U.S. patent application number 11/709338 was filed with the patent office on 2008-04-10 for stabilizing a nucleic acid for nucleic acid sequencing.
This patent application is currently assigned to Helicos BioSciences Corporation. Invention is credited to Philip Richard Buzby.
Application Number | 20080085840 11/709338 |
Document ID | / |
Family ID | 36640913 |
Filed Date | 2008-04-10 |
United States Patent
Application |
20080085840 |
Kind Code |
A1 |
Buzby; Philip Richard |
April 10, 2008 |
Stabilizing a nucleic acid for nucleic acid sequencing
Abstract
The invention provides methods for sequencing a nucleic acid
comprising stabilizing a primer/target nucleic acid duplex on a
substrate. Methods of the invention generally contemplate the use
of a dual-anchored primer/target nucleic acid duplex, or a
stabilizing molecule in a single molecule sequencing reaction.
Inventors: |
Buzby; Philip Richard;
(Brockton, MA) |
Correspondence
Address: |
COOLEY GODWARD KRONISH LLP;ATTN: Patent Group
Suite 1100
777 - 6th Street, NW
WASHINGTON
DC
20001
US
|
Assignee: |
Helicos BioSciences
Corporation
Cambridge
MA
|
Family ID: |
36640913 |
Appl. No.: |
11/709338 |
Filed: |
February 20, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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11027165 |
Dec 30, 2004 |
7220549 |
|
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11709338 |
Feb 20, 2007 |
|
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Current U.S.
Class: |
506/32 |
Current CPC
Class: |
C12Q 1/68 20130101; C12Q
1/6869 20130101; C12Q 1/6869 20130101; C12Q 2535/101 20130101; C12Q
2533/101 20130101; C12Q 2563/131 20130101; C12Q 2563/131 20130101;
C12Q 1/6834 20130101; C12Q 1/6834 20130101 |
Class at
Publication: |
506/032 |
International
Class: |
C40B 50/18 20060101
C40B050/18 |
Claims
1. A method for stabilizing a nucleic acid duplex on a surface, the
method comprising the steps of: exposing a nucleic acid duplex,
wherein each member of said duplex contains a member of a binding
pair such that each of said members is oriented in the same
direction, to a surface comprising a binding partner for each
member of said binding pair, thereby to stabilize said duplex on
said surface.
2. The method of claim 1, wherein each of said members is the same
molecular species.
3. The method of claim 1, wherein each of said members is a
different molecular species.
4. The method of claim 1, wherein said binding pair is selected
from the group consisting of a ligand/receptor pair, a
carbohydrate/lectin pair, and an antigen/antibody pair.
5. The method of claim 1, wherein said binding pair is selected
from the group consisting of biotin/avidin, biotion/streptavidin,
digoxigenin/anti-digoxigenin, and
dinitrophenol/anti-dinitrophenol.
6. The method of claim 1, wherein said duplex is a template/primer
duplex.
7. The method of claim 1, wherein said member is located at the 5'
terminus of said template and the 3' terminus of said primer.
8. The method of claim 1, wherein said member is located at the 3'
terminus of said template and the 5' terminus of said primer.
9. The method of claim 6, further comprising the steps of exposing
a surface-bound duplex to a nucleotide base and a polymerase under
conditions sufficient for said base to be incorporated into said
primer if it is complementary to a corresponding base in said
template.
10. The method of claim 9, further comprising the step of compiling
a nucleic acid sequence of said template by detecting sequential
incorporations of nucleotides into said primer.
11. The method of claim 6, wherein said primer comprises a locked
nucleic acid base.
12. The method of claim 6, wherein said primer comprising a peptide
nucleic acid base.
13. A method for performing a nucleic acid sequencing reaction, the
method comprising the steps of: exposing a mixture comprising a
nucleic acid template, a polymerase, and a primer, wherein said
primer comprises a locked nucleic acid, to a nucleotide under
conditions wherein said nucleotide is capable of incorporation into
said primer.
14. The method of claim 1, wherein a plurality of said duplex is
attached to a substrate such that each duplex is individually
optically resolvable.
15. A surface for nucleic acid sequencing, said surface comprising
a nucleic acid duplex composed of a template and a primer, each of
said template and primer being attached to a member of a binding
pair, such that each of said members is oriented toward said
surface.
16. The surface of claim 15, wherein each duplex is individually
optically resolvable.
17. The surface of claim 14, wherein said surface is a
polyelectrolyte multilayer.
18. The surface of claim 14, wherein said surface is an epoxide
surface.
19. The surface of claim 14, wherein said surface is deposited on a
substrate selected from the group consisting of glass and
silica.
20. The surface of claim 14, wherein binding partners of said
members are covalently attached to said surface.
21. The surface of claim 14, wherein said binding pair is selected
from the group consisting of a ligand/receptor pair, an affinity
binding pair, an antigen/antibody pair, and a carbohydrate/lectin
pair.
22. The surface of claim 21, wherein said pair is selected from the
group consisting of biotin/avidin, digoxigenin/anti-digoxigenin,
and dinitrophenol/anti-dinitrophenol.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of application Ser. No.
11/027,165 filed on Dec. 30, 2004, allowed. The entire contents of
the aforementioned application are now hereby incorporated by
reference.
TECHNICAL FIELD
[0002] The invention provides methods for sequencing a nucleic acid
comprising stabilizing a primer/target nucleic acid duplex attached
to a substrate. Generally, methods of the invention comprise the
use of a dual-anchored primer/target nucleic acid duplex or
stabilizing molecule.
BACKGROUND OF THE INVENTION
[0003] Completion of the human genome has paved the way for
important insights into biologic structure and function. Knowledge
of the human genome has given rise to inquiry into individual
differences, as well as differences within an individual, as the
basis for differences in biological function and dysfunction. For
example, single nucleotide differences between individuals, called
single nucleotide polymorphisms (SNPs), are responsible for
dramatic phenotypic differences. Those differences can be outward
expressions of phenotype or can involve the likelihood that an
individual will get a specific disease or how that individual will
respond to treatment. Moreover, subtle genomic changes have been
shown to be responsible for the manifestation of genetic diseases,
such as cancer. A true understanding of the duplexities in either
normal or abnormal function will require large amounts of specific
sequence information.
