange Process Using Dipeptide Ligands and Butanol as a
Cosolvent
[0700] Core/shell nanocrystals (quantum dots) were prepared by
standard methods, and were washed with acetic acid/toluene several
times, and suspended in hexanes. 10 nmol of core/shell nanocrystals
were suspended in 40 mL hexane. This was mixed with 10 mL of a 300
mM solution of carnosine and 10 mL of 1 M sodium carbonate
solution. n-Butanol (14 mL) was added, and the vessel was flushed
with argon. The mixture was mixed vigorously overnight at room
temperature. The mixture was then heated and allowed to cool to
room temperature. The aqueous phase was then removed and filtered
through a 0.2/0.8 micron syringe filter.
[0701] Excess carnosine was removed by dialyzing against 3.5 L of
25 mM NaCl for one hour. The solution was concentrated to 1 mL
using a 10K MWCO (10,000 molecular weight cut-off) Amicon
centricon. A solution was then prepared with 568 mg of His-Leu
dipeptide plus 212 mg of Gly-His dipeptide in 9 mL sodium carbonate
solution, and this solution was combined with the aqueous solution
of quantum dots. This mixture was stirred overnight at room
temperature. The mixture of water-soluble quantum dots was then
dialyzed against 3.5 L of 25 mM NaCl for one hour.
[0702] To crosslink the peptide ligands (clarify)A solution of 0.5
mM 4-aminobenzophenone in ethanol was then added to the aqueous
quantum dots mixture, and the mixture was irradiated at 365 nm for
4 hours to effect reaction of the aminobenzophenone with the
surface molecules on the quantum dots. To this, 5 mmol of THP
(tris(hydroxymethyl)phosphine) was added, and the mixture was
stirred at RT overnight, to induce crosslinking. Another 5 mmol of
THP was added, and again the mixture was stirred overnight at RT.
Another 5 mmol of THP was added the next day, along with 300
micromoles of PEG1000-COOH. This was mixed overnight at room
temperature, then another 5 mmol of THP was added along with 30
mmol of glycine, and the mixture was stirred overnight at RT.
[0703] The material was purified by dialysis using the 10K MWCO
Amicon centricon, and was washed with 50 mM borate buffer (pH 9).
The final material was dispersed into 50 mM borate buffer to a
final concentration of 2.5 micromolar for storage.
Example 8
Exchange Process using Trithiol Ligands
[0704] A solution of hydrophobic phosphonate-coated quantum dots in
organic solvent (e.g. toluene, chloroform, etc) with a
concentration of between about 0.1 and 10 micromolar quantum dots
was prepared. Approximately 1000 to 1000000 equivalents of a
suitable trithiol ligand was added, optionally as a solution in a
suitable organic solvent (e.g. acetone, methanol, etc). The
reaction mixture was stirred for 1-48 hours and then the solution
was basicified by addition of an organic base (e.g.
tetramethylammonium hydroxide, tetrabutylammonium hydroxide, etc).
After a shorter second stirring period, water or aqueous buffer was
used to extract the dots with hydrophilic ligands. The aqueous
solution was washed with additional organic solvent (e.g. toluene,
chloroform, etc) and purified by filtration.
Example 9
Two-Step Ligand Exchange
Process for Exchanging Phosphonate Ligands with Sulfonate
(Triflate) Ligands
[0705] A nanoparticle comprising a core/shell nanocrystal having
TDPA ligands on its surface is dissolved in dichloromethane, and
excess TMS triflate is added to it. After 1-2 hours at room
temperature, analysis indicates that the TDPA ligands have been
removed, and the nanoparticle remains dispersed in the solvent. It
is dialyzed against dichloromethane using a 10K MWCO (10,000
molecular-weight cut-off) dialysis membrane to remove excess TMS
triflate and the TMS-TDPA produced by the reaction of TMS triflate
with the TDPA ligands. This produces a solution/suspension of
nanoparticles comprising triflate ligands on the surface of
nanocrystals. These triflate-containing nanoparticles are soluble
in many organic solvents, but may not be readily soluble in
hexanes, depending upon the complement of ligands present.
Two-Step Process for Exchanging Sulfonate (Triflate) Ligands with
PEG Conjugated Dithiol (DHLA) Ligands Using n-Butanol as an
Intermediate Ligand and DMF as a Co-solvent
[0706] The triflate-containing nanoparticle solution, described
above, can be contacted with excess n-butanol in acetonitrile,
using DMF as a co-solvent, to provide an intermediate nanoparticle
believed to comprise butanol ligands in place of the triflates
which were on the nanoparticle. This intermediate nanoparticle can
be isolated from the medium, or it can be further modified without
isolation. This intermediate nanoparticle is contacted with an
excess of a dihydrolipoic acid-PEG conjugate of this formula:
##STR00011##
[0707] where n is 1-100.
[0708] The product is a water-soluble, stable nanoparticle. It can
be collected by extraction into a pH 9 buffer, and isolated by
conventional methods, including dialysis with a 10K MWCO dialysis
filter, or by size exclusion (gel filtration) chromatography.
Two-Step Process for Exchanging Sulfonate (Triflate) Ligands with
Nucleophilic Reactanct Group Containing Ligands Using n-Butanol as
an Intermediate Ligand and DMF as a Co-solvent
[0709] The triflate-containing nanoparticle solution from can be
contacted with excess n-butanol in acetonitrile, using DMF as a
co-solvent, to provide an intermediate nanoparticle believed to
comprise butanol ligands in place of the triflates which were on
the nanoparticle. This intermediate nanoparticle can be isolated
from the medium, or it can be further modified without isolation.
To further modify it, it is treated with a new ligand containing at
least one nucleophilic reactant group: suitable ligands include
HS--CH.sub.2--CH.sub.2-PEG; aminomethyl phosphonic acid;
dihydrolipoic acid; omega-thio-alkanoic acids, and
carboxymethylphosphonic acid. The mixture is then treated with
TMEDA (tetramethylethylene diamine), and monitored until triflate
is displaced, then the nanocrystal product is extracted into pH 9
buffer and purified by conventional methods.
Process for Exchanging Sulfonate (Triflate) Ligands with
Carboxylate Functionalized Dithiol (DHLA) Ligands
[0710] The triflate-containing nanoparticle is contacted with neat
dihydrolipoic acid (DHLA) for an hour at room temperature, and is
then dispersed into pH 9 buffer and isolated by conventional
methods. This provides a nanoparticle having carboxylate groups to
provide water solubility, and having two thiol groups binding the
carboxylate to the nanocrystal surface. The product is water
soluble and stable in aqueous buffer. It provides good colloidal
stability, and a moderate quantum yield. This composition
containing DHLA as a ligand contains free carboxyl groups which can
be used to attach other groups such as a PEG moiety, optionally
linked to a functional group or a biomolecule. The same reaction
can be performed to replace triflate groups on a nanoparticle with
thioglycolic acid (HS--CH.sub.2--COOH) ligands. This provides a
highly stabilized nanoparticle which produces a high quantum yield,
but has lower colloidal stability than the product having DHLA on
its surface.
Process for Exchanging Sulfonate (Triflate) Ligands with Amine
Ligands
[0711] The triflate-containing nanoparticle is dispersed in
dichloromethane plus hexanes, and an alkylamine is added. Suitable
alkylamines are preferably primary amines, and include, e.g.,
H.sub.2N--(CH.sub.2).sub.r-PEG (r=2-10), p-aminomethylbenzoic acid,
and lysine ethyl ester. After an hour at room temperature, the
exchange process is completed, and the nanoparticle product can be
isolated by conventional methods.
Process for Pre-treating Phosphonate Coated Nanocrystals with
Toluene Acetic Acid to Remove Impurities Prior to Exchanging with
Sulfonate (Triflate) Ligands
[0712] TDPA-covered nanocrystals were synthesized which emitted
light at 605 nm and had shells of CdS and of ZnS. These when
treated with 200,000 equivalents of TMS triflate in hexanes did not
produce a precipitate. This was attributed to excess TDPA-derived
impurities in the nanocrystals. This was alleviated by dissolving
the nanocrystals in toluene-acetic acid and precipitating them with
methanol, to remove TDPA salts or related by-products. The
resultant TDPA nanocrystals behaved as described above,
demonstrating that impurities were causing the nanocrystals to
behave differently when made with excess TDPA present, and that
those impurities can be removed by precipitation under conditions
better suited to dissolving TDPA-related impurities.
Process for Exchanging Activated (Sulfonate Coated) Nanocrystals
with Dithiol (DHLA) Ligands Using Butanol, DMF or Isopropyl Alcohol
as Dispersants
[0713] Three different methods of depositing the DHLA ligands were
employed, each of which was considerably more rapid than the
classic approach using non-activated dots. In the first approach,
the activated dot powder was dispersed in butanol and stirred with
DHLA, then precipitated with hexane and collected in aqueous
buffer. In the second approach, the activated dot powder was
dispersed in dimethylformamide (DMF) and stirred with DHLA, then
precipitated with toluene and collected in aqueous buffer. In the
third approach, the activated dot powder was stirred as a slurry in
neat DHLA, then dispersed in isopropyl alcohol, precipitated with
hexane, and collected in aqueous buffer and purified with a
filtration membrane.
[0714] These three samples, plus a sample derived from
non-activated dots were diluted to 60 nM for a colloidal stability
challenge, wherein the absorbance is monitored over the course of
days to watch for precipitation. Samples 1 (butanol-mediated), 2
(DMF-mediated), and 4 (classic) all precipitated on day 3 or 4 of
the stability challenge, but sample 3 (neat DHLA) lasted twice as
long, coming out of solution on day 7. HPLC measurements indicated
that the DHLA-coated particles produced from activated dots showed
even less aggregation than the classic DHLA particles made by the
displacement of TOPO or pyridine ligands from nanocrystals. Thus
the invention provided rapid reactions leading to improved
colloidal stability and comparable or lower aggregation levels than
conventional ligand replacement methods of putting DHLA on a
nanocrystal. Similar treatment with other thiol ligands like
mercaptoundecanoic acid (MUA) or the PEGylated thiol also provided
water-dispersible nanocrystals. Reacting triflate-coated
nanoparticles with MUA or PEG-thiol gave particles which were
readily dispersible in water, indicating that ligand exchange had
occurred. The observed quantum yield was over 70% in each case.
Process for Exchanging Activated (Sulfonate Coated) Nanocrystals
with Hydrophilic Phosphonate Ligands
[0715] Triflate-coated dots were dispersed in butanol and then
stirred with phosphonoacetic acid. Triethylamine was added to form
the triethylammonium salt of both the phosphonate and carboxylate
functionalities, and then pH 9 aqueous borate buffer was added to
extract the hydrophilic particles. The result was a bright orange
aqueous dispersion of quantum dots, with no remaining color
observed in the butanol layer. The particles were purified by
centrifugal filtration and the quantum yield was measured to be
72%. Multiple batches of particles were prepared and remained in
solution through room temperature storage for at least eight weeks.
The same method can be successfully employed with DHLA, MUA, and
PEGylated thiol ligands.
Process for Exchanging Activated (Sulfonate Coated) Nanocrystals
with a Variety of Hydrophilic Phosphonate Ligands via Biphasic
Exchange
[0716] Using a biphasic exchange method, dispersing the quantum
dots in organic solvents such as chloroform and the exchangeable
ligands in aqueous solution, quantum dots were made water soluble
and stable after ligand exchange with
N,N-Bis(phosphonomethyl)glycine (1) or phosphonoacetic acid (2). In
a typical bi-phasic ligand exchange experiment, 1 nmol of quantum
dots were dispersed in 1 mL of chloroform and placed in a vial with
2 mL of 300 mM phosphonic acid in basic buffer and the mixture was
rapidly stirred at room temperature for 2 days. Quantum yields as
high as 53% were achieved; however the quantum yields achieved were
dependent on core-shell batch employed, probably as a result of
variable amounts of long-chain alkyl phosphonates remaining on the
nanocrystal surface post-ligand exchange. This demonstrated that
complete removal of TDPA from nanocrystals is important for
successful modification of the surface. Though the dots were
rendered water stable by the above phosphonate-containing ligands,
they were not successfully modified with PEG2000-diamine using
standard EDC condensation chemistry.
##STR00012##
[0717] Nanocrystals coated with compounds 1, 2, or 3 were readily
prepared by this method, as well as nanocrystals having a mixture
of compounds 1 and 2, or 1 and 3, or 2 and 3. In each case, the
nanocrystals were stable, bright and water-soluble. Using mixed
ligands, it was found that PEGylation (with PEG2000-diamine using
standard EDC condensation chemistry) could be achieved with these
phosphonate-containing ligands to produce highly stable, bright,
water soluble nanoparticles. These nanoparticles can be further
stabilized by at least partially cross-linking the ligands using a
diamine such as putrescine, cadaverine, 1,2-diaminoethane,
bis(hexamethylene)triamine, PAMAM dendrimer, and cystamine.
Two-Step Ligand Exchange Process with Tridentate Thiol Ligands
[0718] Triflate exchange step was performed following the procedure
described above. Next, the triflate nanoparticles were dispersed in
organic solvent (e.g. toluene, chloroform, etc) with a
concentration of between about 0.1 and 10 micromolar quantum dots.
Approximately 1000 to 1000000 equivalents of a suitable tridentate
thiol ligand was added, optionally as a solution in a suitable
organic solvent (e.g. acetone, methanol, etc). The reaction mixture
was stirred for 1-48 hours and then the solution was basicified by
addition of an organic base (e.g. tetramethylammonium hydroxide,
tetrabutylammonium hydroxide, etc). After a shorter second stirring
period, water or aqueous buffer was used to extract the dots with
hydrophilic ligands. The aqueous solution was washed with
additional organic solvent (e.g. toluene, chloroform, etc) and
purified by filtration.
Example 10
Functionalized Ligands on Nanoparticles
General Core Reaction Procedure
[0719] Into a 25 mL 3 neck flask with 14/20 joints, 1.575 g of
>99% tri-n-octylphosphine oxide (TOPO) was weighed. To this,
1-1000 micromoles of a bi-functional phosphonate ligand was added.
A stir bar was added to this flask. The flask was connected to an
inert atmosphere manifold and evacuated thoroughly, then refilled
with nitrogen. A solution of a suitable cadmium salt in
tri-n-octylphosphine (TOP) was prepared with a concentration of 0.5
mol Cd per kg solution. A desired amount of cadmium as required for
growth of nanoparticles of a desired size was extracted from this
solution, diluted with 0.9 mL of additional TOP, and added to the
flask. The flask was stirred and heated to .about.200-350.degree.
C. under nitrogen flow. A 1 molar solution of selenium in TOP was
prepared and a desired amount as required for growth of
nanoparticles of a desired size was added to the solution,
optionally with addition of a reaction promoter to achieve desired
levels of particle nucleation. One minute after the reaction was
initiated by adding these final reagents, a 20 microliter sample
was removed from the reaction, mixed with 5 mL of hexane, and an
emission spectrum was collected. This aliquot removal and
measurement process was repeated after 2, 3, 4, 5, 6, 7, 8, 10, 12,
and 14 minutes. After 14 minutes, the reaction was rapidly cooled
and the products were isolated by methods understood in the
art.
Control Core Reaction with tetradecylphosphonic acid [TDPA]
##STR00013##
[0720] The core reaction using TDPA as the phosphonate ligand was
demonstrated as a control reaction. This reaction proceeded with an
initial emission reading at 1 minute of .about.490 nm and
progressing to a final emission reading of .about.544 nm at 14
minutes. The full width at half maximum intensity (FWHM) never got
above 28 nm. The final "growth solution" of the cores was
yellow/light orange in appearance by eye. The aliquoted samples of
this reaction remained dispersed and clear solutions in hexane.
Core Reaction with 11-methoxy-11-oxo-undecylphosphonic acid
##STR00014##
[0721] The reaction using 11-methoxy-11-oxo-undecylphosphonic acid
as the phosphonate ligand proceeded with an initial emission
reading at 1 minute was .about.560 nm; this was redder than the
final emission of the control reaction. The final emission of this
reaction was .about.610 nm. The FWHM of this reaction started at
.about.35 nm and steadily got more broad throughout the reaction
for a final FWHM of .about.50 nm.
[0722] The aliquoted samples were not soluble in hexane, and became
almost instantly flocculated and settled to the bottom of the vials
within minutes.
Core Reaction with 6-ethoxy-6-oxohexylphosphonic acid
##STR00015##
[0723] The core reaction using 6-ethoxy-6-oxohexylphosphonic acid
as the phosphonate ligand had an initial emission reading at 1
minute of .about.560 nm and a final emission reading of .about.606
nm. The FWHM of this reaction started out at 1 minute at .about.43
nm and narrowed to a final FWHM of .about.40.5 nm.
[0724] The solubility of the aliquoted samples was observed. The
hexane samples were immediately cloudy, however the flocculation
did not settle to the bottom of the vials. Six of the aliquoted
samples were centrifuged and the resulting clear, colorless
supernatants were discarded. The pellets were soluble in toluene,
dichloromethane (CH.sub.2Cl.sub.2), dimethylformamide (DMF), and
methanol (MeOH). The pellets were not soluble in water, 50 mM
borate buffer at pH=8.3 or hexane.
[0725] Particles synthesized in the presence of TDPA are soluble in
hexane, toluene, CH.sub.2Cl.sub.2, DMF and hexane. The
6-ethoxy-6-oxohexylphosphonic acid itself is not soluble in hexane,
and neither were the resulting particles from this reaction,
suggesting that the ligand was indeed coating the nanoparticles--a
suggestion which was confirmed with infrared and NMR spectroscopy
indicating the expected ester functionality. Using a solvent system
of toluene as the solubilizing solvent and hexane as a
precipitating solvent, a pellet can be formed along with a clear,
colorless supernatant. The resulting pellet can be re-solubilized
in toluene. This resulting toluene solution allowed an absorbance
spectrum of these cores to be obtained.
[0726] These data suggest that quantum confined nanoparticles have
been formed with 6-ethoxy-6-oxohexylphosphonic acid on the particle
surface. The resulting core particles were taken further into a
shell reaction.
Shell Reaction Procedure using 6-ethoxy-6-oxohexylphosphonic acid
Core precipitation
[0727] Three (3) mL of growth solution cores using
6-ethoxy-6-oxohexylphosphonic acid ligand (prepared according to
the procedure of Example 4) was solubilized into 3 mL toluene in a
250 mL conical bottom centrifuge tube. A total of 135 mL of hexane
was added to precipitate the cores. The tube was centrifuged at
3000 RPM for 5 min. The resulting clear, colorless supernatant was
discarded and the pellet was dispersed into 3 mL of toluene.
Shell Reaction
[0728] Into a 25 mL 3 neck flask with 14/20 joints, 1.4 g of TOPO
was weighed. To this, 1-1000 mg of 6-ethoxy-6-oxohexylphosphonic
acid was added. A stir bar and 1.4 mL of TOP were added to the
flask. The flask was connected to an inert atmosphere manifold and
evacuated thoroughly, then refilled with nitrogen. 2.6 mL of the
toluene solution of cores was added to the flask and the flask was
warmed and evacuated to remove the toluene, then refilled with
nitrogen. Approximately 1 mL of a suitably high-boiling amine was
added to the flask and the flask was heated to 200-350.degree. C.