[0004] An understanding of cancer also requires an understanding of
genomic sequence duplexity. Cancer is a disease that is rooted in
heterogeneous genomic instability. Most cancers develop from a
series of genomic changes, some subtle and some significant, that
occur in a small subpopulation of cells. Knowledge of the sequence
variations that lead to cancer will lead to an understanding of the
etiology of the disease, as well as ways to treat and prevent it.
An essential first step in understanding genomic duplexity is the
ability to perform high-resolution sequencing.
[0005] Various approaches to nucleic acid sequencing exist. One
conventional way to do bulk sequencing is by chain termination and
gel separation, essentially as described by Sanger et al., Proc.
Natl. Acad. Sci., 74(12): 5463-67 (1977). That method relies on the
generation of a mixed population of nucleic acid fragments
representing terminations at each base in a sequence. The fragments
are then run on an electrophoretic gel and the sequence is revealed
by the order of fragments in the gel. Another conventional bulk
sequencing method relies on chemical degradation of nucleic acid
fragments. See, Maxam et al., Proc. Natl. Acad. Sci., 74: 560-564
(1977). Finally, methods have been developed based upon sequencing
by hybridization. See, e.g., Drmanac, et al., Nature Biotech., 16:
54-58 (1998).
[0006] Bulk sequencing techniques are not useful for the
identification of subtle or rare nucleotide changes due to the many
cloning, amplification and electrophoresis steps that complicate
the process of gaining useful information regarding individual
nucleotides. The ability to sequence and gain information from
single molecules obtained from an individual patient is the next
milestone for genomic sequencing. As such, research has evolved
toward methods for rapid sequencing, such as single molecule
sequencing technologies.
[0007] There have been many proposals to develop new sequencing
technologies based on single-molecule measurements, generally
either by observing the interaction of particular proteins with DNA
or by using ultra high resolution scanned probe microscopy. See,
e.g., Rigler, et al., DNA-Sequencing at the Single Molecule Level,
Journal of Biotechnology, 86(3): 161 (2001); Goodwin, P. M., et
al., Application of Single Molecule Detection to DNA Sequencing.
Nucleosides & Nucleotides, 16(5-6): 543-550 (1997); Howorka,
S., et al., Sequence-Specific Detection of Individual DNA Strands
using Engineered Nanopores, Nature Biotechnology, 19(7): 636-639
(2001); Meller, A., et al., Rapid Nanopore Discrimination Between
Single Polynucleotide Molecules, Proceedings of the National
Academy of Sciences of the United States of America, 97(3):
1079-1084 (2000); Driscoll, R. J., et al., Atomic-Scale Imaging of
DNA Using Scanning Tunneling Microscopy. Nature, 346(6281): 294-296
(1990). Unlike conventional sequencing technologies, their speed
and read-length would not be inherently limited by the resolving
power of electrophoretic separation. Other methods proposed for
single molecule sequencing include detecting individual nucleotides
as they are incorporated into a primed template, i.e., sequencing
by synthesis.
[0008] While single molecule techniques have several advantages,
implementation has been problematic. For example, the
reproducibility and accuracy of many single molecule techniques
rely upon the stability of a primer/target nucleic acid duplex
attached to a solid substrate. However, incomplete binding of the
primer to the template, disengagement of the primer from the
template and disengagement of the duplex from the substrate are
frequent occurrences in such single molecule techniques.
[0009] Accordingly, there is a need in the art for methods and
devices for sequencing generally, and single molecule sequencing in
particular, including methods for stabilizing a target nucleic acid
for sequence determination.
BRIEF SUMMARY OF THE INVENTION
[0010] The invention generally provides methods and surfaces for
nucleic acid sequencing comprising stabilized primer/target nucleic
acid duplexes on a surface. Methods of the invention generally
contemplate the use of a primer/target nucleic acid duplex in which
each of the primer and the template contain a molecule having a
binding partner on the substrate. The primer/target is stabilized
on the surface by binding of both the primer and the template to
the surface. Binding pairs for use in the invention are any
molecular pair that can be bound to a surface and attached to a
nucleic acid. Some examples of preferred pairs include
ligand/receptor, affinity pairs, antigen/antibody, and
carbohydrate/lectin. For example, biotin/streptavidin,
digoxigenin/anti-digoxigenin, and dinitrophenol/anti-dinitrophenol
perform well in the invention. Other pairs are apparent to the
skilled artisan based upon the description of the invention
provided below.
[0011] According to the invention, the primer contains a member of
a binding pair at its 5' terminus, and the template contains a
member of a binding pair at its 3' terminus or the primer contains
a member of a binding pair at its 3' terminus and the template
contains a member of a binding pair at its 5' terminus. Thus, the
primer hybridizes to the template, and the two attached binding
pair members are oriented to bind to their respective mates on the
surface.
[0012] The template and primer may contain the same type or species
of binding pair or they may contain separate types or species.
Binding may occur to a single species of binding partner or to
separate members of the same species. For example, in one
embodiment, both the template and the primer are bioinylated at
opposite ends oriented to the surface (i.e., one at the 3' end and
one at the 5' end) and the two biotin molecules adhere to the same
streptavidin molecule (which has capacity to bind four biotins) on
the surface. Alternatively, the two biotins adhere to separate
streptavidin molecules spaced closely together on the surface. In
another embodiment, the primer is attached to a member of a first
binding pair and the template is attached to a member of a second
binding pair. Upon hybridization, the first member attaches to its
mate on the surface, and the second member attaches to its separate
mate on the surface. In either embodiment, the combination of two
separate mating pairs reduces loss of the hybrid due to either the
template or the primer dissociating. It is apparent to the skilled
artisan based upon this disclosure that any combination of binding
pairs works to stabilize hybrid binding to a surface. For example,
template and primer may have attached separate species of binder
that, although distinct, bind to the same surface-bound mate.