Solutions of suitable cadmium and zinc precursors in TOP were
prepared with a concentration of 0.5 mol metal ion per kg of
solution. A solution of 10% trimethylsilylsulfide in TOP by weight
was prepared as well. The metal and sulfur precursor solutions were
added slowly over the course of several hours to minimize
additional nanoparticle nucleation. Sufficient shell precursors
were added to grow a shell of a desired thickness, as can be
calculated by one of ordinary skill in the art. When the desired
shell thickness was reached, the reaction was cooled and the
core/shell nanoparticles were isolated by conventional means.
Aliquots taken during the reaction permitted monitoring of the
progress of the shell reaction. It was observed that the emission
maximum after heating but before addition of shell precursors was
very similar to that of the initial cores (-600 nm), suggesting
that the bi-functional phosphonate was sufficiently strongly
coordinated to the nanoparticle surface to minimize Ostwald
ripening. A red-shift during shell precursor addition of .about.50
nm was typical of a shell as deposited in a reaction employing
TDPA, suggesting that the shell formed as expected. In addition,
the nanoparticle solution became much more intensely emissive, as
would be expected of successful deposition of an insulating shell.
Infrared and NMR spectroscopy confirmed that the functionalized
phosphonates were present on the nanoparticles.
Example 11
Conjugates of Active Polymerase and Nanocrystals
[0729] Preparing Phi29 Polymerase Conjugated with UDG-ugi-C8
Nanoparticles
[0730] His-tagged UDG protein (uracil DNA glycosylase) (2.02 mL,
53.4 .mu.M in 30 mM Tris buffer (pH 8) with 200 mM NaCl, 0.5 mM
EDTA and 1 mM DTT) was mixed with ugi (uracil-DNA glycosylase
inhibitor) (748 .mu.L, 173 .mu.M in 10 mM Tris (pH 7.5) buffer with
100 mM NaCl, 4 mM DTT, 0.5% v/v Tween-20, 0.1 mM EDTA and 50% v/v
glycerol) in 1:1.2 molar ratio (His-tagged-UDG to ugi protein), and
incubated at 4.degree. C. overnight. The resulting
His-tagged-UDG-ugi protein complex was stored at 4.degree. C.
without further purification for future use.
[0731] C8 Nanoparticles (100 .mu.L, 5.3 .mu.M in 50 mM borate
buffer pH 8 with 1.0 M Betaine which is frozen at -20.degree. C.
immediately after synthesis) was thawed and mixed with the
His-tagged-UDG-ugi protein complex (132 .mu.L, 40.0 .mu.M in 30 mM
Tris buffer (pH 8) with 200 mM NaCl, 0.5 mM EDTA and 1 mM DTT) and
389 .mu.L of 100 mM Tris (pH 7.5) buffer with 300 mM NaCl and 1 mM
DTT in a 1:10 molar ratio (nanoparticle to His-tagged-UDG-ugi). The
solution was incubated for 1 hour at 4.degree. C. The resulting
UDG-ugi-nanoparticles solution was mixed with stock His-tagged
HP1-Phi29 mutant polymerase (SEQ ID NO:9) (115 .mu.L, 46 .mu.M in
10 mM Tris (pH 7.5) buffer with 100 mM NaCl, 4 mM DTT, 0.5% v/v
Tween-20, 0.1 mM EDTA and 50% v/v glycerol) in a 1:10 molar ratio
(nanoparticle to polymerase). The conjugation solution was
incubated overnight at 4.degree. C., centrifuged for 5 minutes at
16.8K rcf, purified on Ni.sup.2+-NTA Agarose column using 100 mM
Tris (pH 7.5) buffer with 300 mM NaCl and 1 mM DTT as the eluent.
The purified conjugate solution was centrifuged for 5 minutes at
16.8K rcf, transferred into a 10K MWCO dialysis cassette, then
dialyzed into 50 mM Tris buffer pH7.5 with 150 mM NaCl, 0.2 mM
EDTA, 0.5% v/v Tween-20, 5 mM DTT and 50% v/v glycerol. The
resulting UDG-ugi-nanoparticle-HP1-Phi29 conjugate was assayed to
determine concentration, template extension activity, and active
number of Phi29 per conjugate and DNA binding by FRET signal
detection (see Table 1 below).
TABLE-US-00010 TABLE 1 Conjugate Activity C8-UDG-ugi-HP1 Phi29
mutant 0.50 base/sec/conj Stock HP1 Phi29 mutant 0.10
base/sec/enz
[0732] The FRET signals from the mutant Phi29-nanoparticle
conjugate binding to oligonucleotide 199 labeled at the 3' end with
ALEXA FLUOR 647 (conjugate and C8 dot concentration: 10 nM;
AF647-3'-oligo 199 concentration: 1000 nM) were compared to
non-conjugated C8 nanoparticles. The 605/670 ratio is the
fluorescence intensity at 605 nm divided by fluorescence intensity
at 670 nm with 450 nm excitation for both the conjugate and the
unconjugated C8 nanoparticles. The low 605/670 ratio for the
conjugate indicated the conjugate binding to the dye labeled oligo
and showing FRET signal.
[0733] The active number of polymerases per conjugate for
C8-UDG-ugi-HP1 Phi29 mutant conjugate are shown in the graph and
tables above.
Preparing B103 Polymerase Conjugated with UDG-ugi-C8
Nanoparticles
[0734] His-tagged UDG protein (uracil DNA glycosylase) (2.02 mL,
53.4 .mu.M in 30 mM Tris buffer (pH 8) with 200 mM NaCl, 0.5 mM
EDTA and 1 mM DTT) was mixed with ugi (uracil-DNA glycosylase
inhibitor) (748 .mu.L, 173 .mu.M in 10 mM Tris (pH 7.5) buffer with
100 mM NaCl, 4 mM DTT, 0.5% v/v Tween-20, 0.1 mM EDTA and 50% v/v
glycerol) in 1:1.2 molar ratio (His-tagged-UDG to ugi protein), and
incubated at 4.degree. C. overnight. The resulting
His-tagged-UDG-ugi protein complex was stored at 4.degree. C.
without further purification for future use.
[0735] C8 nanoparticle s (100 .mu.L, 4.5 .mu.M in 50 mM borate
buffer pH 8.0 with 1.0 M Betaine which were frozen at -20.degree.
C. immediately after synthesis) was thawed and mixed with stock
His-tagged HP1-B103 polymerase (SEQ ID NO:4) (40.5 .mu.L, 111 .mu.M
in 10 mM Tris (pH 7.5) buffer with 100 mM NaCl, 4 mM DTT, 0.5% v/v
Tween-20, 0.1 mM EDTA and 50% v/v glycerol) and 309 .mu.L of 100 mM
Tris (pH 7.5) buffer with 300 mM NaCl and 1 mM DTT in a 1:10 molar
ratio (nanoparticle to polymerase). The conjugation solution was
incubated overnight at 4.degree. C. The resulting B103
polymerase-C8 nanoparticle conjugate was mixed with the
His-tagged-UDG-ugi protein complex (112 .mu.L, 40.0 .mu.M in 30 mM
Tris buffer (pH 8) with 200 mM NaCl, 0.5 mM EDTA and 1 mM DTT) in a
1:10 molar ratio (nanoparticle to His-tagged-UDG-ugi). The mixture
was incubated for 5 hours at 4.degree. C. to prepare the
UDG-ugi-nanoparticle s-B103 conjugate. The resulting conjugate
solution was centrifuged for 5 minutes at 16.8K rcf, purified on
Ni.sup.2+-NTA agarose column using 100 mM Tris (pH 7.5) buffer with
300 mM NaCl and 1 mM DTT as the eluent, centrifuged and transferred
into a 10K MWCO dialysis cassette. The conjugate was dialyzed into
50 mM Tris buffer pH 7.5 with 150 mM NaCl, 0.2 mM EDTA, 0.5% v/v
Tween-20, 5 mM DTT and 50% v/v glycerol. The resulting
UDG-ugi-nanoparticle--HP1-B103 conjugate was assayed to determine
concentration, template extension activity, active number of Phi29
per conjugate and DNA binding by FRET (see Table 2 below).
TABLE-US-00011 TABLE 2 Conjugate Activity C8-HP1 B103-UDG-ugi 1.41
base/sec/conj Stock HP1 B103 0.41 base/sec/enz
[0736] The FRET signals from the B103-nanoparticle conjugate
binding to oligonucleotide 199 labeled at the 3' end with ALEXA
FLUOR 647 (conjugate and C8 dot concentration: 10 nM;
AF647-3'-oligo 199 concentration: 1000 nM) were compared to
non-conjugated C8 nanoparticles. The 605/670 ratio is the
fluorescence intensity at 605 nm divided by fluorescence intensity
at 670 nm with 450 nm excitation for both the conjugate and the
unconjugated C8 nanoparticles. The low 605/670 ratio for the
conjugate indicated the conjugate binding to the dye labeled oligo
and showing FRET signal.
[0737] Active number of polymerase per conjugate for C8-HP1
B103-UDG-ugi conjugate are shown in the graph and tables above.
Example 12
FRET Detection of Incorporated Nucleotides Using Polymerase-Dye
Conjugates
Preparing PEG-Biotin Surfaces:
[0738] Glass coverslips surfaces were plasma cleaned and treated
with a mixture of poly-ethyleneglycol (PEG) and biotin-PEG to
produce a low density biotin surface with a PEG coating to prevent
non-specific background of proteins and macromolecules.
Fluidic Chamber Assembly:
[0739] Fluidic cassettes were assembled with glass coverslips to
create fluidic chambers capable of containing approximately 2 .mu.l
of fluid.
Attaching Biotinylated DNA to Low Density Peg-Biotin Surfaces:
[0740] Streptavidin protein was diluted to 200 .mu.pM in Incubation
Buffer (50 mM NaCl; 50 mM Tris-Cl pH=7.5; 0.5% BSA). Diluted
streptavidin was flowed into fluidic chamber and streptavidin was
incubated for 10 minutes. Chambers were washed 1.times. with 1 ml
Incubation Buffer. Biotinylated-DNA templates were diluted to 200
.mu.pM in Incubation Buffer and allowed to bind for 5 minutes.
Surfaces were washed 1.times. with 1 ml Incubation Buffer.
SA-Polymerase Preparation:
[0741] Streptavidin was labeled with Cy3 (Life Technologies).
Streptavidin-Cy3 was mixed with a biotinylated mutant Phi29
(b-Phi29) (SEQ ID NO:12) at a 1:1 ratio of
SA-protein:biotinylated-Phi29 in 1.times.PBS.
SA-Cy3-b-Phi29 Binding to Templates:
[0742] SA-Cy3-b-Phi29 was diluted to 1 nM in binding buffer (50 mM
Tris-Cl; pH=7.5; 0.3% BSA; 100 mM NaCl). Conjugates were flowed
into fluidic chamber which were previously loaded with DNA
templates on the surface. Surfaces were incubated for 5 minutes
with 1 nM SA-Cy3-b-Phi29. Surfaces were washed with 1.times.1 ml
Incubation Buffer.
Fluorescence Imaging:
[0743] The Olympus microscope body was outfitted with a TIRF
objective lens (100.times.; 1.45 NA). The excitation light passes
through an excitation filter (EX FT-543/22), and dichroic mirror
(DM-532) and the sample is epi-illuminated (Coherent) using TIR at
typically 100 W/cm.sup.2. Upon excitation, resulting
epifluorescence emission passes an emission filter (EM FT-540LP)
and the resulting emission is split into three paths (triview)
using 2 dichroic mirrors and the appropriate bandpass filters for
the dye sets of choice. The emission was imaged on a CCD camera.
Images were collected at a frame rate of approximately 30 ms.
Images depict single DNA strands complexed with single
SA-Cy3-b-Phi29 conjugates (donor molecules in this example) and
FRET signals from acceptor species (hexaphosphate nucleotides
labeled with ALEXA FLUORE 647, 676 or 680) bound in the enzyme
active site.
Nucleotide Polymerization with SA-DonorDye-Phi29 or B103
Conjugates:
Homopolymer Template Sequence:
[0744] Hexa-phosphate dye-labeled nucleotides were diluted to 200
nM in extension buffer. (50 mM MOPS pH=7.1; 75 mM potassium acetate
(pH=7.0); 0.3% BSA; 1 mM MnCl.sub.2; 300 nM procatuate dioxygenase;
4 mM 3,4 dihydroxyl benzoic acid; 1 mM 2-nitrobenzoic acid; 400 mM
1,2 phenylenediamine; 100 mM ferrocene monocarboxylic acid; 0.02%
cyclooctratetraene; 6 mM Trolox). Nucleotide mix was flowed into
channel with SA-Cy3-b-Phi29 or SA-Cy3-b-B103 (SEQ ID NO:5) bound to
DNA template and images were recorded for approximately 2 minutes
at approximately 20 ms frame rates.
[0745] As one example, the DNA template sequence extends with the
following sequence (G).sub.15 (A).sub.15(G).sub.15 (A).sub.15 (SEQ
ID NO:39). Using 200 nM hexaphosphate-nucleotide 647-dGTP and 200
nM hexaphosphate-nucleotide 676-dATP, patterns were identified with
spectral signatures for 647 dye emission (G signal) preceding
spectral signatures for 676 dye emission (e.g. the A signal) which
resulted from fluorescence resonance energy transfer (FRET) from
the donor molecule SA-Cy3-b-Phi29, or SA-Cy3-b-B103.
Random Template Sequence:
[0746] Hexa-phosphate nucleotides which were dye-labeled at the
terminal phosphate group were diluted to 200 nM in extension
buffer. (50 mM MOPS pH=7.1; 75 mM potassium acetate (pH=7.0); 0.3%
BSA; 1 mM MnCl.sub.2; 300 nM procatuate dioxygenase; 4 mM 3,4
dihydroxyl benzoic acid; 1 mM 2-nitrobenzoic acid; 400 mM 1,2
phenylenediamine; 100 mM ferrocene monocarboxylic acid; 0.02%
cyclooctratetraene; 6 mM Trolox). Nucleotide mix was flowed into
channel with SA-Cy3-b-Phi29 bound to DNA template and images were
recorded for approximately 2 minutes at approximately 20 ms frame
rates. The DNA template sequence extended with the following
sequence:
Random oligonucleotide:
TABLE-US-00012 (SEQ ID NO: 40)
5'-TTGAACGGATGAGGACCAGACACCACTTGAACGGATGAGGAAAAAA AAAATCA-3'.
[0747] Using 200 nM hexaphosphate-647-dGTP and 200 nM
hexaphosphate-676-dATP, 2 .mu.M dCTP and 2 .mu.M dTTP, patterns
were identified with spectral signatures for 647 dye emission (G
signal) and 676 dye emission (A signal) which resulted from
fluorescence resonance energy transfer (FRET) using SA-Cy3-b-Phi29
or SA-Cy3-b-B103 as the donor molecule, respectively.
Analysis of Homopolymer Sequencing Results.
[0748] The resulting pattern sequencing data acquired using the
methodologies described in Example 12 herein was processed. The
subsequent detected FRET events were filtered and sequences were
aligned.
[0749] In one exemplary experiment, 200 nM hexaphosphate-647-dGTP
and 200 nM hexaphosphate-676-dATP was used along with
SA-Cy3-b-Phi29 as the donor molecule. The alignment algorithm found
55 molecules in the field of view, which clearly demonstrated the
completion of at least 30 base pairs of the full 60 base pair
sequence. As a result, the number of events detected for the first
15 G insertions was approximately 15 and the number of events for
the subsequent 15 A insertions was approximately 15.
[0750] In another exemplary experiment, 200 nM
hexaphosphate-647-dGTP and 200 nM hexaphosphate-676-dATP was used
along with SA-Cy3-b-B103 as the donor molecule.
Analysis of Random Sequencing Results.
[0751] Resulting pattern sequencing data acquired using the
methodologies described in Example 13 herein was processed. The
subsequent detected FRET events were filtered and sequences were
aligned. Results are represented by the aligning the sequence,
whereby light gray blocks represents the G insertion signals and
dark gray indicates A insertion signals. The alignment algorithm
found 95 molecules in the field of view, which demonstrated the
matching of detected events with the actual sequence.
Enzyme Kinetics and Extension Speeds for Polymerase Conjugates:
[0752] Using the conditions and template described above in the
homopolymer sequence example, various SA-Cy3-labeled mutant
polymerase conjugates were tested to determine on-chip single
molecule kinetics and extension speeds.
[0753] Distributions of the start times were shown for all of the
events from all of the molecules which were successfully aligned
with the algorithm from their respective blocks, whereby a block
refers to 1 of the 4 homopolymer stretches. This analysis
demonstrates the nearly uniform extension speed of the population
of SA-Cy3-polymerase conjugates. In addition, the distributions for
event duration from each of the blocks for all of molecules with
correct sequence alignment was also determined and found to provide
strong correlation with stopped-flow experiments.
Example 13
Analysis of Fluorescence Data To Extrapolate Sequence
Information
[0754] To convert the observed fluorescence emissions detected
during the nucleotide incorporation reaction into nucleotide
sequence information, the raw data comprising a movie of observed
emissions was first processed by using a Hidden Markov Model
(HMM)-based algorithm to detect and identify FRET events. The
subsequent detected FRET events were filtered and filtered
sequences were aligned. Each of these two steps, FRET event
detection and sequence analysis, are described in more detail
below. The HMM-based algorithm was used to analyze the data in
Example 14 below.
Detection of FRET Events
[0755] The analysis underlying FRET event detection is designed to
process spatially correlated movie(s) comprising real time sequence
fluorescence emission data, and extract time-series of interest
from those data. A movie typically contains one or more channels
where each channel represents the same spatial location at
different wavelengths. The analysis chain begins with the
submission of one or more movies to the analysis machine via a
comprehensive user interface. The user interface requires the user
to input various parameters which describe the movie(s) (e.g.
channel regions, dye emission properties). Once this data is
submitted the movie(s) are then processed by the image analysis
software where a sliding window of N frames propagates through the
movie calculating a temporal local average of the frames within the
window. At each position of the window in the movie, the local
average image is then further processed and enhanced using well
known image processing algorithms and a record of the maximum
projection of all the local average images is recorded to produce a
global image of the movie. This global image is the input into a
spot identification algorithm which produces a set of spots
identified by a unique spot identification, its x and y location
and its corresponding channel. Each set of spots for a given
channel is then registered to the set of spots in every other
channel. In this way a set of spot tuples is constructed. If a
detected spot in one channel does not have a corresponding detected
spot in another channel, then the position of the undetected spot
using the transformation between the two channels and the location
of the detected spot is inferred. Once a complete set of spot
tuples is constructed the movie is iterated over and at each frame
the amplitude of each spot is calculated and appended to the
appropriate time-series.
[0756] The collection of time-series from a spot tuple consists of
time-series from donor and corresponding acceptor channels. This
collection is called a Vector Time-Series (VTS). The FRET detection
process starts with a data segmentation step using a Markov Chain
Monte-Carlo (MCMC) algorithm. Each segment of VTS is modeled by a
multivariate Gaussian model, with each of the channel modeled by a
mean and a standard deviation. This model establishes a baseline
for each channel, from which quantities such as "Donor Down" and
"Acceptor Up" can be calculated. A Hidden Markov Model (HMM) was
used to model the observed data. The underlying states consist of a
null state, a blink state and a number of FRET states (one for each
acceptor channel). Each state has its emission probability, which
reflects the state's corresponding physical concept. FRET states
are characterized by significant "donor down" and "acceptor up"
signals. Blink state is characterized by significant "donor down"
with no "acceptor up". Null state is characterized by no "donor
down" and no "acceptor up". Given the observed VTS signal, the
emission matrix, and a state transition probability matrix, the
most probable state path can be computed using the Viterbi
algorithm. This state path assigns each of the frames to a state.
Temporally neighboring FRET frames are grouped into FRET events.
For each of the detected FRET events, a list of event features are
calculated, including event duration, signal average, signal to
noise ratio, FRET efficiency, probability of event, color calling
and other features. This list of events and corresponding features
are stored in a file.
[0757] The final stage of the automated analysis generates a report
summarizing the results in the form of a web page containing
summary image, statistics of the spots and FRET detection, together
with line intensity plots and base call plots. See for example,
Watkins et al., "Detection of Intensity Change Points in
Time-Resolved Single-Molecule Measurements" J. Phys. Chem. B.,
109(1):617-628 (2005).
[0758] Using the above process, the movie data obtained from the
sequencing reactions was analyzed to detect and identify FRET
events according to the process described above. The FRET events
were then processed to identify sequences as described below.
Sequence Analysis
[0759] Beginning with the set of detected Forster resonance energy
transfer (FRET) events, a data overview was constructed in the form
of a color image interpreted as a sequencing plot. To generate the
plot, the original FRET event data was pre-processed using a set of
filters constructed by a priori knowledge of the sequence. For each
reaction site (each molecule) an ordered sequence of FRET events
was constructed. The base call letters for each FRET event (e.g.
"A", "C", "G" or "T") were concatenated to form a sequence ASCII
string. The order of letters in the string reflects the temporal
relationship of the events. Given that the expected sequence was
known a priori, a regular expression was then constructed which
represented the full or partial expected sequence or sequence
pattern. Matching against the regular expression (expected
sequence) was then computed for each sequence in the set and the
start and stop indices of the match were recorded. A color plot
image was then constructed where each row corresponds to a sequence
in the set. The plot image was padded to accommodate sequences of
different lengths. A color map of 2*N+1 colors was constructed,
where N denotes the number of possible base calls in each sequence
(N=2 for the plot of this Example). N colors were assigned to the
base characters which fell within the pattern, N colors were
assigned to the base characters which did not fall within the
pattern (muted color), and finally a color was assigned to the
padding (background) of the image. The rows of the image were then
sorted according to the number of base calls in the first part of
the sequence pattern. The rows of the image were also aligned such
that the start of the expected sequence is in the same column for
all rows of the plot.
Example 14
Nucleotide Incorporation with B103 Polymerase-Nanoparticle
Conjugates
TABLE-US-00013 [0760] Template oligonucleotide: (SEQ ID NO: 41) 5'
TTTTGA TT CCCCC TT CCCCC G ACA CGG AGG TTC TAT CAT CGT CAT CGT CAT
CGT CAT CG-Biotin TEG-T-3' Primer oligonucleotide: (SEQ ID NO: 42)
5'-CGATGACGATGACGATGACGATGATAGAACCTCCGTGTC-3' The expected sequence
is: (SEQ ID NO: 55) GGGGGAAGGGGGAA
[0761] A template/primer duplex was formed by mixing 1 .mu.L
template (100 .mu.M) and 0.5 .mu.L of primer (250 .mu.M) in 48.5
.mu.L of buffer composed of 50 mM Tris pH 7.5, 50 mM NaCl and 10 mM
MgCl.sub.2. The mixture was incubated at 98.degree. C. for 2
minutes. The mixture was incubated for 30 minutes at room
temperature.
[0762] Polymerase/Nanoparticle stocks: C8
nanoparticles-UDG-ugi-HP1-Phi29 mutant A, 0.17 .mu.M stock
concentration. C8 nanoparticles-44-UDG-UGi-Phi29 mutant B; 0.38
.mu.M stock concentration. C8 nanoparticles-38-L-B103-UDG-Ugi (SEQ
ID NO:4); 0.38 .mu.M stock concentration.
[0763] Functionalized nanoparticles were diluted to 2 nM in 100
.mu.L of buffer composed of 50 mM MOPS pH 6.8, 200 mM NaCl, and
0.3% BSA.
[0764] The chip was prepared as follows: dd H2O (1 mL/lane). Wash
buffer wash (0.2 mL/lane). Inject lane w/5 nM SA and incubate for
.about.10 minutes. Wash buffer wash (0.4 mL/lane). Inject 200 pM
355/366 duplex for and incubate for .about.5 minutes. Wash buffer
wash (0.4 mL/lane). Polymerase binding buffer wash (0.2 mL/lane).
Mount on scope. Inject 2-5 nM of conjugate and incubate until
desired density is reached (<700 spots/FOV). Polymerase binding
buffer wash (0.2 mL/lane). Inject 1.times. extension mix (w/o
nucleotides) .about.3 min (0.1 mL/lane). Inject 1.times. extension
mix with nucleotides (0.1 mL/lane).
[0765] The template/primer duplex (200 .mu.M) was immobilized on
biotin-embedded, PEG-coated glass slides purchased from
Microsurfaces, Inc. (Bio-01 PEG, Austin, Tex.) using 5 nM
streptavidin. The functionalized nanoparticle (2 nM) were
conjugated to the surface immobilized duplexes. The conjugates were
washed with 100 .mu.L of buffer composed of 50 mM MOPS pH 6.8, 50
mM potassium-OAc, 2 mM MnCl.sub.2, 0.3% BSA, 100 U/mL glucose
oxidase, 10 U/.mu.L Katalase, 10 mM Trolox (dissolved in 24 mM MOPS
pH 6.8), 0.1% Tween-20, 2 mM (Asp).sub.4 (SEQ ID NO: 67), and 0.5%
glucose.
[0766] The extension reaction was initiated by injecting 100 .mu.L
of buffer composed of 50 mM MOPS pH 6.8, 50 mM potassium-OAc, 2 mM
MnCl.sub.2, 0.3% BSA, 100 U/mL glucose oxidase, 10 U/.mu.L
katalase, 10 mM Trolox, 0.1% Tween-20, 2 mM (Asp).sub.4 (SEQ ID NO:
67), 0.5% glucose, 0.2 .mu.M AF647-terminal phosphate labeled dG6P,
and 0.2 .mu.M AF680-terminal phosphate labeled dA6P. Laser
excitation: 405 nm, .about.19 W/cm2, 16 ms.
[0767] As described in Example 13 above, the fluorescent signals
emitted by the nucleotide incorporation reaction were captured in a
movie, and the images were processed using the Hidden Markov Model
(HMM).
Example 15
Nucleotide Incorporation with B103 Polymerase-Nanoparticle
Conjugates
TABLE-US-00014 [0768] Template 404 sequence: (SEQ ID NO: 43)
5'-TGATTTTTTTTTTCCTCATCCGTTCAAGTGGTGTCTGG TCCTCATCCGTTCAAGACA CGG
AGG TTC TAT CAT CGT CAT CGT CAT-biotin TEG-T-3' Primer 317
sequence: (SEQ ID NO: 44) 5' TGA TAG AAC CTC CGT GT 3'
Duplex Template/Primer Preparation:
[0769] The template oligonucleotide, at 100 nanomolar
concentration, and the primer oligonucleotide, at 1 micromolar
concentration, were heated to 98.degree. C. in annealing buffer (50
mM Tris, pH 7.5, 50 mM NaCl) for 5 minutes and allowed to cool to
room temperature.
Flow Chamber Preparation
[0770] PEG/PEG-biotin coated cover slips (MicroSurfaces, Inc.,
Minneapolis, Minn.) were assembled into 9-lane reaction chambers
with laser-cut 3M adhesive and custom fabricated plastic
superstructures with inlet/outlet ports for fluid addition. The
surface was wetted by flowing 1 milliliter of Tris-buffered saline
(50 mM Tris, pH 7.5, 150 mM NaCl) containing 0.1% Tween-20 and 0.5%
bovine serum albumin (Sigma, Cat.# A8577) (TBST-B) into each
chamber and incubating at room temperature for 5 minutes. The
surface was coated with streptavidin by flowing 100 microliters of
5 nM streptavidin, (Zymed, Cat # 43-4302) diluted in TBST-B, and
incubating for 30 minutes at room temperature. The lanes were
washed with 1 milliliter of TBST-B. The duplex template/primer was
diluted to 5 .mu.pM in TBST-B and 100 microliters was flowed into
the reaction chamber and incubated 30 minutes at room temperature.
The lanes were washed with 1 milliliter of TBSB.
Nucleotide Incorporation Reaction
[0771] Polymerase-nanoparticle conjugates are bound to the
templates in the flow chamber for one minute in binding buffer (50
mM MOPS, pH 7.0, 2 mM 4-Aspartate (SEQ ID NO: 67), 50 mM Potassium
Acetate, 0.3% BSA, 2 mM MnCl.sub.2, 0.1% tween-20, 0.5 mg/ml
glucose Oxidase, 10 U/.mu.l Katalase, 10 mM Trolox (in ethanol),
0.5% glucose) at a concentration of 10 nM. The polymerase was
exo-minus Phi29 (SEQ ID NO:9). Excess unbound conjugate is washed
off with 200 microliters of TBST-B. Image acquisition is initiated
and 100 microliters of extension buffer (binding buffer with 200
nanomolar each 680-dG6P and 647-dA6P and 1 micromolar each dTTP and
dCTP) was flowed through the chamber. Images are acquired for 90
seconds and exposure time was 16 ms. 405 nanometer laser power
density was 20 W/cm.sup.2.
[0772] As described in Example 13 above, the fluorescent signals
emitted by the nucleotide incorporation reaction were captured in a
movie, and the images were processed using the Hidden Markov Model
(HMM).
Example 16
Nucleotide Incorporation with B103-Fluorescent Dye Conjugates
Preparing NHS-Ester Surfaces:
[0773] Glass coverslips surfaces were plasma cleaned and treated
with a mixture of poly-ethyleneglycol (PEG) and NHS-ester to
produce a low density NHS-ester surface with a PEG coating to
prevent non-specific background of proteins and macromolecules.
Fluidic Chamber Assembly:
[0774] Fluidic cassettes were assembled with glass coverslips to
create fluidic chambers capable of carrying approximately 2 .mu.l
of fluid.
Attaching Amine Terminated Hairpin DNA to Low Density NHS-Ester
Surfaces:
TABLE-US-00015 [0775] Target DNA hairpin sequence: (SEQ ID NOS 45
and 65, respectively)
5'-TTTTTTTTACCCCCGGGTGACAGGTTXTTCCTGTCACCC-3'
where "X" is an amine group.
[0776] The target DNA was diluted to 500 nM in 1 M NaHCO.sub.3. The
diluted target molecules were flowed into the fluidic chamber and
incubated for 1 hour. Chambers were washed 1.times. with 1 ml
deactivating buffer (ethanolamine). Surfaces were washed 1.times.
with 1 ml incubation buffer (50 mM Tris-Cl, pH=7.5; 50 mM NaCl;
0.3% BSA).
SA-Polymerase Conjugate Preparation:
[0777] Streptavidin was labeled with Cy3. Streptavidin-Cy3 was
mixed with biotinylated-B103 (b-B103-exo minus) (SEQ ID NO:5) at a
1:1 ratio of SA-protein: biotinylated-B103 in 1.times.PBS.
SA-Cy3-b-B103 Binding to Templates:
[0778] The SA-Cy3-b-B103 conjugates were diluted to 1 nM in binding
buffer (50 mM Tris-Cl; pH=7.5; 0.3% BSA; 100 mM NaCl). The
conjugates were flowed into the fluidic chamber which were
previously loaded with DNA templates on the surface. Surfaces were
incubated for 5 minutes with 1 nM SA-Cy3-b-B103. Surfaces were
washed with 1.times.1 ml incubation buffer.
Fluorescence Imaging:
[0779] The microscope body was purchased from Olympus and was
outfitted with a TIRF objective lens (100.times.; 1.45 NA). The
excitation light passes through an excitation filter (EX
FT-543/22), and dichroic mirror (DM-532) and the sample was
epi-illuminated (Coherent) using TIR at typically 100 W/cm.sup.2.
Upon excitation, the resulting epifluorescence emission passed
through an emission filter (EM FT-540LP) and the resulting emission
was split into three paths (tri-view) using 2 dichroic mirrors and
the appropriate bandpass filters for the dye sets of choice. Using
this filter combination, we were able to spectrally resolve 1 donor
dye and 3 acceptor dyes in 3 detection channels.
[0780] In separate experiments, 1 donor dye and 4 different
acceptor dyes could be resolved in 4 detection channels. The
optical detection scheme was as follows: DC1=635, F1 640LP;
DC2=675, F2=688/31; DC3=705, F3=700 LP. The donor dye used in this
case was CY3 and the 4 acceptor dyes are as follows DY634, AF647,
AF676, AF700
[0781] The emissions resulting in each experiment were imaged on a
CCD camera. Images were collected at a frame rate of approximately
20 ms.
Three-Color Nucleotide Incorporation Reaction:
[0782] Hexa-phosphate dye-labeled nucleotides were diluted to 200
nM in extension buffer (50 mM MOPS pH=7.1; 75 mM potassium acetate
(pH=7.0); 0.3% BSA; 1 mM MnCl.sub.2; 300 nM procatuate dioxygenase;
4 mM 3,4 dihydroxyl benzoic acid; 1 mM 2-nitrobenzoic acid; 400
.mu.M 1,2 phenylenediamine; 100 .mu.M ferrocene monocarboxylic
acid; 0.02% cyclooctratetraene; 6 mM TROLOX). Nucleotide mix was
flowed into channel with SA-Cy3-b-B103 bound to DNA template and
images are recorded for approximately 2 minutes at approximately 20
ms frame rates. In this example, the synthesized strand is expected
to have the following sequence: (G).sub.5T(A).sub.8 (SEQ ID NO:
66). Terminal phosphate-labeled nucleotides and 125 nM cold dC6P
were used for the nucleotide incorporation reaction. The labeled
nucleotides included 125 nM 647-dT6P, 125 nM 676-dG6P, 125 nM
700-dA6P. The spectral signatures for the ALEXA FLUOR-676 G signal,
AF-647 T signal, and AF-700 A signal were identified that resulted
from fluorescence resonance energy transfer (FRET) from the Cy3
donor molecule, and corresponded to the correct insertion sequence
pattern.
Analysis of Three-Color Sequencing Results
[0783] Resulting pattern sequencing data was processed using an
alignment algorithm. The alignment algorithm found 100 molecules in
the field of view, which demonstrated completion of the full
14-nucleotide sequence ((G).sub.5T(A).sub.8 (SEQ ID NO: 66), which
represented approximately 20% of the total single molecule donor
population. The consensus sequence was determined using an HMM
alignment algorithm (e.g., see Example 14). By plotting the
accuracy definition (measured as a percentage value) against the
HMM score (X axis), a linear relationship was detected. Various
measurements of accuracy can be devised that can be suitable for
such analysis. In one exemplary experiment, the accuracy was
estimated according to the following equation:
.alpha. ( T , A ) = .beta. - .delta. - .eta. + .lamda. 2 .lamda.
##EQU00002##
[0784] The measurement of accuracy in the above equation is
intended to provide some measure of similarity between some given
template, T, and some alignment, A, of an observed sequence O. It
should be noted that alphabet of T, A, and O are identical. The
length of T is denoted by .lamda., the number of deletions in the
alignment A by .delta., the number of insertions in the alignment
by .eta., and the number of matches in the alignment by .beta..
Equation (1) is normalized by .lamda., such that a an accuracy of 1
indicates a total agreement, and an accuracy of 0 indicates no
agreement between T and A. The above definition of accuracy is
provided as an example only and is in no way intended to limit the
disclosure to any particular theory or definition of accuracy;
alternative definitions of accuracy are also possible and it may be
suitable to use such alternative definitions in some contexts.
[0785] The accuracy in this system using an HMM alignment threshold
of 0 was estimated to be approximately 80%.
Four-Color Nucleotide Incorporation Reaction:
TABLE-US-00016 [0786] Template molecule: (SEQ ID NO: 46)
TTTTTCCCCGACGATGCCTCCCC g ACA Cgg Agg TTC TAT CAT CgT CAT CgT CAT
CgT CAT Cg-Biotin TEG-T-3 Primer for the template: (SEQ ID NO: 47)
5' TGA TAG AAC CTC CGT GTC 3'
[0787] In this example, the synthesized strand is expected to have
the following sequence:
TABLE-US-00017 GGGGAGGCATCGTCGGGAAAA (SEQ ID NO: 48)
Nucleotide Incorporation Reaction:
[0788] Hexa-phosphate dye-labeled nucleotides were diluted to 200
nM in extension buffer (50 mM MOPS pH=7.1; 75 mM potassium acetate
(pH=7.0); 0.3% BSA; 1 mM MnCl.sub.2; 300 nM procatuate dioxygenase;
4 mM 3,4 dihydroxyl benzoic acid; 1 mM 2-nitrobenzoic acid; 400
.mu.M 1,2 phenylenediamine; 100 .mu.M ferrocene monocarboxylic
acid; 0.02% cyclooctratetraene; 6 mM TROLOX). Nucleotide mix was
flowed into channel with SA-Cy3-b-B103 bound to DNA template and
images are recorded for approximately 2 minutes at approximately 20
ms frame rates.
[0789] The terminal phosphate-labeled nucleotides used for the
nucleotide incorporation reaction included 125 nM DY634-dA6P, 125
nM AF647-dT6P, 125 nM AF676-dG6P, 125 nM AF700-dC6P. The spectral
signatures for the DY-634 A signal, and the ALEXA FLUOR G, T and C
signals (AF-676 G signal, AF-647 T signal, and AF-700 C signal)
were identified that resulted from fluorescence resonance energy
transfer (FRET) from the Cy3 donor molecule, and corresponded to
the correct insertion sequence pattern. 4-color sequence alignment
was obtained by visual inspection.
[0790] The observed FRET event durations for various SA-Cy3-b-B103
conjugates, the event count distributions, and the observed
extension speeds of various SA-Cy3-b-B103 conjugates were
calculated.
Example 17
Nucleotide Incorporation Reactions
TABLE-US-00018 [0791] Template oligonucleotide: (SEQ ID NO: 49)
5'-TTTTTCCCCGCGTAACTCTTTACCCC g ACA Cgg Agg TTC TAT CA-3' Primer
oligonucleotide: (SEQ ID NO: 50) 5'-TGATAGAACCTCCGTGTC-3'
[0792] A duplex was formed by mixing 1 .mu.L template (100 .mu.M)
and 4 .mu.L of primer (50 .mu.M) in 21 .mu.L of buffer composed of
50 mM Tris pH 7.5, 50 mM NaCl and 10 mM MgCl.sub.2. The mixture was
incubated at 98.degree. C. for 2 minutes. The mixture was incubated
for 30 minutes at room temperature.
[0793] Dye-polymerase conjugate:
Cy3(9.3)--SAV-biotin-(HBP1)-(B103H370R)-(exo) (SEQ ID NO:3), 0.60
.mu.M stock concentration. The polymerase is HBP1-B103(exo-)
conjugated to Cy3 via streptavidin/biotin.
[0794] The polymerase conjugate was diluted to 0.75 nM in 100 .mu.L
of buffer composed of 50 mM MOPS pH 7.03, 100 mM NaCl, and 0.3%
BSA. The duplex (200 .mu.M) was immobilized on biotin-embedded,
PEG-coated glass slides purchased from Microsurfaces, Inc. (Bio-01
PEG, Austin, Tex.) using 0.3 nM streptavidin. The dye-polymerase
conjugate (0.75 nM) was conjugated to the surface-immobilized
duplexes. The conjugate was washed with 100 .mu.L of buffer
composed of 50 mM MOPS pH 7.1, 50 mM KOAc pH 6.85, 2 mM Trolox,
0.02% cyclo-octatetraene (COT), 200 U/mL glucose oxidase, 19.9
U/.mu.L katalase and 0.4% glucose.