[0013] The invention comprises methods for sequencing nucleic acids
using stabilized, support-bound primer/template hybrids as
described above. In a preferred embodiment, methods of the
invention comprise template-dependent sequencing by synthesis using
a polymerase capable of adding nucleotides to the primer in a
template-dependent fashion. The invention is particularly useful
for single molecule nucleic acid sequencing in which
primer/template duplex is attached to a substrate such that the
duplex is individually optically resolvable. Individual strand
sequence is determined by detecting ordered template-dependent
nucleotide incorporation into the primer and compiling a sequence
of the template based upon the order of incorporated
nucleotides.
[0014] The invention also provides for the use of a stabilizing
molecule in template-dependent sequencing. Stabilizing molecules
useful in the invention include, for example, locked nucleic acid
("LNA") analogs and peptide nucleic acid ("PNA") analogs.
Generally, a stabilizing molecule increases the affinity and
specificity of the primer/target nucleic acid bond, and increases
the melting temperature of the primer/target nucleic acid duplex or
the specificity of incorporation of a nucleotide into the primer in
a sequencing by synthesis reaction. An example of a locked nucleic
acid is shown in FIG. 4. Both locked nucleic acid and peptide
nucleic acid analogs increase the melting temperature of the
primer/template duplex and, therefore, confer stability on the
hybrid, whether or not the anchoring strategies described above are
used.
[0015] Polymerases useful in the invention include any polymerizing
agent capable of catalyzing a template-dependent addition of a
nucleotide or nucleotide analog to a primer. Depending on the
characteristics of the target nucleic acid, a DNA polymerase, an
RNA polymerase, or a reverse transcriptase can be used. According
to one aspect of the invention, a thermophilic polymerase is used,
such as ThermoSequenase.TM., 9.degree.N.TM., Taq, Tfl, Tth, Tli,
Therminator, or Pfu. In one embodiment, the invention provides for
the primer/target nucleic acid duplex to be exposed to the
polymerase and nucleotide at a temperature between about 30.degree.
and about 80.degree. C. A preferred polymerase is a Klenow fragment
having reduced 3'-5' exonuclease activity.
[0016] Nucleotides useful in the invention include any nucleotide
or nucleotide analog, whether naturally-occurring or synthetic. For
example, preferred nucleotides are adenine, cytosine, guanine,
uracil, or thymine bases; xanthine or hypoxanthine, 5-bromouracil,
2-aminopurine, deoxyinosine, or methylated cytosine, such as
5-methylcytosine, and N4-methoxydeoxycytosine. Also included are
bases of polynucleotide mimetics, such as methylated nucleic acids,
e.g., 2'-O-methRNA, peptide nucleic acids, modified peptide nucleic
acids, locked nucleic acids and any other structural moiety that
can act substantially like a nucleotide or base, for example, by
exhibiting base-complementarity with one or more bases that occur
in DNA or RNA and/or being capable of base-complementary
incorporation, and includes chain-terminating analogs.
[0017] Nucleotides for primer addition according to the invention
preferably comprise a detectable label. Labeled nucleotides include
any nucleotide that has been modified to include a label that is
directly or indirectly detectable. Preferred labels include
optically-detectable labels, including fluorescent labels or
fluorophores, such as fluorescein, rhodamine, derivatized rhodamine
dyes, such as TAMRA, phosphor, polymethadine dye, fluorescent
phosphoramidite, Texas Red, green fluorescent protein, acridine,
cyanine, cyanine 5 dye, cyanine 3 dye,
5-(2'-aminoethyl)-aminonaphthalene-1-sulfonic acid (EDANS), BODIPY,
120 ALEXA or a derivative or modification of any of the foregoing,
and also include such labeling systems as hapten labeling.
Accordingly, methods of the invention further provide for exposing
the primer/target nucleic acid duplex to a digoxigenin, a
fluorescein, an alkaline phosphatase or a peroxidase.
[0018] In one embodiment, fluorescence resonance energy transfer
(FRET) is used to determine the base type incorporated into the
primer. Fluorescence resonance energy transfer in the context of
sequencing is described generally in Braslavasky et al., Sequence
Information can be Obtained from Single DNA Molecules, Proc. Nat'l
Acad. Sci., 100: 3960-3964 (2003), incorporated by reference
herein. Essentially, in one embodiment, a donor fluorophore is
attached to either the primer, polymerase, or template. Nucleotides
added for incorporation into the primer comprise an acceptor
fluorophore that is activated by the donor when the two are in
proximity. Activation of the acceptor causes it to emit a
characteristic wavelength of light and also quenches the donor. In
this way, incorporation of a nucleotide in the primer sequence is
detected by detection of acceptor emission. Of course, nucleotides
labeled with a donor fluorophore also are useful in methods of the
invention; FRET-based methods of the invention only require that a
donor and acceptor fluorophore pair are used, a labeled nucleotide
may comprise one fluorophore and either the template or the
polymerase may comprise the other. Such labeling techniques result
in a coincident fluorescent emission of the labels of the
nucleotide and the labeled template or polymerase, or
alternatively, the fluorescent emission of only one of the
labels.
[0019] In a preferred embodiment, after detection, the label is
rendered undetectable by removing the label from the nucleotide or
extended primer, neutralizing the label, or masking the label. In
certain embodiments, methods according to the invention provide for
neutralizing a label by photobleaching. This is accomplished by
focusing a laser with a short laser pulse, for example, for a short
duration of time with increasing laser intensity. In other
embodiments, a label is photocleaved. For example, a
light-sensitive label bound to a nucleotide is photocleaved by
focusing a particular wavelength of light on the label. Generally,
it may be preferable to use lasers having differing wavelengths for
exciting and photocleaving. Labels also can be chemically cleaved.
Labels may be removed from a substrate using reagents, such as NaOH
or other appropriate buffer reagent.
[0020] Preferred substrates include glass, silica, and others with
the optical properties described herein. Surfaces for sequencing
according to the invention may be coated with, for example, an
epoxide, polytetrafluoroethylene or a derivative of
polytetrafluoroethylene, such as silanized polytetrafluoroethylene,
a polyelectrolyte multilayer (PEM), or the equivalent.
[0021] Primers useful in the invention hybridize to template in a
manner that allows template-dependent sequencing-by-synthesis.
Depending on the target nucleic acid, the primer may comprise DNA,
RNA or a mixture of both. The invention also teaches the use of
stabilizing molecules used in connection with the primer or the
primer/template duplex, such as locked nucleic acid or peptide
nucleic acid analogs. According to the invention, the melting
temperature of the primer/target nucleic acid duplex may be
increased from about 3.degree. to about 8.degree. C. per PNA or LNA
base included in the primer. In one embodiment, the primer
comprises a locked nucleic acid base on its 3' terminus. The primer
may comprise any portion of PNA or LNA bases, such as between about
10% and about 50%, more than about 50%, or less than about 10%,
20%, 30%, 40%, 50% or 60% of the total nucleic acid residues in the
primer. The PNA or LNA bases may be consecutive in the primer or
may be interspersed throughout the primer. In a preferred
embodiment, PNA or LNA bases are spaced apart at a distance of at
least one turn of the helix when the primer is hybridized to
template. The use of LNA or PNA analogs allows primers to be
shorter than would be the case to achieve similar melting
temperatures using conventional nucleic acids. According to one
embodiment of the invention, the primer comprises fewer than 25
nucleic acids.
[0022] Methods of the invention are suitable for de novo
sequencing, re-sequencing, sequence analysis, DNA fingerprinting,
polymorphism identification, for example single nucleotide
polymorphisms (SNP) detection, as well as for research and clinical
applications in genetics. Applied to RNA sequences, methods
according to the invention also identify alternate splice sites,
enumerate copy number, measure gene expression, identify unknown
RNA molecules present in cells at low copy number, annotate genomes
by determining which sequences are actually transcribed, determine
phylogenic relationships, and elucidate differentiation of cells.
Methods and surfaces of the invention are useful in diagnostic,
therapeutic, prognostic (including drug selection), and
developmental applications.
[0023] As will be appreciated by one skilled in the art, individual
features of the invention may be used separately or in any
combination. A detailed description of embodiments of the invention
is provided below. Other embodiments of the invention are apparent
upon review of the detailed description that follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 depicts a dual biotinylation reaction securing a
primer and target nucleic acid to a substrate.
[0025] FIG. 2 depicts a primer and target nucleic acid biotinylated
to align complementary nucleotides.
[0026] FIG. 3 depicts three primers comprising locked nucleic acid
bases.
[0027] FIG. 4 depicts the structure of a locked nucleic acid
base.
[0028] FIG. 5 shows the relative stability of dual biotin duplex on
a streptavidinated PEM.
DETAILED DESCRIPTION OF THE INVENTION
[0029] The results of single molecule sequencing are influenced by
the stability of substrate-bound primer/target nucleic acid
duplexes. Typically, single molecule sequencing comprises repeated
single base extension reactions followed by one or more wash steps.
Sequencing occurs on single strands spaced apart such that each
strand is individually optically resolvable. Spatial and temporal
stability of the individual strands are important in order to
preserve the integrity of the sequencing process. One way in which
the spatial stability of a single molecule array can be disrupted
is if the template and/or primer become disassociated with the
surface. For example, template/primer hybridization is a dynamic
process. Primer melts off template at a low, but detectable rate.
Once melting occurs, at least some portion of primer will be
unavailable to re-anneal with template. That is not necessarily a
problem in a bulk sequencing reaction in which numerous copies of
each template are available for sequencing. However, in single
molecule sequencing, in which individual strands are sequenced,
loss of any strand can have a significant effect on the result.
Methods and surfaces of the invention address this problem by
placing stabilizing binding partners on each of the template and
primer and, optionally, utilizing stabilizing molecules that confer
an increased melting temperature on the primer/template hybrid.
I. GENERAL CONSIDERATIONS
[0030] Substrates
[0031] Generally, a substrate may be made of any suitable material
that allows single molecules to be individually optically
resolvable. Substrates for use according to the invention can be
two- or three-dimensional and can comprise a planar surface (e.g.,
a glass slide) or can be shaped. Appropriate substrates include
glass (e.g., controlled pore glass (CPG)), quartz, plastic (such as
polystyrene (low cross-linked and high cross-linked polystyrene),
polycarbonate, polypropylene and poly(methymethacrylate)), acrylic
copolymer, polyamide, silica, metal (e.g.,
alkanethiolate-derivatized gold), cellulose, nylon, latex, dextran,
gel matrix (e.g., silica gel), polyacrolein, or composites.
[0032] Preferably, a substrate used according to the invention
includes a biocompatible or biologically inert material that is
transparent to light and optically that (i.e., with a minimal
micro-roughness rating). Specially manufactured, or chemically
derivatized, low background fluorescence substrates (e.g., glass
slides) are also contemplated according to the invention.
Substrates may be prepared and analyzed on either the top or bottom
surface of the planar substrate (i.e., relative to the orientation
of the substrate in the detection system).
[0033] The invention also includes three-dimensional substrates
such as spheres, tubes (e.g., capillary tubes), microwells,
microfluidic devices, or any other structure suitable for anchoring
a nucleic acid. For example, a substrate can be a microparticle, a
bead, a membrane, a slide, a plate, a micromachined chip, and the
like. Substrates can include planar arrays or matrices capable of
having regions that include populations of target nucleic acids or
primers. Examples include nucleoside-derivatized CPG and
polystyrene slides; derivatized magnetic slides; polystyrene
grafted with polyethylene glycol; and the like.
[0034] Factors for selecting substrates include, for example, the
material, porosity, size, and shape. Other important factors to be
considered in selecting appropriate substrates include size
uniformity, efficiency as a synthesis support, and the substrate's
optical properties, e.g., clear smooth substrates (free from
defects) provide instrumentational advantages when detecting
incorporation of nucleotides in single molecules (e.g., nucleic
acids.).
[0035] Substrates are coated with a surface that facilitates
nucleic acid binding and that reduces background. Preferred
coatings are epoxides, silanized epoxides, biotinylated epoxides,
streptavidinated epoxides, polyelecrolyte multilayers, including
those that are derivatized for nucleic acid attachment (e.g.,
biotinylated, streptavidinated, or coated with a binding partner on
the template/primer.