[0795] The extension reactions were initiated by injecting 100
.mu.L of one of the following buffers composed of 50 mM MOPS pH
7.1, 50 mM KOAc pH 6.85, 2 mM Trolox, 0.02% cyclo-octatetraene
(COT), 200 U/mL glucose oxidase, 19.9 U/.mu.L katalase, 0.4%
glucose, 0.5 mM MnCl.sub.2 and one of the three following
nucleotides combinations: (1) 120 nM 647G (AF647-terminal phosphate
labeled dG6P), 150 nM 676A (AF676-terminal phosphate labeled dA6P),
3 .mu.M dTTP; (2) 24 nM 647G (AF647-terminal phosphate labeled
dG6P), 24 nM 676A (AF676-terminal phosphate labeled dA6P), 24 nM
dTTP; and (3) 30 nM 647T (AF647-terminal phosphate labeled dT6P),
24 nM 676G (AF676-terminal phosphate labeled dG6P), 24 nM 700A
(AF700-terminal phosphate labeled dA6P).
[0796] The HMM-based algorithm described in Example 14 was used to
analyze the data.
Example 18
Nucleotide Incorporation Reactions
TABLE-US-00019 [0797] Target oligonucleotide: (SEQ ID NO: 49)
5'-TTTTTCCCCGCGTAACTCTTTACCCC g ACA Cgg Agg TTC TAT CA-3' Primer
oligonucleotide: (SEQ ID NO: 50) 5'-TGATAGAACCTCCGTGTC-3'
[0798] A DNA duplex was formed by mixing 1 .mu.L template (100
.mu.M) and 4 .mu.L of primer (50 .mu.M) in 21 .mu.L of buffer (50
mM Tris pH 7.5, 50 mM NaCl and 10 mM MgCl.sub.2) and incubated at
98.degree. C. for 2 minutes. And the mixture was incubated for 30
minutes at room temperature.
[0799] A dye-conjugated, exo minus, B103 mutant polymerase
(B103-H370R) (SEQ ID NO:3) (60 .mu.M stock concentration) was used.
The polymerase-dye conjugate was diluted to 0.75 nM in 100 .mu.L of
buffer (50 mM MOPS pH 7.03, 100 mM NaCl, and 0.3% BSA).
[0800] The DNA duplex (200 .mu.M) was immobilized on
biotin-embedded, PEG-coated glass slides purchased from
Microsurfaces, Inc. (Bio-01 PEG, Austin, Tex.) using 0.3 nM
streptavidin. The polymerase-dye conjugate (0.75 nM) was reacted
with the surface-immobilized DNA duplexes. The DNA
duplex/polymerase complex was washed with 100 .mu.L of buffer (50
mM ACES pH 7.1, 50 mM KOAc pH 6.85, 2 mM Trolox, 0.02%
cyclo-octatetraene (COT), 200 U/mL glucose oxidase, 19.9 U/.mu.L
katalase and 0.4% glucose).
[0801] The extension reactions were initiated by injecting 100
.mu.L of one of the following buffers composed of 50 mM MOPS pH
7.1, 50 mM KOAc pH 6.85, 2 mM Trolox, 0.02% cyclo-octatetraene
(COT), 200 U/mL glucose oxidase, 19.9 U/.mu.L katalase, 0.4%
glucose, 0.5 mM MnCl.sub.2 following nucleotides combinations: 30
nM 647T (AF647-terminal phosphate labeled dT6P), 24 nM 676G
(AF676-terminal phosphate labeled dG6P), 24 nM 700A (AF700-terminal
phosphate labeled dA6P).
[0802] The HMM-based algorithm described in Example 14 was used to
analyze the data.
Example 19
Nucleotide Incorporation with Polymerase-Tripod Nanoparticle
Conjugates
Preparing Tripod Nanoparticle-His-B103-H370R(exo-) Polymerase
Conjugates
[0803] Tripod Nanocrystals (50 .mu.L, 2.7 .mu.M in 50 mM borate
buffer pH 8.0) were mixed with stock His-tagged HP1-B103H370R
exo-polymerase (SEQ ID NO:3) (25 .mu.L, 16 .mu.M in 10 mM Tris (pH
7.5) buffer with 100 mM NaCl, 4 mM DTT, 0.5% v/v Tween-20, 0.1 mM
EDTA and 50% v/v glycerol) and 40 .mu.L of 100 mM Tris (pH 7.5)
buffer with 300 mM NaCl and 1 mM DTT in a 1:3 molar ratio
(nanocrystal to polymerase). The conjugation solution was incubated
overnight at 4.degree. C. The resulting conjugate solution was
centrifuged for 5 minutes at 16.8K rcf, purified on Ni.sup.2+-NTA
Agarose column using 100 mM Tris (pH 7.5) buffer with 300 mM NaCl
and 1 mM DTT as the eluent, centrifuged and transferred into a 10K
MWCO dialysis cassette. The conjugate was dialyzed into 50 mM Tris
buffer pH7.5 with 150 mM NaCl, 0.2 mM EDTA, 0.5% v/v Tween-20, 5 mM
DTT and 50% v/v glycerol. The resulting
Tripod-nanocrystal-HP1-B103H370R exo-conjugate was assayed to
determine concentration, template extension activity, active number
of Phi29 per conjugate and DNA binding by FRET. In a DNA extension
assay, the Tripod-nanoparticle-HP1-B103-H370R (exo-) conjugates
exhibited 0.35 base/sec/conjugate, and the stock HP1-B104-H370R
(exo-) polymerase exhibited 0.29 base/sec/enzyme.
Nucleotide Incorporation using the Conjugates
[0804] To a 50 .mu.L solution of 10 .mu.M template DNA:
Biotin-5'-TTTTTCCCCGCGTAACTCTTTACCCCgACACggAggTTCTATCA-3'-amine)
(SEQ ID NO:49), was mixed in 50 .mu.L of 50 .mu.M primer DNA
(5'-TGATAGAACCTCCGTGTC-3' (SEQ ID NO:50)). The mixture was heated
at 98.degree. C. for 1 minute and chilled on ice. The annealed
template/primer was diluted to 200 .mu.pM using 500 mM borate
buffer (pH 8.2), and injected into lanes of a microfluidic device
with coverslip containing NHS ester reactive groups on the surface,
incubated at room temperature for 10 minutes. The coverslip surface
was deactivated by incubating with 50 mM glycine in 500 mM borate
buffer (pH 8.2) for 10 minutes, washed with 50 mM Tris buffer (pH
7.5) with 50 mM NaCl, 0.5% BSA and 0.05% Tween-20.
[0805] The microfluidic device was secured on a TIRF (total
internal reflection fluorescence) microscope. The TIRF microscope
was setup on TIRF mode with power density at .about.15 W/cm.sup.2
for the 405 nm excitation laser. Nanoparticle-polymerase conjugate
solution (10 nM in GO-Cat OSS buffer system, 50 mM MOPS buffer pH
7.2 with 50 mM KOAc, 0.1% Tween-20, 10 mM Trolox, 0.3% BSA, 0.5
mg/mL glucose oxidase, 10 unit/.mu.L catalase, 2 mM tetra-aspartic
acid (SEQ ID NO: 67) and 0.5% freshly added glucose) was injected
into a lane of the microfluidic, incubated at room temperature for
.about.1 minute, then washed with the GO-Cat OSS buffer system (50
mM MOPS buffer pH 7.2 with 50 mM KOAc, 0.1% Tween-20, 10 mM Trolox,
0.3% BSA, 0.5 mg/mL glucose oxidase, 10 unit/.mu.L catalase, 2 mM
tetra-aspartic acid (SEQ ID NO: 67) and 0.5% freshly added
glucose). The successive nucleotide incorporation was captured on a
movie, which was recorded for 100 seconds at 30 ms per frame rate
on a new FOV (field of view) when injecting into the lane of a
primer extension reaction mixture (e.g. 150 nM dG6P-C6-AF647, 150
nM dA6P-C6-AF680, 1000 nM dTTP, 1000 nM dCTP and 0.5 mM MnCl.sub.2
in GO-Cat OSS buffer system containing freshly added 0.5% glucose.
The movie was analyzed to identify the incorporated nucleotides
using time series extraction and base calling software.
Example 20
Reagent Exchange Reactions
TABLE-US-00020 [0806] Target molecule: (SEQ ID NO: 51) 5'TTTTGA
TTTTTTTTTTTT CCCCCCCCCCCC TTTTTTTTTTTT CCCCCCCCCCCC g ACA Cgg Agg
TTC TAT CAT CgT CAT CgT CAT CgT CAT Cg-amine-3' Primer molecule A
for cycle 1: (SEQ ID NO: 52) 5' TGA TAG AAC CTC CGT GTC 3' Primer
molecule B for cycle 2: (SEQ ID NO: 53) 5' TGA TAG AAC CTC YGT GTC
3' (Y = amino modifier C6, C is base, labeled with AF647)
1.times.TBST/BSA Wash buffer: 50 mM Tris pH 7.5; 50 mM NaCl; 0.05%
Tween-20; 0.5% BSA. 1.times. Polymerase binding buffer: 50 mM MOPS
pH 6.8; 100 mM NaCl; 0.1% BSA.
Pre-Extension mix G.O./Cat OSS:
[0807] 50 mM MOPS pH 7.2 w/KOH; 50 mM potassium acetate (KOAc) pH
7.0; 2 mM Trolox (dissolved 24 mM MOPS pH 6.8; stored at
-20.degree. C.); 0.2% cyclooctratetraene; 100 U/mL glucose oxidase;
10 U/.mu.L Catalase; 0.4% glucose.
Extension mix G.O./Cat OSS:
[0808] 50 mM MOPS pH 7.2 w/KOH; 50 mM KOAc pH 7.0; 2 mM Trolox
(dissolved 24 mM MOPS pH 6.8; stored at -20.degree. C.); 0.2%
cyclooctratetraene; 100 U/mL glucose oxidase; 10 U/.mu.L Catalase;
0.4% glucose; 0.6 mM MnCl.sub.2; 100 nM AF647-dG6P; 100 nM
AF676-dA6P.
Covalent-DNA Immobilization and Chip Preparation:
[0809] Coverslips from MicroSurfaces, Inc. were prepared as
follows. The lane was injected with 300 .mu.pM the target molecules
primed with primer A, dissolved in 500 mM borate pH 8.2 and
incubated for approximately 5 minutes. The reaction was terminated
with 0.1 mL wash (500 mM Borate, pH 8.2). NHS deactivation was
conducted using Deactivation buffer supplied by Micro Surfaces,
Inc., by injecting 0.08 mL/lane and incubated for more than 5
minutes. The chip was washed with 1.times.TBST/BSA (1 mL/lane). The
chip was mounted on the scope.
Cycle 1 Nucleotide Incorporation Reaction:
[0810] Polymerase binding buffer wash (0.3 mL/lane) was injected.
2-5 nM of the polymerase conjugate (Cy3-SA-Phi29 mutant) was
injected and incubated until desired density was reached
(.about.900 spots/FOV).
[0811] Polymerase binding buffer wash (0.2 mL/lane) was injected.
Pre-extension mix (without nucleotides) .about.3-5 mM (0.1 mL/lane)
was injected. 1.times. extension mix with nucleotides (0.1 mL/lane)
was injected. For cycle 1, AF647-dG6P and AF676-dA6P terminal
phosphate labeled nucleotides were used. Cycle 1 donors were mapped
visually.
Removal of Polymerase and Synthesized Strand:
[0812] The polymerase used in cycle 1 was removed using 6.3 M
guanidine isothiocyanate, 160 mM Tris pH 9.7, and 2.6 mM EGTA. The
synthesized strand was removed using 25% Formamide, 50 mM NaOH.
Cycle 2 Exchanged Polymerase and Primer:
[0813] For cycle 2, 500 nM of fresh, AF647-labeled primer B was
added in 1.times. TBST/BSA, and incubated for 5 minutes.
[0814] Polymerase binding buffer wash (0.3 mL/lane) was injected.
Approximately 2-5 nM of the polymerase conjugate (Cy3-SA-Phi29
mutant) was injected.
[0815] Polymerase binding buffer wash (0.2 mL/lane) was injected.
Pre-extension mix (without nucleotides) .about.3-5 mM (0.1 mL/lane)
was injected. 1.times. extension mix with nucleotides (0.1 mL/lane)
was injected. For cycle 2, AF676-dG6P and AF700-dA6P terminal
phosphate labeled nucleotides were used. Cycle 2 donors were mapped
visually (same field of view as for cycle 1). Time traces of the
fluorescent acceptor signals for cycle 1 and 2 were obtained. The
number of donors mapped in the same field of view for cycle 1 and 2
were analyzed.
Example 21
Reagent Exchange Reactions
[0816] In a first cycle, nucleotide incorporation reactions were
conducted on a target nucleic acid molecule with 3 types of
nucleotides. The reagents were exchanged, and a second cycle of
nucleotide incorporation reactions were conducted using 4 types of
nucleotides. Accordingly, reagent exchange reactions were performed
to continue nucleotide incorporation reactions on the same target
nucleic acid molecules.
[0817] In this experiment, the nucleotide incorporation reaction
proceeded towards the solid surface.
TABLE-US-00021 Target hairpin oligonucleotide: (SEQ ID NOS 54 and
65, respectively) 5'TTTTTCCCCGACGATGCCTCCCCTTTTTTTTACCCCCGGGTGACA
GGTTXTTCCTGTCACCC-3',
where X=amino modifier C6 dT; 5' biotin. Polymerase conjugated to a
Cy3 dye: Cy3(9.3)-SAV-biotin(HBP1)(B104H370R)(exo.sup.-), 0.60
.mu.M stock concentration.
[0818] The polymerase-dye conjugate was diluted to 0.75 nM in 100
.mu.L of buffer (50 mM MOPS pH 7.03, 100 mM NaCl, and 0.3%
BSA).
[0819] The hairpin oligonucleotide (300 pM) was surface-immobilized
on biotin-embedded, PEG-coated glass slides purchased from
Microsurfaces, Inc. (Bio-01 PEG, Austin, Tex.) using 0.5 nM
streptavidin. The polymerase-dye conjugate (0.75 nM) was reacted
with the surface-immobilized hairpin oligonucleotide (i.e., target
nucleic acid molecule) to produce a polymerase/target complex. The
complex was washed with 100 .mu.L of a pre-extension buffer (50 mM
ACES pH 7.1, 50 mM KOAc pH 6.85, 2 mM Trolox, 0.02%
cyclo-octatetraene (COT), 200 U/mL glucose oxidase, 19.9 U/.mu.L
katalase and 0.4% glucose).
[0820] The first cycle was initiated by injecting 100 .mu.L of
extension buffer: 50 mM ACES pH 7.1, 50 mM KOAc pH 6.85, 2 mM
Trolox, 0.02% cyclo-octatetraene (COT), 200 U/mL glucose oxidase,
19.9 U/.mu.L katalase, 0.4% glucose, 0.5 mM MnCl.sub.2 supplemented
with the following nucleotides combinations: 25 nM 647T
(AF647-terminal phosphate labeled dT6P), 50 nM 676G (AF676-terminal
phosphate labeled dG6P), and 25 nM 700A (AF700-terminal phosphate
labeled dA6P).
[0821] A reagent exchange reaction was performed to remove the
polymerase, using 200 .mu.L a solution (4.8 M guanidine
isothiocyanate and 200 mM Tris pH 9.7). The immobilized hairpin
oligonucleotide was washed with 200 .mu.L wash buffer (50 mM Tris
pH 7.5, 50 mM NaCl, and 0.3% BSA).
[0822] In the second cycle, a fresh supply of the polymerase-dye
conjugate (Cy3(9.3)-SAV-biotin(HBP1)(B104H370R)(exo.sup.-)) was
reacted with the immobilized hairpin oligonucleotide and washed
with pre-extension buffer (50 mM ACES pH 7.1, 50 mM KOAc pH 6.85, 2
mM Trolox, 0.02% cyclo-octatetraene (COT), 200 U/mL glucose
oxidase, 19.9 U/.mu.L katalase and 0.4% glucose). The second
extension was conducted using the extension buffer supplemented
with the nucleotide combination: 25 nM 634A (Dy634-terminal
phosphate labeled dA6P) 25 nM 647T (AF647-terminal phosphate
labeled dT6P), 50 nM 676G (AF676-terminal phosphate labeled dG6P),
and 25 nM 700C (AF700-terminal phosphate labeled dC6P).
[0823] The full length of the expected extendible sequence is:
TABLE-US-00022 (SEQ ID NO: 56)
GGGGGTAAAAAAAAGGGGAGGCATCGTCGGGGAAAAA
[0824] In the first cycle, the nucleotide incorporation reaction
was expected to produce the underlined sequence shown above. In the
second cycle, the nucleotide incorporation reaction was expected to
produce the non-underlined sequence shown above. A time trace of
fluorescent signals from cycle 1 and 2 reactions was obtained.
Example 22
Reagent Exchange Reactions
[0825] Reagent exchange reactions were conducted using a polymerase
labeled with a fluorescent donor dye, 4 types of nucleotides each
labeled with a different fluorescent acceptor dye, and 2 types of
non-hydrolyzable nucleotides (unlabeled).
TABLE-US-00023 Template oligonucleotide: (SEQ ID NO: 49)
5'-TTTTTCCCCGCGTAACTCTTTACCCC g ACA Cgg Agg TTC TAT CA-3' Primer
oligonucleotide: (SEQ ID NO: 47) 5'-TGATAGAACCTCCGTGTC-3'
[0826] A duplex was formed by mixing 1 .mu.L template (100 .mu.M)
and 4 .mu.L of primer (50 .mu.M) in 21 .mu.L of buffer (50 mM Tris
pH 7.5, 50 mM NaCl and 10 mM MgCl.sub.2). The mixture was incubated
at 98.degree. C. for 2 minutes. The mixture was then incubated for
30 minutes at room temperature.
[0827] Polymerase conjugated to a Cy3 dye:
Cy3(9.3)-SAV-biotin(HBP1)(B104H370R)(exo.sup.-), 0.60 .mu.M stock
concentration.
[0828] The polymerase-dye conjugate was diluted to 0.75 nM in 100
.mu.L of buffer (50 mM MOPS pH 7.03, 100 mM NaCl, and 0.3%
BSA).
[0829] The duplex (300 .mu.M) was immobilized on biotin-embedded,
PEG-coated glass slides purchased from Microsurfaces, Inc. (Bio-01
PEG, Austin, Tex.) using 0.5 nM streptavidin.
[0830] The nucleotide incorporation and reagent exchange reactions
were repeated 5.times. on the same target DNA molecules according
to the following protocol:
[0831] The polymerase-dye conjugate (0.75 nM) was reacted with the
surface-immobilized DNA duplex (i.e., target nucleic acid molecule)
to produce a polymerase/target complex. The complex was washed with
100 .mu.L of a pre-extension buffer (50 mM ACES pH 7, 50 mM KOAc pH
6.85, 2 mM Trolox, 0.02% cyclo-octatetraene (COT), 200 U/mL glucose
oxidase, 19.9 U/.mu.L katalase and 0.4% glucose).
[0832] The extension reactions were initiated by injecting 100
.mu.L of extension buffer (50 mM ACES pH 7, 50 mM KOAc pH 6.85, 2
mM Trolox, 0.02% cyclo-octatetraene (COT), 200 U/mL glucose
oxidase, 19.9 U/.mu.L katalase, 0.4% glucose, 0.5 mM MnCl.sub.2, 25
nM 634C (Dy634-terminal phosphate labeled dC6P), 38 nM 647T
(AF647-terminal phosphate labeled dT6P), 32 nM 676G (AF676-terminal
phosphate labeled dG6P), 38 nM 700A (AF700-terminal phosphate
labeled dA6P), 25 nM dApCpp
(2'-Deoxy-adenosine-5'-[(.alpha.,.beta.)-methyleno]triphosphate,
sodium salt) (Jena Bioscience, Germany) and 25 nM dUpCpp
(2'-Deoxy-uridine-5'-[(.alpha.,.beta.)-methyleno]triphosphate,
Sodium salt) (Jena Bioscience, Germany).
[0833] Upon completion of each extension cycle, the polymerase-dye
conjugate was removed with a 200 .mu.L solution (4.8 M guanidine
isothiocyanate and 200 mM Tris pH 9.7). The immobilized target DNA
(now having synthesized strands) was washed with 200 .mu.L wash
buffer (50 mM Tris pH 7.5, 50 mM NaCl, and 0.3% BSA). The target
DNA and synthesized strands were separated using a solution (25%
v/v formamide and 50 mM NaOH). The strand separation reaction was
terminated by injecting 200 .mu.L of wash buffer (50 mM Tris pH
7.5, 50 mM NaCl, and 0.3% BSA).
[0834] The immobilized target DNA molecules were re-hybridized with
primers by injecting 100 .mu.L of 500 nM the primer dissolved in
wash buffer and incubated for 30 minutes. The re-hybridization was
terminated by injecting 200 .mu.L of wash buffer (50 mM Tris pH
7.5, 50 mM NaCl, and 0.3% BSA). A time trace of fluorescent signals
from cycle 2 and 3 reactions were obtained.
Example 23
Nucleotide Incorporation of B103 Polymerase
Stopped Flow Analysis
[0835] 1) B103 Polymerase: Stopped-Flow Measurements of
t.sub.po1
TABLE-US-00024 Template C sequence: 5'-CGTTAACCGCCCGCTCCTTTGCAAC-3'
(SEQ ID NO: 57) Primer sequence: 5'-GTTGCAAAGGAGCGGGCG-3' (SEQ ID
NO: 58)
[0836] The kinetics of nucleotide incorporation by B103 (exo.sup.-)
(SEQ ID NO:5) and an H370R mutant (SEQ ID NO:3) DNA polymerases
were measured in an Applied Photophysics SX20 stopped-flow
spectrometer by monitoring changes in fluorescence from a duplex
Alexa fluor 546 dye-labeled-DNA template following the mixing of
the enzyme-DNA complex with dye-labeled nucleotides (AF647-C6-dG6P)
in the reaction buffer containing 50 mM Tris-HCl, pH 7.5, 50 mM
NaCl, 4 mM DTT, 0.2% BSA, and 2 mM MnCl.sub.2. The reactions
included 330 nM recombinant DNA polymerase, 100 nM template/primer
duplex, and 7 .mu.M labeled nucleotides.
[0837] The averaged (5 traces) stopped-flow fluorescence traces
(>1.5 ms) were fitted with a double exponential equation (1) to
extrapolate the rates of the nucleotide binding and product
release,
Fluorescence=A.sub.1e.sup.-k1*t+A.sub.2*e.sup.-kpo1*t+C (equation
1)
[0838] where A.sub.1 and A.sub.2 represent corresponding
fluorescence amplitudes, C is an offset constant, and k1 and kpo1
are the observed rate constants for the fast and slow phases of the
fluorescence transition, respectively. The dye-labeled nucleotides
comprise terminal-phosphate-labeled nucleotides having an alkyl
linker with a functional amine group attached to the dye. The
stopped-flow techniques for measuring t.sub.po1 (1/k.sub.po1)
followed the techniques described by M P Roettger (2008
Biochemistry 47:9718-9727; M. Bakhtina 2009 Biochemistry
48:3197-320).
2) B103 Polymerase: Stopped-Flow Measurements of t.sub.-1
TABLE-US-00025 Template C sequence: (SEQ ID NO: 59) 5'-CAGTAACGG
AGT TGG TTG GAC GGC TGC GAG GC-3' Dideoxy-primer sequence: (SEQ ID
NO: 60) 5'-GCC TCG CAG CCG TCC AAC CAA CTC ddC-3'
[0839] The rate of the nucleotide dissociation (k.sub.1) from the
ternary complex of [enzyme.cndot.DNA.cndot.nucleotide] was measured
in an Applied Photophysics SX20 stopped-flow spectrometer by
monitoring changes in fluorescence from in fluorescence from a
duplex Alexa fluor 546 dye-labeled-DNA template following the
mixing of the [enzyme.cndot.DNA.cndot.labeled nucleotide] ternary
complex with 50 .mu.M cognate non-labeled deoxynucleoside
triphosphate in a buffer containing 50 mM Tris-HCl (pH 7.5), 50 mM
NaCl, 4 mM DTT, 0.2% BSA, and 2 mM MnCl.sub.2.
[0840] The ternary complexes were prepared using: 330 nM
polymerase, 100 nM template/primer duplex, and 7 .mu.M terminal
phosphate-labeled nucleotides (AF647-C6-dG6P).
[0841] The averaged stopped-flow fluorescence traces (>1.5 msec)
were fitted with a single exponential equation (2) to extrapolate
the rate of the nucleotide dissociation (k.sub.-1) from the
[enzyme.cndot.DNA.cndot.nucleotide] ternary complex.
Fluorescence=A.sub.1e.sup.-k-1*t+C (equation 2)
[0842] where A.sub.1 represents the corresponding fluorescence
amplitude, C is an offset constant, and k.sub.-1 and the observed
rate constants for the fluorescence transition. The stopped-flow
techniques for measuring t.sub.-1 (1/k.sub.-1) followed the
techniques described by M. Bakhtina (2009 Biochemistry
48:3197-3208). The results of the stopped-flow experiments are
listed in the table below.
TABLE-US-00026 Summary of the t.sub.pol and t.sub.-1 measurements
Polymerase t.sub.pol t.sub.-1 B103 (exo-) 14 16 H370R 17 43 H370Y
15 12 E371R 11 17 E371Y 11 7 K372R 14 12 K380R 783 17 D507G 11 13
D507H 7 16 K509Y 10 20 Ph-29 (exo-) 11 27 T373R 15 81 T373Y 14 45
Sequence CWU 1
1
671572PRTBacillus phage B103 1Met Pro Arg Lys Met Phe Ser Cys Asp
Phe Glu Thr Thr Thr Lys Leu1 5 10 15Asp Asp Cys Arg Val Trp Ala Tyr
Gly Tyr Met Glu Ile Gly Asn Leu 20 25 30Asp Asn Tyr Lys Ile Gly Asn
Ser Leu Asp Glu Phe Met Gln Trp Val 35 40 45Met Glu Ile Gln Ala Asp
Leu Tyr Phe His Asn Leu Lys Phe Asp Gly 50 55 60Ala Phe Ile Val Asn
Trp Leu Glu His His Gly Phe Lys Trp Ser Asn65 70 75 80Glu Gly Leu
Pro Asn Thr Tyr Asn Thr Ile Ile Ser Lys Met Gly Gln 85 90 95Trp Tyr
Met Ile Asp Ile Cys Phe Gly Tyr Lys Gly Lys Arg Lys Leu 100 105
110His Thr Val Ile Tyr Asp Ser Leu Lys Lys Leu Pro Phe Pro Val Lys
115 120 125Lys Ile Ala Lys Asp Phe Gln Leu Pro Leu Leu Lys Gly Asp
Ile Asp 130 135 140Tyr His Ala Glu Arg Pro Val Gly His Glu Ile Thr
Pro Glu Glu Tyr145 150 155 160Glu Tyr Ile Lys Asn Ala Ile Glu Ile
Ile Ala Arg Ala Leu Asp Ile 165 170 175Gln Phe Lys Gln Gly Leu Asp
Arg Met Thr Ala Gly Ser Asp Ser Leu 180 185 190Lys Gly Phe Lys Asp
Ile Leu Ser Thr Lys Lys Phe Asn Lys Val Phe 195 200 205Pro Lys Leu
Ser Leu Pro Met Asp Lys Glu Ile Arg Arg Ala Tyr Arg 210 215 220Gly
Gly Phe Thr Trp Leu Asn Asp Lys Tyr Lys Glu Lys Glu Ile Gly225 230
235 240Glu Gly Met Val Phe Asp Val Asn Ser Leu Tyr Pro Ser Gln Met
Tyr 245 250 255Ser Arg Pro Leu Pro Tyr Gly Ala Pro Ile Val Phe Gln
Gly Lys Tyr 260 265 270Glu Lys Asp Glu Gln Tyr Pro Leu Tyr Ile Gln
Arg Ile Arg Phe Glu 275 280 285Phe Glu Leu Lys Glu Gly Tyr Ile Pro
Thr Ile Gln Ile Lys Lys Asn 290 295 300Pro Phe Phe Lys Gly Asn Glu
Tyr Leu Lys Asn Ser Gly Ala Glu Pro305 310 315 320Val Glu Leu Tyr
Leu Thr Asn Val Asp Leu Glu Leu Ile Gln Glu His 325 330 335Tyr Glu
Met Tyr Asn Val Glu Tyr Ile Asp Gly Phe Lys Phe Arg Glu 340 345
350Lys Thr Gly Leu Phe Lys Glu Phe Ile Asp Lys Trp Thr Tyr Val Lys
355 360 365Thr His Glu Lys Gly Ala Lys Lys Gln Leu Ala Lys Leu Met
Leu Asn 370 375 380Ser Leu Tyr Gly Lys Phe Ala Ser Asn Pro Asp Val
Thr Gly Lys Val385 390 395 400Pro Tyr Leu Lys Glu Asp Gly Ser Leu
Gly Phe Arg Val Gly Asp Glu 405 410 415Glu Tyr Lys Asp Pro Val Tyr
Thr Pro Met Gly Val Phe Ile Thr Ala 420 425 430Trp Ala Arg Phe Thr
Thr Ile Thr Ala Ala Gln Ala Cys Tyr Asp Arg 435 440 445Ile Ile Tyr
Cys Asp Thr Asp Ser Ile His Leu Thr Gly Thr Glu Val 450 455 460Pro
Glu Ile Ile Lys Asp Ile Val Asp Pro Lys Lys Leu Gly Tyr Trp465 470
475 480Ala His Glu Ser Thr Phe Lys Arg Ala Lys Tyr Leu Arg Gln Lys
Thr 485 490 495Tyr Ile Gln Asp Ile Tyr Ala Lys Glu Val Asp Gly Lys
Leu Ile Glu 500 505 510Cys Ser Pro Asp Glu Ala Thr Thr Thr Lys Phe
Ser Val Lys Cys Ala 515 520 525Gly Met Thr Asp Thr Ile Lys Lys Lys
Val Thr Phe Asp Asn Phe Arg 530 535 540Val Gly Phe Ser Ser Thr Gly
Lys Pro Lys Pro Val Gln Val Asn Gly545 550 555 560Gly Val Val Leu
Val Asp Ser Val Phe Thr Ile Lys 565 5702572PRTBacillus phage B103
2Met Pro Arg Lys Met Phe Ser Cys Asp Phe Glu Thr Thr Thr Lys Leu1 5
10 15Asp Asp Cys Arg Val Trp Ala Tyr Gly Tyr Met Glu Ile Gly Asn
Leu 20 25 30Asp Asn Tyr Lys Ile Gly Asn Ser Leu Asp Glu Phe Met Gln
Trp Val 35 40 45Met Glu Ile Gln Ala Asp Leu Tyr Phe His Asn Leu Lys
Phe Asp Gly 50 55 60Ala Phe Ile Val Asn Trp Leu Glu His His Gly Phe
Lys Trp Ser Asn65 70 75 80Glu Gly Leu Pro Asn Thr Tyr Asn Thr Ile
Ile Ser Lys Met Gly Gln 85 90 95Trp Tyr Met Ile Asp Ile Cys Phe Gly
Tyr Lys Gly Lys Arg Lys Leu 100 105 110His Thr Val Ile Tyr Asp Ser
Leu Lys Lys Leu Pro Phe Pro Val Lys 115 120 125Lys Ile Ala Lys Asp
Phe Gln Leu Pro Leu Leu Lys Gly Asp Ile Asp 130 135 140Tyr His Ala
Glu Arg Pro Val Gly His Glu Ile Thr Pro Glu Glu Tyr145 150 155
160Glu Tyr Ile Lys Asn Asp Ile Glu Ile Ile Ala Arg Ala Leu Asp Ile
165 170 175Gln Phe Lys Gln Gly Leu Asp Arg Met Thr Ala Gly Ser Asp
Ser Leu 180 185 190Lys Gly Phe Lys Asp Ile Leu Ser Thr Lys Lys Phe
Asn Lys Val Phe 195 200 205Pro Lys Leu Ser Leu Pro Met Asp Lys Glu
Ile Arg Arg Ala Tyr Arg 210 215 220Gly Gly Phe Thr Trp Leu Asn Asp
Lys Tyr Lys Glu Lys Glu Ile Gly225 230 235 240Glu Gly Met Val Phe
Asp Val Asn Ser Leu Tyr Pro Ser Gln Met Tyr 245 250 255Ser Arg Pro
Leu Pro Tyr Gly Ala Pro Ile Val Phe Gln Gly Lys Tyr 260 265 270Glu
Lys Asp Glu Gln Tyr Pro Leu Tyr Ile Gln Arg Ile Arg Phe Glu 275 280
285Phe Glu Leu Lys Glu Gly Tyr Ile Pro Thr Ile Gln Ile Lys Lys Asn
290 295 300Pro Phe Phe Lys Gly Asn Glu Tyr Leu Lys Asn Ser Gly Ala
Glu Pro305 310 315 320Val Glu Leu Tyr Leu Thr Asn Val Asp Leu Glu
Leu Ile Gln Glu His 325 330 335Tyr Glu Met Tyr Asn Val Glu Tyr Ile
Asp Gly Phe Lys Phe Arg Glu 340 345 350Lys Thr Gly Leu Phe Lys Glu
Phe Ile Asp Lys Trp Thr Tyr Val Lys 355 360 365Thr His Glu Lys Gly
Ala Lys Lys Gln Leu Ala Lys Leu Met Leu Asn 370 375 380Ser Leu Tyr
Gly Lys Phe Ala Ser Asn Pro Asp Val Thr Gly Lys Val385 390 395
400Pro Tyr Leu Lys Glu Asp Gly Ser Leu Gly Phe Arg Val Gly Asp Glu
405 410 415Glu Tyr Lys Asp Pro Val Tyr Thr Pro Met Gly Val Phe Ile
Thr Ala 420 425 430Trp Ala Arg Phe Thr Thr Ile Thr Ala Ala Gln Ala
Cys Tyr Asp Arg 435 440 445Ile Ile Tyr Cys Asp Thr Asp Ser Ile His
Leu Thr Gly Thr Glu Val 450 455 460Pro Glu Ile Ile Lys Asp Ile Val
Asp Pro Lys Lys Leu Gly Tyr Trp465 470 475 480Ala His Glu Ser Thr
Phe Lys Arg Ala Lys Tyr Leu Arg Gln Lys Thr 485 490 495Tyr Ile Gln
Asp Ile Tyr Ala Lys Glu Val Asp Gly Lys Leu Ile Glu 500 505 510Cys
Ser Pro Asp Glu Ala Thr Thr Thr Lys Phe Ser Val Lys Cys Ala 515 520
525Gly Met Thr Asp Thr Ile Lys Lys Lys Val Thr Phe Asp Asn Phe Arg
530 535 540Val Gly Phe Ser Ser Thr Gly Lys Pro Lys Pro Val Gln Val
Asn Gly545 550 555 560Gly Val Val Leu Val Asp Ser Val Phe Thr Ile
Lys 565 5703608PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 3Met Ser His His His His His His Ser
Met Ser Gly Leu Asn Asp Ile1 5 10 15Phe Glu Ala Gln Lys Ile Glu Trp
His Glu Gly Ala Pro Gly Ala Arg 20 25 30Gly Ser Lys His Met Pro Arg
Lys Met Phe Ser Cys Asp Phe Glu Thr 35 40 45Thr Thr Lys Leu Asp Asp
Cys Arg Val Trp Ala Tyr Gly Tyr Met Glu 50 55 60Ile Gly Asn Leu Asp
Asn Tyr Lys Ile Gly Asn Ser Leu Asp Glu Phe65 70 75 80Met Gln Trp
Val Met Glu Ile Gln Ala Asp Leu Tyr Phe His Asn Leu 85 90 95Lys Phe
Asp Gly Ala Phe Ile Val Asn Trp Leu Glu His His Gly Phe 100 105
110Lys Trp Ser Asn Glu Gly Leu Pro Asn Thr Tyr Asn Thr Ile Ile Ser
115 120 125Lys Met Gly Gln Trp Tyr Met Ile Asp Ile Cys Phe Gly Tyr
Lys Gly 130 135 140Lys Arg Lys Leu His Thr Val Ile Tyr Asp Ser Leu
Lys Lys Leu Pro145 150 155 160Phe Pro Val Lys Lys Ile Ala Lys Asp
Phe Gln Leu Pro Leu Leu Lys 165 170 175Gly Asp Ile Asp Tyr His Ala
Glu Arg Pro Val Gly His Glu Ile Thr 180 185 190Pro Glu Glu Tyr Glu
Tyr Ile Lys Asn Ala Ile Glu Ile Ile Ala Arg 195 200 205Ala Leu Asp
Ile Gln Phe Lys Gln Gly Leu Asp Arg Met Thr Ala Gly 210 215 220Ser
Asp Ser Leu Lys Gly Phe Lys Asp Ile Leu Ser Thr Lys Lys Phe225 230
235 240Asn Lys Val Phe Pro Lys Leu Ser Leu Pro Met Asp Lys Glu Ile
Arg 245 250 255Arg Ala Tyr Arg Gly Gly Phe Thr Trp Leu Asn Asp Lys
Tyr Lys Glu 260 265 270Lys Glu Ile Gly Glu Gly Met Val Phe Asp Val
Asn Ser Leu Tyr Pro 275 280 285Ser Gln Met Tyr Ser Arg Pro Leu Pro
Tyr Gly Ala Pro Ile Val Phe 290 295 300Gln Gly Lys Tyr Glu Lys Asp
Glu Gln Tyr Pro Leu Tyr Ile Gln Arg305 310 315 320Ile Arg Phe Glu
Phe Glu Leu Lys Glu Gly Tyr Ile Pro Thr Ile Gln 325 330 335Ile Lys