[0036] Surfaces
[0037] Surfaces used to attach duplexes according to the invention
can be any surface to which a binding partner is capable of
attaching. For sequencing, surfaces should be free of debris,
especially debris capable of fluorescing. Also, surfaces should be
stable and transparent to light. Preferred surfaces are epoxy
surfaces and polyelectrolyte multilayer surfaces. Either of those
surfaces is easily derivatized as described in the art for
attachment of binding pairs. For example, epoxide surfaces are
derivatized with silane or other species capable of receiving
binding partners. In certain embodiments, binding pair members
attached to template/primer hybrids attach directly to the surface
via a molecule embedded in the surface that is not the normal
binding partner for the binding pair member. Polyelectrolyte
multilayer surfaces are formed from a variety of alternating layers
of positive and negative charge. Preferred polyelectrolyte
multilayer surfaces are described in detail below.
[0038] Target Nucleic Acids
[0039] A target nucleic acid for analysis may be obtained from a
patient sample, e.g., from blood, urine, cerebrospinal fluid,
seminal fluid, saliva, breast nipple aspirate, sputum, stool and
biopsy tissue. Any tissue or body fluid specimen may be used
according to methods of the invention.
[0040] A target nucleic acid can come from a variety of sources.
For example, nucleic acids can be naturally occurring DNA or RNA
isolated from any source, recombinant molecules, cDNA, or synthetic
analogs, as known in the art. For example, the target nucleic acid
may be genomic DNA, genes, gene fragments, exons, introns,
regulatory elements (such as promoters, enhancers, initiation and
termination regions, expression regulatory factors, expression
controls, and other control regions), DNA comprising one or more
single-nucleotide polymorphisms (SNPs), allelic variants, and other
mutations. Also included is the full genome of one or more cells,
for example cells from different stages of diseases such as cancer.
The target nucleic acid may also be mRNA, tRNA, rRNA, ribozymes,
splice variants, antisense RNA, or siRNA. Also contemplated
according to the invention are RNA with a recognition site for
binding a polymerase, transcripts of a single cell, organelle or
microorganism, and all or portions of RNA complements of one or
more cells, for example, cells from different stages of development
or differentiation, and cells from different species. Nucleic acids
can be obtained from any cell of a person, animal, plant, bacteria,
or virus, including pathogenic microbes or other cellular
organisms. Individual nucleic acids can be isolated for
analysis.
[0041] Stabilizing Molecules and Primers
[0042] Methods of the invention also contemplate using a
stabilizing molecule in a sequencing-by-synthesis reaction. The
stabilizing molecule strengthens the primer/template bond and
increases the specificity of incorporation of nucleotides into the
primer, the melting temperature of the primer/target nucleic acid
duplex, or both. A stabilizing molecule may comprise nucleotide or
internucleotide analogs, or covalently bound minor groove binders
or intercalators that enhance hybridization avidity or specificity
of the primer to a target nucleic acid. The internucleotide analogs
can comprise one or more of a phosphate ester analog such as alkyl
phosphonates, phosphoroamidates, alkylphosphotriesters,
phosphorothioates and phosphorodithioates.
[0043] In one aspect, the stabilizing molecule comprises a minor
groove binder. Minor groove binders are described in detail in U.S.
Pat. No. 6,084,102 which is incorporated by reference in its
entirety herein. Generally, a minor groove binder has a molecular
weight of approximately 150 to approximately 2000 daltons, and
typically covalently attaches to at least one of the nucleotides in
a duplex. It incorporates into a duplex to strengthen the
template/primer bond, thus increasing hybridization stability.
[0044] In one embodiment, a stabilizing molecule may comprise a
conformationally restricted nucleotide analog such as a peptide
nucleic acid base, a locked nucleic acid base or an oxetane
modified base (for a discussion of base constraining oxetane
modifications, see U.S. Published Patent Application No.
20040142946, the disclosure of which is incorporated by reference
herein). A peptide nucleic acid is a nucleic acid analog in which
the backbone comprises synthetic peptide like linkages (amide
bonds) usually formed from N-(2-amino-ethyl)-glycine units,
resulting in an achiral and uncharged molecule. PNA hybridizes with
complementary nucleic acids with high affinity and specificity, and
forms PNA/DNA and PNA/RNA duplexes having greater thermal and
chemical stability than counterpart DNA/DNA duplexes.
[0045] A locked nucleic acid is a bicyclic nucleic acid analog that
contains one or more 2'-O, 4'-C methylene linkage(s), which
effectively locks the furanose ring in a C3'-endo conformation.
This methylene linkage "bridge" restricts the flexibility of the
ribofuranose ring and locks the structure into a rigid bicyclic
formation. Because of its unique structural conformation, locked
nucleic acids demonstrate a much greater affinity and specificity
to their complementary nucleic acids than do natural DNA
counterparts and increases the thermal and chemical stability of a
primer/target nucleic acid duplex. LNAs will hybridize to
complementary nucleic acids even under adverse conditions, such as
under low salt concentrations and in the presence of chaotropic
agents. According to one aspect of the invention, locked nucleic
acids increase the melting point of the primer/target nucleic acid
duplex by about 3.degree. to about 8.degree. C. per locked nucleic
acid base incorporated in the primer. FIG. 4 shows an example of
the structure of a locked nucleic acid base.
[0046] Depending on the target nucleic acid, the primer may
comprise DNA, RNA or a mixture of both. Locked nucleic acid bases
may be interspersed throughout a strand of a primer, as shown in
FIG. 3, or may be placed consecutively or singularly in
predetermined locations. The amount and placement of the locked
nucleic acid bases depends on the desired characteristics of the
primer. In one embodiment, the primer comprises a locked nucleic
acid base at its 3' terminus. The primer may comprise any portion
of locked nucleic acid bases, such as between about 10% and about
50%, more than about 50%, or less than about 10%, 20%, 30%, 40%,
50% or 60% of the total bases in the primer.
[0047] In general, primer length is selected to facilitate
hybridization to a sufficiently complementary region of the
template nucleic acid downstream of the region to be analyzed. The
exact lengths of the primers depend on many factors, including
temperature and source of primer. Placement of locked nucleic acid
bases throughout a primer allows for an increased melting
temperature of the primer/target nucleic acid duplex during a
sequencing reaction. This also allows the primer length to remain
short compared to a primer that does not contain locked nucleic
acid bases. Embodiments of this invention include primers with 20
bases or less, which incorporate from 1 to 12 or more locked
nucleic acid bases. For example, a 20 base primer which includes 12
locked nucleic acid bases may yield a melting temperature of
between about 80.degree. to 90.degree. C. According to one
embodiment of the invention, the primer comprises less than about
30, 25, 20, 15, 10, or 5 bases.