Lys Asn Pro Phe Phe Lys Gly Asn Glu Tyr Leu Lys Asn Ser 340 345
350Gly Ala Glu Pro Val Glu Leu Tyr Leu Thr Asn Val Asp Leu Glu Leu
355 360 365Ile Gln Glu His Tyr Glu Met Tyr Asn Val Glu Tyr Ile Asp
Gly Phe 370 375 380Lys Phe Arg Glu Lys Thr Gly Leu Phe Lys Glu Phe
Ile Asp Lys Trp385 390 395 400Thr Tyr Val Lys Thr Arg Glu Lys Gly
Ala Lys Lys Gln Leu Ala Lys 405 410 415Leu Met Leu Asn Ser Leu Tyr
Gly Lys Phe Ala Ser Asn Pro Asp Val 420 425 430Thr Gly Lys Val Pro
Tyr Leu Lys Glu Asp Gly Ser Leu Gly Phe Arg 435 440 445Val Gly Asp
Glu Glu Tyr Lys Asp Pro Val Tyr Thr Pro Met Gly Val 450 455 460Phe
Ile Thr Ala Trp Ala Arg Phe Thr Thr Ile Thr Ala Ala Gln Ala465 470
475 480Cys Tyr Asp Arg Ile Ile Tyr Cys Asp Thr Asp Ser Ile His Leu
Thr 485 490 495Gly Thr Glu Val Pro Glu Ile Ile Lys Asp Ile Val Asp
Pro Lys Lys 500 505 510Leu Gly Tyr Trp Ala His Glu Ser Thr Phe Lys
Arg Ala Lys Tyr Leu 515 520 525Arg Gln Lys Thr Tyr Ile Gln Asp Ile
Tyr Ala Lys Glu Val Asp Gly 530 535 540Lys Leu Ile Glu Cys Ser Pro
Asp Glu Ala Thr Thr Thr Lys Phe Ser545 550 555 560Val Lys Cys Ala
Gly Met Thr Asp Thr Ile Lys Lys Lys Val Thr Phe 565 570 575Asp Asn
Phe Arg Val Gly Phe Ser Ser Thr Gly Lys Pro Lys Pro Val 580 585
590Gln Val Asn Gly Gly Val Val Leu Val Asp Ser Val Phe Thr Ile Lys
595 600 6054600PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 4Met Asn His Leu Val His His His His
His His Ile Glu Gly Arg His1 5 10 15Met Glu Leu Gly Thr Leu Glu Gly
Ser Met Lys His Met Pro Arg Lys 20 25 30Met Phe Ser Cys Asp Phe Glu
Thr Thr Thr Lys Leu Asp Asp Cys Arg 35 40 45Val Trp Ala Tyr Gly Tyr
Met Glu Ile Gly Asn Leu Asp Asn Tyr Lys 50 55 60Ile Gly Asn Ser Leu
Asp Glu Phe Met Gln Trp Val Met Glu Ile Gln65 70 75 80Ala Asp Leu
Tyr Phe His Asn Leu Lys Phe Asp Gly Ala Phe Ile Val 85 90 95Asn Trp
Leu Glu His His Gly Phe Lys Trp Ser Asn Glu Gly Leu Pro 100 105
110Asn Thr Tyr Asn Thr Ile Ile Ser Lys Met Gly Gln Trp Tyr Met Ile
115 120 125Asp Ile Cys Phe Gly Tyr Lys Gly Lys Arg Lys Leu His Thr
Val Ile 130 135 140Tyr Asp Ser Leu Lys Lys Leu Pro Phe Pro Val Lys
Lys Ile Ala Lys145 150 155 160Asp Phe Gln Leu Pro Leu Leu Lys Gly
Asp Ile Asp Tyr His Ala Glu 165 170 175Arg Pro Val Gly His Glu Ile
Thr Pro Glu Glu Tyr Glu Tyr Ile Lys 180 185 190Asn Asp Ile Glu Ile
Ile Ala Arg Ala Leu Asp Ile Gln Phe Lys Gln 195 200 205Gly Leu Asp
Arg Met Thr Ala Gly Ser Asp Ser Leu Lys Gly Phe Lys 210 215 220Asp
Ile Leu Ser Thr Lys Lys Phe Asn Lys Val Phe Pro Lys Leu Ser225 230
235 240Leu Pro Met Asp Lys Glu Ile Arg Arg Ala Tyr Arg Gly Gly Phe
Thr 245 250 255Trp Leu Asn Asp Lys Tyr Lys Glu Lys Glu Ile Gly Glu
Gly Met Val 260 265 270Phe Asp Val Asn Ser Leu Tyr Pro Ser Gln Met
Tyr Ser Arg Pro Leu 275 280 285Pro Tyr Gly Ala Pro Ile Val Phe Gln
Gly Lys Tyr Glu Lys Asp Glu 290 295 300Gln Tyr Pro Leu Tyr Ile Gln
Arg Ile Arg Phe Glu Phe Glu Leu Lys305 310 315 320Glu Gly Tyr Ile
Pro Thr Ile Gln Ile Lys Lys Asn Pro Phe Phe Lys 325 330 335Gly Asn
Glu Tyr Leu Lys Asn Ser Gly Ala Glu Pro Val Glu Leu Tyr 340 345
350Leu Thr Asn Val Asp Leu Glu Leu Ile Gln Glu His Tyr Glu Met Tyr
355 360 365Asn Val Glu Tyr Ile Asp Gly Phe Lys Phe Arg Glu Lys Thr
Gly Leu 370 375 380Phe Lys Glu Phe Ile Asp Lys Trp Thr Tyr Val Lys
Thr His Glu Lys385 390 395 400Gly Ala Lys Lys Gln Leu Ala Lys Leu
Met Leu Asn Ser Leu Tyr Gly 405 410 415Lys Phe Ala Ser Asn Pro Asp
Val Thr Gly Lys Val Pro Tyr Leu Lys 420 425 430Glu Asp Gly Ser Leu
Gly Phe Arg Val Gly Asp Glu Glu Tyr Lys Asp 435 440 445Pro Val Tyr
Thr Pro Met Gly Val Phe Ile Thr Ala Trp Ala Arg Phe 450 455 460Thr
Thr Ile Thr Ala Ala Gln Ala Cys Tyr Asp Arg Ile Ile Tyr Cys465 470
475 480Asp Thr Asp Ser Ile His Leu Thr Gly Thr Glu Val Pro Glu Ile
Ile 485 490 495Lys Asp Ile Val Asp Pro Lys Lys Leu Gly Tyr Trp Ala
His Glu Ser 500 505 510Thr Phe Lys Arg Ala Lys Tyr Leu Arg Gln Lys
Thr Tyr Ile Gln Asp 515 520 525Ile Tyr Ala Lys Glu Val Asp Gly Lys
Leu Ile Glu Cys Ser Pro Asp 530 535 540Glu Ala Thr Thr Thr Lys Phe
Ser Val Lys Cys Ala Gly Met Thr Asp545 550 555 560Thr Ile Lys Lys
Lys Val Thr Phe Asp Asn Phe Arg Val Gly Phe Ser 565 570 575Ser Thr
Gly Lys Pro Lys Pro Val Gln Val Asn Gly Gly Val Val Leu 580 585
590Val Asp Ser Val Phe Thr Ile Lys 595 6005608PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
5Met Ser His His His His His His Ser Met Ser Gly Leu Asn Asp Ile1 5
10 15Phe Glu Ala Gln Lys Ile Glu Trp His Glu Gly Ala Pro Gly Ala
Arg 20 25 30Gly Ser Lys His Met Pro Arg Lys Met Phe Ser Cys Asp Phe
Glu Thr 35 40 45Thr Thr Lys Leu Asp Asp Cys Arg Val Trp Ala Tyr Gly
Tyr Met Glu 50 55 60Ile Gly Asn Leu Asp Asn Tyr Lys Ile Gly Asn Ser
Leu Asp Glu Phe65 70 75
80Met Gln Trp Val Met Glu Ile Gln Ala Asp Leu Tyr Phe His Asn Leu
85 90 95Lys Phe Asp Gly Ala Phe Ile Val Asn Trp Leu Glu His His Gly
Phe 100 105 110Lys Trp Ser Asn Glu Gly Leu Pro Asn Thr Tyr Asn Thr
Ile Ile Ser 115 120 125Lys Met Gly Gln Trp Tyr Met Ile Asp Ile Cys
Phe Gly Tyr Lys Gly 130 135 140Lys Arg Lys Leu His Thr Val Ile Tyr
Asp Ser Leu Lys Lys Leu Pro145 150 155 160Phe Pro Val Lys Lys Ile
Ala Lys Asp Phe Gln Leu Pro Leu Leu Lys 165 170 175Gly Asp Ile Asp
Tyr His Ala Glu Arg Pro Val Gly His Glu Ile Thr 180 185 190Pro Glu
Glu Tyr Glu Tyr Ile Lys Asn Ala Ile Glu Ile Ile Ala Arg 195 200
205Ala Leu Asp Ile Gln Phe Lys Gln Gly Leu Asp Arg Met Thr Ala Gly
210 215 220Ser Asp Ser Leu Lys Gly Phe Lys Asp Ile Leu Ser Thr Lys
Lys Phe225 230 235 240Asn Lys Val Phe Pro Lys Leu Ser Leu Pro Met
Asp Lys Glu Ile Arg 245 250 255Arg Ala Tyr Arg Gly Gly Phe Thr Trp
Leu Asn Asp Lys Tyr Lys Glu 260 265 270Lys Glu Ile Gly Glu Gly Met
Val Phe Asp Val Asn Ser Leu Tyr Pro 275 280 285Ser Gln Met Tyr Ser
Arg Pro Leu Pro Tyr Gly Ala Pro Ile Val Phe 290 295 300Gln Gly Lys
Tyr Glu Lys Asp Glu Gln Tyr Pro Leu Tyr Ile Gln Arg305 310 315
320Ile Arg Phe Glu Phe Glu Leu Lys Glu Gly Tyr Ile Pro Thr Ile Gln
325 330 335Ile Lys Lys Asn Pro Phe Phe Lys Gly Asn Glu Tyr Leu Lys
Asn Ser 340 345 350Gly Ala Glu Pro Val Glu Leu Tyr Leu Thr Asn Val
Asp Leu Glu Leu 355 360 365Ile Gln Glu His Tyr Glu Met Tyr Asn Val
Glu Tyr Ile Asp Gly Phe 370 375 380Lys Phe Arg Glu Lys Thr Gly Leu
Phe Lys Glu Phe Ile Asp Lys Trp385 390 395 400Thr Tyr Val Lys Thr
His Glu Lys Gly Ala Lys Lys Gln Leu Ala Lys 405 410 415Leu Met Leu
Asn Ser Leu Tyr Gly Lys Phe Ala Ser Asn Pro Asp Val 420 425 430Thr
Gly Lys Val Pro Tyr Leu Lys Glu Asp Gly Ser Leu Gly Phe Arg 435 440
445Val Gly Asp Glu Glu Tyr Lys Asp Pro Val Tyr Thr Pro Met Gly Val
450 455 460Phe Ile Thr Ala Trp Ala Arg Phe Thr Thr Ile Thr Ala Ala
Gln Ala465 470 475 480Cys Tyr Asp Arg Ile Ile Tyr Cys Asp Thr Asp
Ser Ile His Leu Thr 485 490 495Gly Thr Glu Val Pro Glu Ile Ile Lys
Asp Ile Val Asp Pro Lys Lys 500 505 510Leu Gly Tyr Trp Ala His Glu
Ser Thr Phe Lys Arg Ala Lys Tyr Leu 515 520 525Arg Gln Lys Thr Tyr
Ile Gln Asp Ile Tyr Ala Lys Glu Val Asp Gly 530 535 540Lys Leu Ile
Glu Cys Ser Pro Asp Glu Ala Thr Thr Thr Lys Phe Ser545 550 555
560Val Lys Cys Ala Gly Met Thr Asp Thr Ile Lys Lys Lys Val Thr Phe
565 570 575Asp Asn Phe Arg Val Gly Phe Ser Ser Thr Gly Lys Pro Lys
Pro Val 580 585 590Gln Val Asn Gly Gly Val Val Leu Val Asp Ser Val
Phe Thr Ile Lys 595 600 6056575PRTBacillus phage phi29 6Met Lys His
Met Pro Arg Lys Met Tyr Ser Cys Asp Phe Glu Thr Thr1 5 10 15Thr Lys
Val Glu Asp Cys Arg Val Trp Ala Tyr Gly Tyr Met Asn Ile 20 25 30Glu
Asp His Ser Glu Tyr Lys Ile Gly Asn Ser Leu Asp Glu Phe Met 35 40
45Ala Trp Val Leu Lys Val Gln Ala Asp Leu Tyr Phe His Asn Leu Lys
50 55 60Phe Asp Gly Ala Phe Ile Ile Asn Trp Leu Glu Arg Asn Gly Phe
Lys65 70 75 80Trp Ser Ala Asp Gly Leu Pro Asn Thr Tyr Asn Thr Ile
Ile Ser Arg 85 90 95Met Gly Gln Trp Tyr Met Ile Asp Ile Cys Leu Gly
Tyr Lys Gly Lys 100 105 110Arg Lys Ile His Thr Val Ile Tyr Asp Ser
Leu Lys Lys Leu Pro Phe 115 120 125Pro Val Lys Lys Ile Ala Lys Asp
Phe Lys Leu Thr Val Leu Lys Gly 130 135 140Asp Ile Asp Tyr His Lys
Glu Arg Pro Val Gly Tyr Lys Ile Thr Pro145 150 155 160Glu Glu Tyr
Ala Tyr Ile Lys Asn Asp Ile Gln Ile Ile Ala Glu Ala 165 170 175Leu
Leu Ile Gln Phe Lys Gln Gly Leu Asp Arg Met Thr Ala Gly Ser 180 185
190Asp Ser Leu Lys Gly Phe Lys Asp Ile Ile Thr Thr Lys Lys Phe Lys
195 200 205Lys Val Phe Pro Thr Leu Ser Leu Gly Leu Asp Lys Glu Val
Arg Tyr 210 215 220Ala Tyr Arg Gly Gly Phe Thr Trp Leu Asn Asp Arg
Phe Lys Glu Lys225 230 235 240Glu Ile Gly Glu Gly Met Val Phe Asp
Val Asn Ser Leu Tyr Pro Ala 245 250 255Gln Met Tyr Ser Arg Leu Leu
Pro Tyr Gly Glu Pro Ile Val Phe Glu 260 265 270Gly Lys Tyr Val Trp
Asp Glu Asp Tyr Pro Leu His Ile Gln His Ile 275 280 285Arg Cys Glu
Phe Glu Leu Lys Glu Gly Tyr Ile Pro Thr Ile Gln Ile 290 295 300Lys
Arg Ser Arg Phe Tyr Lys Gly Asn Glu Tyr Leu Lys Ser Ser Gly305 310
315 320Gly Glu Ile Ala Asp Leu Trp Leu Ser Asn Val Asp Leu Glu Leu
Met 325 330 335Lys Glu His Tyr Asp Leu Tyr Asn Val Glu Tyr Ile Ser
Gly Leu Lys 340 345 350Phe Lys Ala Thr Thr Gly Leu Phe Lys Asp Phe
Ile Asp Lys Trp Thr 355 360 365Tyr Ile Lys Thr Thr Ser Glu Gly Ala
Ile Lys Gln Leu Ala Lys Leu 370 375 380Met Leu Asn Ser Leu Tyr Gly
Lys Phe Ala Ser Asn Pro Asp Val Thr385 390 395 400Gly Lys Val Pro
Tyr Leu Lys Glu Asn Gly Ala Leu Gly Phe Arg Leu 405 410 415Gly Glu
Glu Glu Thr Lys Asp Pro Val Tyr Thr Pro Met Gly Val Phe 420 425
430Ile Thr Ala Trp Ala Arg Tyr Thr Thr Ile Thr Ala Ala Gln Ala Cys
435 440 445Tyr Asp Arg Ile Ile Tyr Cys Asp Thr Asp Ser Ile His Leu
Thr Gly 450 455 460Thr Glu Ile Pro Asp Val Ile Lys Asp Ile Val Asp
Pro Lys Lys Leu465 470 475 480Gly Tyr Trp Ala His Glu Ser Thr Phe
Lys Arg Ala Lys Tyr Leu Arg 485 490 495Gln Lys Thr Tyr Ile Gln Asp
Ile Tyr Met Lys Glu Val Asp Gly Lys 500 505 510Leu Val Glu Gly Ser
Pro Asp Asp Tyr Thr Asp Ile Lys Phe Ser Val 515 520 525Lys Cys Ala
Gly Met Thr Asp Lys Ile Lys Lys Glu Val Thr Phe Glu 530 535 540Asn
Phe Lys Val Gly Phe Ser Arg Lys Met Lys Pro Lys Pro Val Gln545 550
555 560Val Pro Gly Gly Val Val Leu Val Asp Asp Thr Phe Thr Ile Lys
565 570 5757599PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 7Met Gly Leu Arg Arg Ala Ser Leu His
His Leu Leu Gly Gly Gly Gly1 5 10 15Ser Gly Gly Gly Gly Ser Ala Ala
Ala Gly Ser Ala Ala Arg Lys Met 20 25 30Tyr Ser Cys Asp Phe Glu Thr
Thr Thr Lys Val Glu Asp Cys Arg Val 35 40 45Trp Ala Tyr Gly Tyr Met
Asn Ile Glu Asp His Ser Glu Tyr Lys Ile 50 55 60Gly Asn Ser Leu Asp
Glu Phe Met Ala Trp Val Leu Lys Val Gln Ala65 70 75 80Asp Leu Tyr
Phe His Asn Leu Lys Phe Asp Gly Ala Phe Ile Ile Asn 85 90 95Trp Leu
Glu Arg Asn Gly Phe Lys Trp Ser Ala Asp Gly Leu Pro Asn 100 105
110Thr Tyr Asn Thr Ile Ile Ser Arg Met Gly Gln Trp Tyr Met Ile Asp
115 120 125Ile Cys Leu Gly Tyr Lys Gly Lys Arg Lys Ile His Thr Val
Ile Tyr 130 135 140Asp Ser Leu Lys Lys Leu Pro Phe Pro Val Lys Lys
Ile Ala Lys Asp145 150 155 160Phe Lys Leu Thr Val Leu Lys Gly Asp
Ile Asp Tyr His Lys Glu Arg 165 170 175Pro Val Gly Tyr Lys Ile Thr
Pro Glu Glu Tyr Ala Tyr Ile Lys Asn 180 185 190Asp Ile Gln Ile Ile
Ala Glu Ala Leu Leu Ile Gln Phe Lys Gln Gly 195 200 205Leu Asp Arg
Met Thr Ala Gly Ser Asp Ser Leu Lys Gly Phe Lys Asp 210 215 220Ile
Ile Thr Thr Lys Lys Phe Lys Lys Val Phe Pro Thr Leu Ser Leu225 230
235 240Gly Leu Asp Lys Glu Val Arg Tyr Ala Tyr Arg Gly Gly Phe Thr
Trp 245 250 255Leu Asn Asp Arg Phe Lys Glu Lys Glu Ile Gly Glu Gly
Met Val Phe 260 265 270Asp Val Asn Ser Leu Tyr Pro Ala Gln Met Tyr
Ser Arg Leu Leu Pro 275 280 285Tyr Gly Glu Pro Ile Val Phe Glu Gly
Lys Tyr Val Trp Asp Glu Asp 290 295 300Tyr Pro Leu His Ile Gln His
Ile Arg Cys Glu Phe Glu Leu Lys Glu305 310 315 320Gly Tyr Ile Pro
Thr Ile Gln Ile Lys Arg Ser Arg Phe Tyr Lys Gly 325 330 335Asn Glu
Tyr Leu Lys Ser Ser Gly Gly Glu Ile Ala Asp Leu Trp Leu 340 345
350Ser Asn Val Asp Leu Glu Leu Met Lys Glu His Tyr Asp Leu Tyr Asn
355 360 365Val Glu Tyr Ile Ser Gly Leu Lys Phe Lys Ala Thr Thr Gly
Leu Phe 370 375 380Lys Asp Phe Ile Asp Lys Trp Thr Tyr Ile Lys Thr
Thr Ser Glu Gly385 390 395 400Ala Ile Lys Gln Leu Ala Lys Leu Met
Leu Asn Ser Leu Tyr Gly Lys 405 410 415Phe Ala Ser Asn Pro Asp Val
Thr Gly Lys Val Pro Tyr Leu Lys Glu 420 425 430Asn Gly Ala Leu Gly
Phe Arg Leu Gly Glu Glu Glu Thr Lys Asp Pro 435 440 445Val Tyr Thr
Pro Met Gly Val Phe Ile Thr Ala Trp Ala Arg Tyr Thr 450 455 460Thr
Ile Thr Ala Ala Gln Ala Cys Tyr Asp Arg Ile Ile Tyr Cys Asp465 470
475 480Thr Asp Ser Ile His Leu Thr Gly Thr Glu Ile Pro Asp Val Ile
Lys 485 490 495Asp Ile Val Asp Pro Lys Lys Leu Gly Tyr Trp Ala His
Glu Ser Thr 500 505 510Phe Lys Arg Ala Lys Tyr Leu Arg Gln Lys Thr
Tyr Ile Gln Asp Ile 515 520 525Tyr Met Lys Glu Val Asp Gly Lys Leu
Val Glu Gly Ser Pro Asp Asp 530 535 540Tyr Thr Asp Ile Lys Phe Ser
Val Lys Cys Ala Gly Met Thr Asp Lys545 550 555 560Ile Lys Lys Glu
Val Thr Phe Glu Asn Phe Lys Val Gly Phe Ser Arg 565 570 575Lys Met
Lys Pro Lys Pro Val Gln Val Pro Gly Gly Val Val Leu Val 580 585
590Asp Asp Thr Phe Thr Ile Lys 5958596PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
8Met His His His His His His Leu Leu Gly Gly Gly Gly Ser Gly Gly1 5
10 15Gly Gly Ser Ala Ala Ala Gly Ser Ala Ala Arg Lys Met Tyr Ser
Cys 20 25 30Asp Phe Glu Thr Thr Thr Lys Val Glu Asp Cys Arg Val Trp
Ala Tyr 35 40 45Gly Tyr Met Asn Ile Glu Asp His Ser Glu Tyr Lys Ile
Gly Asn Ser 50 55 60Leu Asp Glu Phe Met Ala Trp Val Leu Lys Val Gln
Ala Asp Leu Tyr65 70 75 80Phe His Asn Leu Lys Phe Asp Gly Ala Phe
Ile Ile Asn Trp Leu Glu 85 90 95Arg Asn Gly Phe Lys Trp Ser Ala Asp
Gly Leu Pro Asn Thr Tyr Asn 100 105 110Thr Ile Ile Ser Arg Met Gly
Gln Trp Tyr Met Ile Asp Ile Cys Leu 115 120 125Gly Tyr Lys Gly Lys
Arg Lys Ile His Thr Val Ile Tyr Asp Ser Leu 130 135 140Lys Lys Leu
Pro Phe Pro Val Lys Lys Ile Ala Lys Asp Phe Lys Leu145 150 155
160Thr Val Leu Lys Gly Asp Ile Asp Tyr His Lys Glu Arg Pro Val Gly
165 170 175Tyr Lys Ile Thr Pro Glu Glu Tyr Ala Tyr Ile Lys Asn Ala
Ile Gln 180 185 190Ile Ile Ala Glu Ala Leu Leu Ile Gln Phe Lys Gln
Gly Leu Asp Arg 195 200 205Met Thr Ala Gly Ser Asp Ser Leu Lys Gly
Phe Lys Asp Ile Ile Thr 210 215 220Thr Lys Lys Phe Lys Lys Val Phe
Pro Thr Leu Ser Leu Gly Leu Asp225 230 235 240Lys Glu Val Arg Tyr
Ala Tyr Arg Gly Gly Phe Thr Trp Leu Asn Asp 245 250 255Arg Phe Lys
Glu Lys Glu Ile Gly Glu Gly Met Val Phe Asp Val Asn 260 265 270Ser
Leu Tyr Pro Ala Gln Met Tyr Ser Arg Leu Leu Pro Tyr Gly Glu 275 280
285Pro Ile Val Phe Glu Gly Lys Tyr Val Trp Asp Glu Asp Tyr Pro Leu
290 295 300His Ile Gln His Ile Arg Cys Glu Phe Glu Leu Lys Glu Gly
Tyr Ile305 310 315 320Pro Thr Ile Gln Ile Lys Arg Ser Arg Phe Tyr
Lys Gly Asn Glu Tyr 325 330 335Leu Lys Ser Ser Gly Gly Glu Ile Ala
Asp Leu Trp Leu Ser Asn Val 340 345 350Asp Leu Glu Leu Met Lys Glu
His Tyr Asp Leu Tyr Asn Val Glu Tyr 355 360 365Ile Ser Gly Leu Lys
Phe Lys Ala Thr Thr Gly Leu Phe Lys Asp Phe 370 375 380Ile Asp Lys
Trp Thr Tyr Ile Lys Thr Thr Ser Glu Gly Ala Ile Lys385 390 395
400Gln Leu Ala Lys Leu Met Leu Asn Ser Leu Tyr Gly Lys Phe Ala Ser
405 410 415Asn Pro Asp Val Thr Gly Lys Val Pro Tyr Leu Lys Glu Asn
Gly Ala 420 425 430Leu Gly Phe Arg Leu Gly Glu Glu Glu Thr Lys Asp
Pro Val Tyr Thr 435 440 445Pro Met Gly Val Phe Ile Thr Ala Trp Ala
Arg Tyr Thr Thr Ile Thr 450 455 460Ala Ala Gln Ala Cys Tyr Asp Arg
Ile Ile Tyr Cys Asp Thr Asp Ser465 470 475 480Ile His Leu Thr Gly
Thr Glu Ile Pro Asp Val Ile Lys Asp Ile Val 485 490 495Asp Pro Lys
Lys Leu Gly Tyr Trp Ala His Glu Ser Thr Phe Lys Arg 500 505 510Ala
Lys Tyr Leu Arg Gln Lys Thr Tyr Ile Gln Asp Ile Tyr Met Lys 515 520
525Glu Val Asp Gly Lys Leu Val Glu Gly Ser Pro Asp Asp Tyr Thr Asp
530 535 540Ile Lys Phe Ser Val Lys Cys Ala Gly Met Thr Asp Lys Ile
Lys Lys545 550 555 560Glu Val Thr Phe Glu Asn Phe Lys Val Gly Phe
Ser Arg Lys Met Lys 565 570 575Pro Lys Pro Val Gln Val Pro Gly Gly
Val Val Leu Val Asp Asp Thr 580 585 590Phe Thr Ile Lys
5959600PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 9Met Asn His Leu Val His His His His His His
Ile Glu Gly Arg His1 5 10 15Met Glu Leu Gly Thr Leu Glu Gly Ser Met
Lys His Met Pro Arg Lys 20 25 30Met Tyr Ser Cys Ala Phe Glu Thr Thr
Thr Lys Val Glu Asp Cys Arg 35 40 45Val Trp Ala Tyr Gly Tyr Met Asn
Ile Glu Asp His Ser Glu Tyr Lys 50 55 60Ile Gly Asn Ser Leu Asp Glu
Phe Met Ala Trp Val Leu Lys Val Gln65 70 75 80Ala Asp Leu Tyr Phe
His Asn Leu Lys Phe Ala Gly Ala Phe Ile Ile 85 90 95Asn Trp Leu Glu
Arg Asn Gly Phe Lys Trp Ser Ala Asp Gly Leu Pro 100 105 110Asn Thr
Tyr Asn Thr Ile Ile Ser Arg Met Gly Gln Trp Tyr Met Ile 115 120
125Asp Ile Cys Leu Gly Tyr Lys
Gly Lys Arg Lys Ile His Thr Val Ile 130 135 140Tyr Asp Ser Leu Lys
Lys Leu Pro Phe Pro Val Lys Lys Ile Ala Lys145 150 155 160Asp Phe
Lys Leu Thr Val Leu Lys Gly Asp Ile Asp Tyr His Lys Glu 165 170
175Arg Pro Val Gly Tyr Lys Ile Thr Pro Glu Glu Tyr Ala Tyr Ile Lys
180 185 190Asn Asp Ile Gln Ile Ile Ala Glu Ala Leu Leu Ile Gln Phe
Lys Gln 195 200 205Gly Leu Asp Arg Met Thr Ala Gly Ser Asp Ser Leu
Lys Gly Phe Lys 210 215 220Asp Ile Ile Thr Thr Lys Lys Phe Lys Lys
Val Phe Pro Thr Leu Ser225 230 235 240Leu Gly Leu Asp Lys Glu Val
Arg Tyr Ala Tyr Arg Gly Gly Phe Thr 245 250 255Trp Leu Asn Asp Arg
Phe Lys Glu Lys Glu Ile Gly Glu Gly Met Val 260 265 270Phe Asp Val
Asn Ser Leu Tyr Pro Ala Gln Met Tyr Ser Arg Leu Leu 275 280 285Pro
Tyr Gly Glu Pro Ile Val Phe Glu Gly Lys Tyr Val Trp Asp Glu 290 295
300Asp Tyr Pro Leu His Ile Gln His Ile Arg Cys Glu Phe Glu Leu
Lys305 310 315 320Glu Gly Tyr Ile Pro Thr Ile Gln Ile Lys Arg Ser
Arg Phe Tyr Lys 325 330 335Gly Asn Glu Tyr Leu Lys Ser Ser Gly Gly
Glu Ile Ala Asp Leu Trp 340 345 350Leu Ser Asn Val Asp Leu Glu Leu
Met Lys Glu His Tyr Asp Leu Tyr 355 360 365Asn Val Glu Tyr Ile Ser
Gly Leu Lys Phe Lys Ala Thr Thr Gly Leu 370 375 380Phe Lys Asp Phe
Ile Asp Lys Trp Thr Tyr Ile Lys Thr Thr Ser Glu385 390 395 400Gly
Ala Ile Lys Gln Leu Ala Lys Leu Met Leu Asn Ser Leu Tyr Gly 405 410
415Lys Phe Ala Ser Asn Pro Asp Val Thr Gly Lys Val Pro Tyr Leu Lys
420 425 430Glu Asn Gly Ala Leu Gly Phe Arg Leu Gly Glu Glu Glu Thr
Lys Asp 435 440 445Pro Val Tyr Thr Pro Met Gly Val Phe Ile Thr Ala
Trp Ala Arg Tyr 450 455 460Thr Thr Ile Thr Ala Ala Gln Ala Cys Tyr
Asp Arg Ile Ile Tyr Cys465 470 475 480Asp Thr Asp Ser Ile His Leu
Thr Gly Thr Glu Ile Pro Asp Val Ile 485 490 495Lys Asp Ile Val Asp
Pro Lys Lys Leu Gly Tyr Trp Ala His Glu Ser 500 505 510Thr Phe Lys
Arg Ala Lys Tyr Leu Arg Gln Lys Thr Tyr Ile Gln Asp 515 520 525Ile
Tyr Met Lys Glu Val Asp Gly Lys Leu Val Glu Gly Ser Pro Asp 530 535
540Asp Tyr Thr Asp Ile Lys Phe Ser Val Lys Cys Ala Gly Met Thr
Asp545 550 555 560Lys Ile Lys Lys Glu Val Thr Phe Glu Asn Phe Lys
Val Gly Phe Ser 565 570 575Arg Lys Met Lys Pro Lys Pro Val Gln Val
Pro Gly Gly Val Val Leu 580 585 590Val Asp Asp Thr Phe Thr Ile Lys
595 60010600PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 10Met Asn His Leu Val His His His
His His His Ile Glu Gly Arg His1 5 10 15Met Glu Leu Gly Thr Leu Glu
Gly Ser Met Lys His Met Pro Arg Lys 20 25 30Met Tyr Ser Cys Ala Phe
Glu Thr Thr Thr Lys Val Glu Asp Cys Arg 35 40 45Val Trp Ala Tyr Gly
Tyr Met Asn Ile Glu Asp His Ser Glu Tyr Lys 50 55 60Ile Gly Asn Ser
Leu Asp Glu Phe Met Ala Trp Val Leu Lys Val Gln65 70 75 80Ala Asp
Leu Tyr Phe His Asn Leu Lys Phe Ala Gly Ala Phe Ile Ile 85 90 95Asn
Trp Leu Glu Arg Asn Gly Phe Lys Trp Ser Ala Asp Gly Leu Pro 100 105
110Asn Thr Tyr Asn Thr Ile Ile Ser Arg Met Gly Gln Trp Tyr Met Ile
115 120 125Asp Ile Cys Leu Gly Tyr Lys Gly Lys Arg Lys Ile His Thr
Val Ile 130 135 140Tyr Asp Ser Leu Lys Lys Leu Pro Phe Pro Val Lys
Lys Ile Ala Lys145 150 155 160Asp Phe Lys Leu Thr Val Leu Lys Gly
Asp Ile Asp Tyr His Lys Glu 165 170 175Arg Pro Val Gly Tyr Lys Ile
Thr Pro Glu Glu Tyr Ala Tyr Ile Lys 180 185 190Asn Asp Ile Gln Ile
Ile Ala Glu Ala Leu Leu Ile Gln Phe Lys Gln 195 200 205Gly Leu Asp
Arg Met Thr Ala Gly Ser Asp Ser Leu Lys Gly Phe Lys 210 215 220Asp
Ile Ile Thr Thr Lys Lys Phe Lys Lys Val Phe Pro Thr Leu Ser225 230
235 240Leu Gly Leu Asp Lys Glu Val Arg Tyr Ala Tyr Arg Gly Gly Phe
Thr 245 250 255Trp Leu Asn Asp Arg Phe Lys Glu Lys Glu Ile Gly Glu
Gly Met Val 260 265 270Phe Asp Val Asn Ser Leu Tyr Pro Ala Gln Met
Tyr Ser Arg Leu Leu 275 280 285Pro Tyr Gly Glu Pro Ile Val Phe Glu
Gly Lys Tyr Val Trp Asp Glu 290 295 300Asp Tyr Pro Leu His Ile Gln
His Ile Arg Cys Glu Phe Glu Leu Lys305 310 315 320Glu Gly Tyr Ile
Pro Thr Ile Gln Ile Lys Arg Ser Arg Phe Tyr Lys 325 330 335Gly Asn
Glu Tyr Leu Lys Ser Ser Gly Gly Glu Ile Ala Asp Leu Trp 340 345
350Leu Ser Asn Val Asp Leu Glu Leu Met Lys Glu His Tyr Asp Leu Tyr
355 360 365Asn Val Glu Tyr Ile Ser Gly Leu Lys Phe Lys Ala Thr Thr
Gly Leu 370 375 380Phe Lys Asp Phe Ile Asp Lys Trp Thr Tyr Ile Lys
Thr Thr Ser Glu385 390 395 400Gly Ala Ile Lys Ala Leu Ala Lys Leu
Met Leu Asn Ser Leu Tyr Gly 405 410 415Lys Phe Ala Ser Asn Pro Asp
Val Thr Gly Lys Val Pro Tyr Leu Lys 420 425 430Glu Asn Gly Ala Leu
Gly Phe Arg Leu Gly Glu Glu Glu Thr Lys Asp 435 440 445Pro Val Tyr
Thr Pro Met Gly Val Phe Ile Thr Ala Trp Ala Arg Tyr 450 455 460Thr
Thr Ile Thr Ala Ala Gln Ala Cys Tyr Asp Arg Ile Ile Tyr Cys465 470
475 480Asp Thr Asp Ser Ile His Leu Thr Gly Thr Glu Ile Pro Asp Val
Ile 485 490 495Lys Asp Ile Val Asp Pro Lys Lys Leu Gly Tyr Trp Ala
His Glu Ser 500 505 510Thr Phe Lys Arg Ala Lys Tyr Leu Arg Gln Lys
Thr Tyr Ile Gln Asp 515 520 525Ile Tyr Met Lys Glu Val Asp Gly Lys
Leu Val Glu Gly Ser Pro Asp 530 535 540Asp Tyr Thr Asp Ile Lys Phe
Ser Val Lys Cys Ala Gly Met Thr Asp545 550 555 560Lys Ile Lys Lys
Glu Val Thr Phe Glu Asn Phe Lys Val Gly Phe Ser 565 570 575Arg Lys
Met Lys Pro Lys Pro Val Gln Val Pro Gly Gly Val Val Leu 580 585
590Val Asp Asp Thr Phe Thr Ile Lys 595 60011600PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
11Met Asn His Leu Val His His His His His His Ile Glu Gly Arg His1
5 10 15Met Glu Leu Gly Thr Leu Glu Gly Ser Met Lys His Met Pro Arg
Lys 20 25 30Met Tyr Ser Cys Ala Phe Glu Thr Thr Thr Lys Val Glu Asp
Cys Arg 35 40 45Val Trp Ala Tyr Gly Tyr Met Asn Ile Glu Asp His Ser
Glu Tyr Lys 50 55 60Ile Gly Asn Ser Leu Asp Glu Phe Met Ala Trp Val
Leu Lys Val Gln65 70 75 80Ala Asp Leu Tyr Phe His Asn Leu Lys Phe
Ala Gly Ala Phe Ile Ile 85 90 95Asn Trp Leu Glu Arg Asn Gly Phe Lys
Trp Ser Ala Asp Gly Leu Pro 100 105 110Asn Thr Tyr Asn Thr Ile Ile
Ser Arg Met Gly Gln Trp Tyr Met Ile 115 120 125Asp Ile Cys Leu Gly
Tyr Lys Gly Lys Arg Lys Ile His Thr Val Ile 130 135 140Tyr Asp Ser
Leu Lys Lys Leu Pro Phe Pro Val Lys Lys Ile Ala Lys145 150 155
160Asp Phe Lys Leu Thr Val Leu Lys Gly Asp Ile Asp Tyr His Lys Glu
165 170 175Arg Pro Val Gly Tyr Lys Ile Thr Pro Glu Glu Tyr Ala Tyr
Ile Lys 180 185 190Asn Asp Ile Gln Ile Ile Ala Glu Ala Leu Leu Ile
Gln Phe Lys Gln 195 200 205Gly Leu Asp Arg Met Thr Ala Gly Ser Asp
Ser Leu Lys Gly Phe Lys 210 215 220Asp Ile Ile Thr Thr Lys Lys Phe
Lys Lys Val Phe Pro Thr Leu Ser225 230 235 240Leu Gly Leu Asp Lys
Glu Val Arg Tyr Ala Tyr Arg Gly Gly Phe Thr 245 250 255Trp Leu Asn
Asp Arg Phe Lys Glu Lys Glu Ile Gly Glu Gly Met Val 260 265 270Phe
Asp Val Asn Ser Leu Tyr Pro Ala Gln Met Tyr Ser Arg Leu Leu 275 280
285Pro Tyr Gly Glu Pro Ile Val Phe Glu Gly Lys Tyr Val Trp Asp Glu
290 295 300Asp Tyr Pro Leu His Ile Gln His Ile Arg Cys Glu Phe Glu
Leu Lys305 310 315 320Glu Gly Tyr Ile Pro Thr Ile Gln Ile Lys Arg
Ser Arg Phe Tyr Lys 325 330 335Gly Asn Glu Tyr Leu Lys Ser Ser Gly
Gly Glu Ile Ala Asp Leu Trp 340 345 350Leu Ser Asn Val Asp Leu Glu
Leu Met Lys Glu His Tyr Asp Leu Tyr 355 360 365Asn Val Glu Tyr Ile
Ser Gly Leu Lys Phe Lys Ala Thr Thr Gly Leu 370 375 380Phe Lys Asp
Phe Ile Asp Lys Trp Thr Tyr Ile Lys Thr Thr Ser Glu385 390 395
400Gly Ala Ile Lys Gln Leu Ala Lys Leu Met Leu Asn Gly Leu Tyr Gly
405 410 415Lys Phe Ala Ser Asn Pro Asp Val Thr Gly Lys Val Pro Tyr
Leu Lys 420 425 430Glu Asn Gly Ala Leu Gly Phe Arg Leu Gly Glu Glu
Glu Thr Lys Asp 435 440 445Pro Val Tyr Thr Pro Met Gly Val Phe Ile
Thr Ala Trp Ala Arg Tyr 450 455 460Thr Thr Ile Thr Ala Ala Gln Ala
Cys Tyr Asp Arg Ile Ile Tyr Cys465 470 475 480Asp Thr Asp Ser Ile
His Leu Thr Gly Thr Glu Ile Pro Asp Val Ile 485 490 495Lys Asp Ile
Val Asp Pro Lys Lys Leu Gly Tyr Trp Ala His Glu Ser 500 505 510Thr
Phe Lys Arg Ala Lys Tyr Leu Arg Gln Lys Thr Tyr Ile Gln Asp 515 520
525Ile Tyr Met Lys Glu Val Asp Gly Lys Leu Val Glu Gly Ser Pro Asp
530 535 540Asp Tyr Thr Asp Ile Lys Phe Ser Val Lys Cys Ala Gly Met
Thr Asp545 550 555 560Lys Ile Lys Lys Glu Val Thr Phe Glu Asn Phe
Lys Val Gly Phe Ser 565 570 575Arg Lys Met Lys Pro Lys Pro Val Gln
Val Pro Gly Gly Val Val Leu 580 585 590Val Asp Asp Thr Phe Thr Ile
Lys 595 60012608PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 12Met Ser His His His His His His
Ser Met Ser Gly Leu Asn Asp Ile1 5 10 15Phe Glu Ala Gln Lys Ile Glu
Trp His Glu Gly Ala Pro Gly Ala Arg 20 25 30Gly Ser Lys His Met Pro
Arg Lys Met Tyr Ser Cys Ala Phe Glu Thr 35 40 45Thr Thr Lys Val Glu
Asp Cys Arg Val Trp Ala Tyr Gly Tyr Met Asn 50 55 60Ile Glu Asp His
Ser Glu Tyr Lys Ile Gly Asn Ser Leu Asp Glu Phe65 70 75 80Met Ala
Trp Val Leu Lys Val Gln Ala Asp Leu Tyr Phe His Asn Leu 85 90 95Lys