[0048] FIG. 3 shows three exemplary primers (DXS 17, 7G7A, and
377), each containing nine locked nucleic acids and comprising a
sequence complementary to a known primer attachment site of a
target nucleic acid. While FIG. 3 shows primers of known sequences
complementary to a known region of a template, primers useful in
the invention also include primers comprising a random sequences.
Useful primers also include primers comprising a sequence that is
complementary to a known priming region that has been ligated to a
target nucleic acid.
[0049] Primers can be synthetically made using conventional nucleic
acid synthesis techniques. For example, primers are synthesized on
an automated DNA synthesizer (e.g., Applied Biosystems, Inc.,
Foster City, Calif.) using standard chemistries, such as
phosphoramidite chemistry, and the like. Alternative chemistries,
e.g., resulting in non-natural backbone groups, such as
phosphorothioate, phosphoramidate, and the like, may also be
employed provided that, for example, the resulting oligonucleotides
are compatible with the polymerizing agent. The primers can also be
ordered commercially from a variety of companies which specialize
in custom nucleic acids such as Operon, Inc. (Alameda, Calif.).
Primers comprising locked nucleic acids are purchased commercially
(Proligo.TM. LLC, Boulder, Colo.) or prepared as needed by methods
known in the art.
[0050] The foregoing methods confer a significant advantage in
single molecule reactions, in which one is tracking nucleotide
incorporation into individual template/primer duplexes. Single
molecule techniques provide the ability to observe discrete
differences within and between individuals in terms of nucleotide
sequence. Disruption of a hybrid impairs the ability to obtain full
advantage from single molecule techniques because the loss of a
hybrid represents the loss of significant information content
relative to a bulk reaction in which there exist numerous copies of
each hybrid pair. Methods of the invention maximize the ability to
keep hybrid pairs intact and attached to substrate.
[0051] Primer Hybridization
[0052] Conditions for hybridizing primers to target nucleic acids
are known in the art. The annealing reaction is performed under
conditions which are stringent enough to ensure sequence
specificity, yet sufficiently permissive to allow formation of
stable hybrids at an acceptable rate. The temperature and length of
time required for primer annealing depend upon several factors
including the base composition, length and concentration of the
primer, and the nature of the solvent used, e.g., the concentration
of cosolvents such as DMSO (dimethylsulfoxide), formamide, or
glycerol, and counterions such as magnesium. Typically,
hybridization (annealing) is carried out at a temperature that is
approximately 5 to 10.degree. C. below the melting temperature of
the primer/target nucleic acid duplex in the annealing solvent.
Annealing temperatures may be modified based on the amount of
locked nucleic acid included in the primer, based on manufacturer's
recommendation and methods known in the art.
[0053] Primer Extension and Labeling
[0054] During primer extension, the primer/target nucleic acid
duplex is exposed to a polymerase, and at least one nucleotide or
nucleotide analog under conditions that allow for incorporation of
the nucleotide into the primer. Polymerases useful in the invention
include any polymerizing agent capable of catalyzing a
template-dependant addition of a nucleotide to a primer, such as,
Klenow, Vent ThermoSequenase.TM., 9.degree.N.TM., Therminator, Taq,
Tfl, Tth, Tli, Pfu, and others. According to one aspect of the
invention, a thermophilic polymerase is used. In one embodiment,
the invention provides for the primer/target nucleic acid duplex to
be exposed to the polymerase and nucleotide at a temperature
between about 30.degree. and about 80.degree. C., or at least about
50.degree., 60.degree., or 70.degree. C.
[0055] Nucleotides useful in the invention include any nucleotide
or nucleotide analog, whether naturally-occurring or synthetic. For
example, preferred nucleotides are adenine, cytosine, guanine,
uracil, or thymine bases; xanthine or hypoxanthine, 5-bromouracil,
2-aminopurine, deoxyinosine, or methylated cytosine, such as
5-methylcytosine, and N4-methoxydeoxycytosine. Also included are
bases of polynucleotide mimetics, such as methylated nucleic acids,
e.g., 2'-O-methRNA, peptide nucleic acids, modified peptide nucleic
acids, locked nucleic acids, oxetane-modified bases and any other
structural moiety that can act substantially like a nucleotide or
base, for example, by exhibiting base-complementarity with one or
more bases that occur in DNA or RNA and/or being capable of
base-complementary incorporation, and includes chain-terminating
analogs.
[0056] Nucleotides particularly useful in the invention comprise
detectable labels. Labeled nucleotides include any nucleotide that
has been modified to include a label that is directly or indirectly
detectable. Preferred labels include optically-detectable labels,
including fluorescent labels or fluorophores, such as fluorescein,
rhodamine, derivatized rhodamine dyes, such as TAMRA, phosphor,
polymethadine dye, fluorescent phosphoramidite, Texas Red, green
fluorescent protein, acridine, cyanine, cyanine 5 dye, cyanine 3
dye, 5-(2'-aminoethyl)-aminonaphthalene-1-sulfonic acid (EDANS),
BODIPY, 120 ALEXA or a derivative or modification of any of the
foregoing, and also include such labeling systems as hapten
labeling. Accordingly, methods of the invention further provide for
exposing the primer/target nucleic acid duplex to a digoxigenin, a
fluorescein, an alkaline phosphatase or a peroxidase.
[0057] Other suitable fluorescent labels include, but are not
limited to, 4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic
acid; acridine and derivatives: acridine, acridine isothiocyanate;
4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate;
N-(4-anilino-1-naphthyl)maleimide; anthranilamide; Brilliant
Yellow; coumarin and derivatives; coumarin,
7-amino-4-methylcoumarin (AMC, Coumarin 120),
7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanine dyes;
cyanosine; 4',6-diaminidino-2-phenylindole (DAPI);
5'5''-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);
7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin;
diethylenetriamine pentaacetate;
4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid;
4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid;
5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS,
dansylchloride); 4-dimethylaminophenylazophenyl-4'-isothiocyanate
(DABITC); eosin and derivatives; eosin, eosin isothiocyanate,
erythrosin and derivatives; erythrosin B, erythrosin,
isothiocyanate; ethidium; fluorescein derivatives;
5-carboxyfluorescein (FAM),
5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),
2',7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE),
fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144;
IR1446; Malachite Green isothiocyanate; 4-methylumbelliferoneortho
cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;
B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives:
pyrene, pyrene butyrate, succinimidyl 1-pyrene; butyrate quantum
dots; Reactive Red 4 (Cibacron.TM. Brilliant Red 3B-A) rhodamine
and derivatives: 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine
(R6G), lissamine rhodamine B sulfonyl chloride rhodamine (Rhod),
rhodamine B, rhodamine 123, rhodamine X isothiocyanate,
sulforhodamine B, sulforhodamine; tetramethyl rhodamine;
tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic
acid; terbium chelate derivatives; Cy5.5; Cy7; IRD 700; IRD 800; La
Jolta Blue; phthalo cyanine; and naphthalo cyanine.
[0058] In a preferred embodiment, after detection, the label is
rendered undetectable by removing the label from the nucleotide or
extended primer, neutralizing the label, or masking the label. In
certain embodiments, methods according to the invention provide for
neutralizing a label by photobleaching. This is accomplished by
focusing a laser with a short laser pulse, for example, for a short
duration of time with increasing laser intensity. In other
embodiments, a label is photocleaved. For example, a
light-sensitive label bound to a nucleotide is photocleaved by
focusing a particular wavelength of light on the label. Generally,
it may be preferable to use lasers having differing wavelengths for
exciting and photocleaving. Labels also can be chemically cleaved.
Labels may be removed from a substrate using reagents, such as NaOH
or other appropriate buffer reagent.
[0059] Further, the primer or the target nucleic acid can also
include a detectable label. When the labeled primer and/or target
nucleic acid are attached to the substrate, the label facilitates
locating the bound molecule through imaging. The primer or target
nucleic acid can be labeled with a fluorescent labeling moiety
(e.g., Cy3 or Cy5), or any other means used to label nucleotides.
The detectable label used to label the primer or target nucleic
acid can be different from the label used on the nucleotides or
nucleotide analogs in the subsequent extension reactions.
Additionally, once the molecule has been localized, it may be
desirable to render the label undetectable prior to the nucleotide
incorporation detection steps by methods such as washing or
photobleaching.
[0060] A nucleotide analog according to the invention can be
modified to remove, cap or modify the 3' hydroxyl group. As such,
in certain embodiments, methods of the invention can include, for
example, the step of removing the 3' hydroxyl group from the
incorporated nucleotide or nucleotide analog. By removing the 3'
hydroxyl group from the incorporated nucleotide in the primer,
further extension is halted or impeded. In certain embodiments, the
modified nucleotide can be engineered so that the 3' hydroxyl group
can be removed and/or added by chemical methods. Alternatively, a
nucleotide analog can be modified to include a moiety that is
sufficiently large to prevent or sterically hinder further chain
elongation by interfering with the polymerase, thereby halting
incorporation of additional nucleotides or nucleotide analogs.
Subsequent removal of the moiety, or at least the steric-hindering
portion of the moiety, can concomitantly reverse chain termination
and allow chain elongation to proceed. In some embodiments, the
moiety also can be a label. As such, in those embodiments,
chemically cleaving or photocleaving the blocking moiety may also
chemically-bleach or photobleach the label, respectively.
[0061] Detection of Incorporated Nucleotides
[0062] Incorporation of a nucleotide or a nucleotide analog and
their locations on the surface of a substrate can be detected with
single molecule sensitivity according to the invention. In some
aspects of the invention, single molecule resolution is achieved by
anchoring a target nucleic acid at a low concentration to a
substrate, and then imaging nucleotide incorporation with for
example, with total internal reflection fluorescence
microscopy.
[0063] A number of detection methods are available for use in
single molecule analysis. Methods for visualizing single molecules
within nucleic acids labeled with an intercalating dye include, for
example, fluorescence microscopy. For example, the fluorescent
spectrum and lifetime of a single molecule excited-state can be
measured. Standard detectors such as a photomultiplier tube or
avalanche photodiode can be used. Full field imaging with a
two-stage image intensified COD camera also can be used.
Additionally, low noise cooled CCD can also be used to detect
single fluorescent molecules.
[0064] The detection system for the signal may depend upon the
labeling moiety used, which can be defined by the chemistry
available. For optical signals, a combination of an optical fiber
or CCD can be used in the detection step. In the embodiments where
the substrate is itself transparent to the radiation used, it is
possible to have an incident light beam pass through the substrate
with the detector located opposite the substrate from the primer.
For electromagnetic labels, various forms of spectroscopy systems
can be used. Various physical orientations for the detection system
are available and known in the art.
[0065] A number of approaches can be used to detect incorporation
of fluorescently-labeled nucleotides into a single molecule.
Optical systems include near-field scanning microscopy, far-field
confocal microscopy, wide-field epi-illumination, light scattering,
dark field microscopy, photoconversion, single and/or multiphoton
excitation, spectral wavelength discrimination, fluorophore
identification, evanescent wave illumination, and total internal
reflection fluorescence (TIRF) microscopy. In general, methods
involve detection of laser-activated fluorescence using a
microscope equipped with a camera, sometimes referred to as
high-efficiency photon detection system. Suitable photon detection
systems include, but are not limited to, photodiodes and
intensified CCD cameras. For example, as illustrated in FIG. 2, an
intensified charge couple device (ICCD) camera can be used. The use
of an ICCD camera to image individual fluorescent dye molecules in
a fluid near a surface provides numerous advantages. For example,
with an ICCD optical setup, it is possible to acquire a sequence of
images (movies) of fluorophores.
[0066] Certain embodiments of the invention are described in the
following examples, which are not meant to be limiting.