Phe Ala Gly Ala Phe Ile Ile Asn Trp Leu Glu Arg Asn Gly Phe 100 105
110Lys Trp Ser Ala Asp Gly Leu Pro Asn Thr Tyr Asn Thr Ile Ile Ser
115 120 125Arg Met Gly Gln Trp Tyr Met Ile Asp Ile Cys Leu Gly Tyr
Lys Gly 130 135 140Lys Arg Lys Ile His Thr Val Ile Tyr Asp Ser Leu
Lys Lys Leu Pro145 150 155 160Phe Pro Val Lys Lys Ile Ala Lys Asp
Phe Lys Leu Thr Val Leu Lys 165 170 175Gly Asp Ile Asp Tyr His Lys
Glu Arg Pro Val Gly Tyr Lys Ile Thr 180 185 190Pro Glu Glu Tyr Ala
Tyr Ile Lys Asn Asp Ile Gln Ile Ile Ala Glu 195 200 205Ala Leu Leu
Ile Gln Phe Lys Gln Gly Leu Asp Arg Met Thr Ala Gly 210 215 220Ser
Asp Ser Leu Lys Gly Phe Lys Asp Ile Ile Thr Thr Lys Lys Phe225 230
235 240Lys Lys Val Phe Pro Thr Leu Ser Leu Gly Leu Asp Lys Glu Val
Arg 245 250 255Tyr Ala Tyr Arg Gly Gly Phe Thr Trp Leu Asn Asp Arg
Phe Lys Glu 260 265 270Lys Glu Ile Gly Glu Gly Met Val Phe Asp Val
Asn Ser Leu Tyr Pro 275 280 285Ala Gln Met Tyr Ser Arg Leu Leu Pro
Tyr Gly Glu Pro Ile Val Phe 290 295 300Glu Gly Lys Tyr Val Trp Asp
Glu Asp Tyr Pro Leu His Ile Gln His305 310 315 320Ile Arg Cys Glu
Phe Glu Leu Lys Glu Gly Tyr Ile Pro Thr Ile Gln 325 330 335Ile Lys
Arg Ser Arg Phe Tyr Lys Gly Asn Glu Tyr Leu Lys Ser Ser 340 345
350Gly Gly Glu Ile Ala Asp Leu Trp Leu Ser Asn Val Asp Leu Glu Leu
355 360 365Met Lys Glu His Tyr Asp Leu Tyr Asn Val Glu Tyr Ile Ser
Gly Leu 370 375 380Lys Phe Lys Ala Thr Thr Gly Leu Phe Lys Asp Phe
Ile Asp Lys Trp385 390 395 400Thr Tyr Ile Lys Thr Thr Ser Glu Gly
Ala Ile Lys Gln Leu Ala Lys 405 410 415Leu Met Leu Asn Ser Leu Tyr
Gly Lys Phe Ala Ser Asn Pro Asp Val 420 425 430Thr Gly Lys Val Pro
Tyr Leu Lys Glu Asn Gly Ala Leu Gly Phe Arg 435 440 445Leu Gly Glu
Glu Glu Thr Lys Asp Pro Val Tyr Thr Pro Met Gly Val 450 455 460Phe
Ile Thr Ala Trp Ala Arg Tyr Thr Thr Ile Thr Ala Ala Gln Ala465 470
475 480Cys Tyr Asp Arg Ile Ile Tyr Cys Asp Thr Asp Ser Ile His Leu
Thr 485 490 495Gly Thr Glu Ile Pro Asp Val Ile Lys Asp Ile Val Asp
Pro Lys Lys 500 505 510Leu Gly Tyr Trp Ala His Glu Ser Thr Phe Lys
Arg Ala Lys Tyr Leu 515 520 525Arg Gln Lys Thr Tyr Ile Gln Asp Ile
Tyr Met Lys Glu Val Asp Gly 530 535 540Lys Leu Val Glu Gly Ser Pro
Asp Asp Tyr Thr Asp Ile Lys Phe Ser545 550 555 560Val Lys Cys Ala
Gly Met Thr Asp Lys Ile Lys Lys Glu Val Thr Phe 565 570 575Glu Asn
Phe Lys Val Gly Phe Ser Arg Lys Met Lys Pro Lys Pro Val 580 585
590Gln Val Pro Gly Gly Val Val Leu Val Asp Asp Thr Phe Thr Ile Lys
595 600 60513903PRTEnterobacteria phage RB69 13Met Lys Glu Phe Tyr
Leu Thr Val Glu Gln Ile Gly Asp Ser Ile Phe1 5 10 15Glu Arg Tyr Ile
Asp Ser Asn Gly Arg Glu Arg Thr Arg Glu Val Glu 20 25 30Tyr Lys Pro
Ser Leu Phe Ala His Cys Pro Glu Ser Gln Ala Thr Lys 35 40 45Tyr Phe
Asp Ile Tyr Gly Lys Pro Cys Thr Arg Lys Leu Phe Ala Asn 50 55 60Met
Arg Asp Ala Ser Gln Trp Ile Lys Arg Met Glu Asp Ile Gly Leu65 70 75
80Glu Ala Leu Gly Met Asp Asp Phe Lys Leu Ala Tyr Leu Ser Asp Thr
85 90 95Tyr Asn Tyr Glu Ile Lys Tyr Asp His Thr Lys Ile Arg Val Ala
Asn 100 105 110Phe Asp Ile Glu Val Thr Ser Pro Asp Gly Phe Pro Glu
Pro Ser Gln 115 120 125Ala Lys His Pro Ile Asp Ala Ile Thr His Tyr
Asp Ser Ile Asp Asp 130 135 140Arg Phe Tyr Val Phe Asp Leu Leu Asn
Ser Pro Tyr Gly Asn Val Glu145
150 155 160Glu Trp Ser Ile Glu Ile Ala Ala Lys Leu Gln Glu Gln Gly
Gly Asp 165 170 175Glu Val Pro Ser Glu Ile Ile Asp Lys Ile Ile Tyr
Met Pro Phe Asp 180 185 190Asn Glu Lys Glu Leu Leu Met Glu Tyr Leu
Asn Phe Trp Gln Gln Lys 195 200 205Thr Pro Val Ile Leu Thr Gly Trp
Asn Val Glu Ser Phe Asp Ile Pro 210 215 220Tyr Val Tyr Asn Arg Ile
Lys Asn Ile Phe Gly Glu Ser Thr Ala Lys225 230 235 240Arg Leu Ser
Pro His Arg Lys Thr Arg Val Lys Val Ile Glu Asn Met 245 250 255Tyr
Gly Ser Arg Glu Ile Ile Thr Leu Phe Gly Ile Ser Val Leu Asp 260 265
270Tyr Ile Asp Leu Tyr Lys Lys Phe Ser Phe Thr Asn Gln Pro Ser Tyr
275 280 285Ser Leu Asp Tyr Ile Ser Glu Phe Glu Leu Asn Val Gly Lys
Leu Lys 290 295 300Tyr Asp Gly Pro Ile Ser Lys Leu Arg Glu Ser Asn
His Gln Arg Tyr305 310 315 320Ile Ser Tyr Asn Ile Ile Asp Val Tyr
Arg Val Leu Gln Ile Asp Ala 325 330 335Lys Arg Gln Phe Ile Asn Leu
Ser Leu Asp Met Gly Tyr Tyr Ala Lys 340 345 350Ile Gln Ile Gln Ser
Val Phe Ser Pro Ile Lys Thr Trp Asp Ala Ile 355 360 365Ile Phe Asn
Ser Leu Lys Glu Gln Asn Lys Val Ile Pro Gln Gly Arg 370 375 380Ser
His Pro Val Gln Pro Tyr Pro Gly Ala Phe Val Lys Glu Pro Ile385 390
395 400Pro Asn Arg Tyr Lys Tyr Val Met Ser Phe Asp Leu Thr Ser Leu
Tyr 405 410 415Pro Ser Ile Ile Arg Gln Val Asn Ile Ser Pro Glu Thr
Ile Ala Gly 420 425 430Thr Phe Lys Val Ala Pro Leu His Asp Tyr Ile
Asn Ala Val Ala Glu 435 440 445Arg Pro Ser Asp Val Tyr Ser Cys Ser
Pro Asn Gly Met Met Tyr Tyr 450 455 460Lys Asp Arg Asp Gly Val Val
Pro Thr Glu Ile Thr Lys Val Phe Asn465 470 475 480Gln Arg Lys Glu
His Lys Gly Tyr Met Leu Ala Ala Gln Arg Asn Gly 485 490 495Glu Ile
Ile Lys Glu Ala Leu His Asn Pro Asn Leu Ser Val Asp Glu 500 505
510Pro Leu Asp Val Asp Tyr Arg Phe Asp Phe Ser Asp Glu Ile Lys Glu
515 520 525Lys Ile Lys Lys Leu Ser Ala Lys Ser Leu Asn Glu Met Leu
Phe Arg 530 535 540Ala Gln Arg Thr Glu Val Ala Gly Met Thr Ala Gln
Ile Asn Arg Lys545 550 555 560Leu Leu Ile Asn Ser Leu Tyr Gly Ala
Leu Gly Asn Val Trp Phe Arg 565 570 575Tyr Tyr Asp Leu Arg Asn Ala
Thr Ala Ile Thr Thr Phe Gly Gln Met 580 585 590Ala Leu Gln Trp Ile
Glu Arg Lys Val Asn Glu Tyr Leu Asn Glu Val 595 600 605Cys Gly Thr
Glu Gly Glu Ala Phe Val Leu Tyr Gly Asp Thr Asp Ser 610 615 620Ile
Tyr Val Ser Ala Asp Lys Ile Ile Asp Lys Val Gly Glu Ser Lys625 630
635 640Phe Arg Asp Thr Asn His Trp Val Asp Phe Leu Asp Lys Phe Ala
Arg 645 650 655Glu Arg Met Glu Pro Ala Ile Asp Arg Gly Phe Arg Glu
Met Cys Glu 660 665 670Tyr Met Asn Asn Lys Gln His Leu Met Phe Met
Asp Arg Glu Ala Ile 675 680 685Ala Gly Pro Pro Leu Gly Ser Lys Gly
Ile Gly Gly Phe Trp Thr Gly 690 695 700Lys Lys Arg Tyr Ala Leu Asn
Val Trp Asp Met Glu Gly Thr Arg Tyr705 710 715 720Ala Glu Pro Lys
Leu Lys Ile Met Gly Leu Glu Thr Gln Lys Ser Ser 725 730 735Thr Pro
Lys Ala Val Gln Lys Ala Leu Lys Glu Cys Ile Arg Arg Met 740 745
750Leu Gln Glu Gly Glu Glu Ser Leu Gln Glu Tyr Phe Lys Glu Phe Glu
755 760 765Lys Glu Phe Arg Gln Leu Asn Tyr Ile Ser Ile Ala Ser Val
Ser Ser 770 775 780Ala Asn Asn Ile Ala Lys Tyr Asp Val Gly Gly Phe
Pro Gly Pro Lys785 790 795 800Cys Pro Phe His Ile Arg Gly Ile Leu
Thr Tyr Asn Arg Ala Ile Lys 805 810 815Gly Asn Ile Asp Ala Pro Gln
Val Val Glu Gly Glu Lys Val Tyr Val 820 825 830Leu Pro Leu Arg Glu
Gly Asn Pro Phe Gly Asp Lys Cys Ile Ala Trp 835 840 845Pro Ser Gly
Thr Glu Ile Thr Asp Leu Ile Lys Asp Asp Val Leu His 850 855 860Trp
Met Asp Tyr Thr Val Leu Leu Glu Lys Thr Phe Ile Lys Pro Leu865 870
875 880Glu Gly Phe Thr Ser Ala Ala Lys Leu Asp Tyr Glu Lys Lys Ala
Ser 885 890 895Leu Phe Asp Met Phe Asp Phe 9001425PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 14Met
Asn His Leu Val His His His His His His Ile Glu Gly Arg His1 5 10
15Met Glu Leu Gly Thr Leu Glu Gly Ser 20 251534PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
15Met Ser His His His His His His Ser Met Ser Gly Leu Asn Asp Ile1
5 10 15Phe Glu Ala Gln Lys Ile Glu Trp His Glu Gly Ala Pro Gly Ala
Arg 20 25 30Gly Ser1627PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 16Met His His His His His His
Leu Leu Gly Gly Gly Gly Ser Gly Gly1 5 10 15Gly Gly Ser Ala Ala Ala
Gly Ser Ala Ala Arg 20 25179PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 17Arg Arg Ala Thr Ser Asn Val
Phe Ala1 5188PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 18Arg Lys Ala Ser Gly Pro Pro Val1
5197PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 19Leu Arg Arg Ala Ser Leu Gly1 52017PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 20Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ala Ala Gly Ser Ala1 5 10
15Ala2114PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 21Gly Ala Ala Ala Lys Gly Ala Ala Ala Lys Gly Ser
Ala Ala1 5 10226PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 22Pro Lys Pro Gln Gln Phe1
5237PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 23Pro Lys Pro Gln Gln Phe Met1 52436DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 24tttttttgca ggtgacaggt ttttcctgtc acctgc
362535DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 25ttatctttgt gggtgacagg tttttcctgt caccc
352639DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 26tttttttgcc cccagggtga caggtttttc
ctgtcaccc 392732DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 27tttttgcggg tgacaggttt
ttcctgtcac cc 322836DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 28tttttttgca ggtgacaggt
ttttcctgtc acctgc 36 2919DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 29ggtactaagc ggccgcatg
193030DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 30taaagccccc ccatgcggcc gcttagtacc
303130DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 31taaagttttt tcatgcggcc gcttagtacc
303226DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 32ggtactaagc ggccgcatga aaaaaa
263317DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 33tgatagaacc tccgtgt 173441DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 34ggaacacgga ggttctatca tcgtcatcgt catcgtcatc g
413519DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 35ggtactaagc ggccgcatg 193626DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 36ttttacccat gcggccgctt agtacc 263716DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
37ggtactaagc ggccgc 163826DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 38ttttacccat
gcggccgctt agtacc 263960DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 39gggggggggg
gggggaaaaa aaaaaaaaaa gggggggggg gggggaaaaa aaaaaaaaaa
604053DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 40ttgaacggat gaggaccaga caccacttga
acggatgagg aaaaaaaaaa tca 534159DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 41ttttgattcc
cccttccccc gacacggagg ttctatcatc gtcatcgtca tcgtcatcg
594239DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 42cgatgacgat gacgatgacg atgatagaac ctccgtgtc
394384DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 43tgattttttt tttcctcatc cgttcaagtg
gtgtctggtc ctcatccgtt caagacacgg 60aggttctatc atcgtcatcg tcat
844417DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 44tgatagaacc tccgtgt 174526DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 45ttttttttac ccccgggtga caggtt 264662DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 46tttttccccg acgatgcctc cccgacacgg aggttctatc
atcgtcatcg tcatcgtcat 60cg 624718DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 47tgatagaacc tccgtgtc
184821DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 48ggggaggcat cgtcgggaaa a
214944DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 49tttttccccg cgtaactctt taccccgaca
cggaggttct atca 445018DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 50tgatagaacc tccgtgtc
185193DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 51ttttgatttt ttttttttcc cccccccccc
tttttttttt ttcccccccc ccccgacacg 60gaggttctat catcgtcatc gtcatcgtca
tcg 935218DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 52tgatagaacc tccgtgtc 185312DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
53tgatagaacc tc 125449DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 54tttttccccg
acgatgcctc cccttttttt tacccccggg tgacaggtt 495514DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 55gggggaaggg ggaa 145637DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 56gggggtaaaa aaaaggggag gcatcgtcgg ggaaaaa
375725DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 57cgttaaccgc ccgctccttt gcaac
255818DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 58gttgcaaagg agcgggcg 185932DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 59cagtaacgga gttggttgga cggctgcgag gc
326025DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 60gcctcgcagc cgtccaacca actcc 256115PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 61Gly
Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu1 5 10
15626PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 62Leu Arg Arg Ala Ser Leu1 5636PRTArtificial
SequenceDescription of Artificial Sequence Synthetic 6xHis tag
63His His His His His His1 56434DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 64ttatctttgt
gggtgacagg tttttcctgt cacc 346512DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 65ttcctgtcac cc
126614DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 66gggggtaaaa aaaa 14674PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 67Asp
Asp Asp Asp1
* * * * *