II. EXAMPLES
Example 1
Dual Biotinylation
[0067] General methods of the invention were demonstrated using
biotin/avidin binding pairs. When a biotin-streptavidin linkage is
used to anchor a primer and a target nucleic acid to a substrate,
the primer and target nucleic acid are biotinylated, while the
surface of the substrate is coated with streptavidin. Because
streptavidin is a tetramer, it is possible that both template and
primer will bind to the same surface streptavidin. However, the
dual biotin labels may bind to adjacent streptavidin molecules as
well.
[0068] Two experiments were done to determine the binding stability
of the dual biotin constructs. A first experiment was conducted in
order to determine the stability of dual biotin duplex on a
polyelectrolyte multilayer (PEM) surface. This experiment was done
using covalent streptavidin attachment to a PEM surface. The PEM
surfaces were prepared as follows. Polyethyleneimine (PEI) and
pollyallylamine (PAA, Sigma, St. Louis, Mo.) were dissolved
separately by stirring in MilliQ water and the pH was adjusted to
8.0 with dilute HCl. The solutions were filtered using a 0.22 .mu.M
filter flask and stored at 4.degree. C. Clean glass slides were
then alternatively immersed for 10 minutes in the PEI and PM
solutions four times each with an 8 minute rinse using MilliQ water
between each immersion. After the last rinse, the slides were kept
immersed in water. The slides were then transferred to MES buffer
(2-[N-morpholino]ethanesulfonic acid), pH 5.5 for EDC-induced
crosslinking of the PEM. A 10 mM solution of
1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC)
was prepared in MES buffer, filtered and added to the solution
containing the PEM-coated slides for 1 hour at room temperature.
Slides were then rinsed in MES buffer and stored.
[0069] Next, the PEM surfaces were amine-derivatized by treatment
with 10 mM NHS and 10 mM EDC in 0.1 M MES buffer, pH 6.0, OSM NaCl
(coupling buffer) for 15 minutes. The surfaces were then rinsed
twice in the coupling buffer and incubated for 1 hour in 0.1 mg/ml
Streptavidin Plus (SA-20, Prozyme) in the coupling buffer. The
resulting streptavidinated surfaces were rinsed in a 200.mu.l
solution of dual-biotin duplex in which the primer had the
sequence: 5'-Biotin-TEG-M AAA CCC CTT ATG CAC TTA TCC TTT ACT (SEQ
ID NO: 1) and the template had the sequence: 5'-TCA GCT GCG TCA GCT
AGC GAC AGT AAA GGA TAA GTG CAT MG GGG TTT TT-TEG-Biotin (SEQ ID
NO: 2; primer binding region bolded and underlined). At its 3' end,
the terminal thymidine was labeled with cyanine-5 dye and the
adenine two positions 5' of the terminus was labeled with a
cyanine-3 dye. Surfaces were soaked for 5 minutes and then rinsed
once in 20 mM Tris, pH 8.0, 50 mM NaCl, 0.01% Triton (Rinse
Buffer). The surfaces were then rinsed 5 times in 3.times.SSC-0.1%
Triton, with a 10 minute soak in the last rinse. Finally, the
surfaces were rinsed in two changes of the Rinse Buffer. The
resulting surface had dual-biotin primer/target nucleic acid duplex
bound to streptavidin. A second set of slides was rinsed with the
dual biotin duplexes as described above, but was also challenged
with 100 nM unlabeled biotin. All slides were imaged over a 1000
.mu.m.sup.2 area and analyzed in ImagePro (Media Cybernetics, San
Diego) using a dark image background subtraction algorithm.
[0070] Visualization of surface-bound duplexes was accomplished
using fluorescence resonance energy transfer (FRET), with the
cyanine-5 labeled thymidine as the donor and the cyanine-3 labeled
adenine as the acceptor. Slides were placed on a Nikon Eclipse
TE-2000 inverted microscope with a total internal reflection
objective. The dual-biotin duplex slides showed 309.7 counts/pixel
and the biotin-challenged slides showed 10.1 counts/pixel. These
results indicate that the dual-biotin duplexes were binding to
streptavidin on the PEM in a specific manner, as the cold biotin
was able to compete away duplex binding.
[0071] In a separate experiment, the dual biotin duplexes referred
to above were first bound to a streptavidinated PEM surface as
described above. The surface was then exposed to 100 nM unlabeled
biotin at 52.degree. C. for 10 minutes. As a control,
streptavidinated PEMs were incubated in parallel and then rinsed
with dual-biotin duplex. All slides then were washed, imaged, and
analyzed as described above. The data are shown in FIG. 5. The
results indicate that the dual-biotin duplexes on a
streptavidinated PEM surface are surface-stable.
[0072] Another experiment shows the stability of dual-biotin
duplexes in single molecule sequencing. For this experiment,
streptavidinated slides are prepared on a PEM as described above.
The slides then are biotinylated. Fresh biotin-long chain
polyethyloxide-amine (Biotin-LC-PEO, Pierce) is prepared in MES
buffer (50 mg Biotin in 2.5 ml MES). A 5 ml aliquot of the EDC
solution described above is combined with 5 ml of the Biotin-LC-PEO
solution and diluted in MES buffer to a total volume of 96 ml by
adding 86 ml of MES buffer to a 2.5 mM final EDC-biotin
concentration. The PEM-coated slides are then immersed in that
solution in a 100 ml beaker and incubated for 60 minutes at room
temperature. The slides are then rinsed in MES buffer with gentle
agitation for 10 seconds. Immersion and rinsing are repeated four
times in clean 100 ml volumes. Slides are then incubated in the
final bath for 10 minutes. The resulting biotin-coated slides are
stored in Tris-NaCl buffer prior to streptavidination.
[0073] Streptavidin-Plus (SA20, Prozyme) is dissolved in a solution
of 10 mM NaCl buffer at 0.14 mg/ml and stirred 10 minutes at room
temperature to thoroughly dissolve flakes. The resulting solution
is filtered with a 0.22 .mu.M filter. Biotinylated slides described
above are placed in this solution in a 100 ml beaker with a stir
bar and stirred for 15 minutes at room temperature. The slides are
then rinsed in 100 ml Tris-NaCl buffer with gentle agitation for 10
seconds. The rinse process is repeated 5 times in clean 100 ml
volumes of 3.times.SSC-0.1% Triton, incubating in the final bath
for 10 minutes. Finally, the slides are transferred to a fresh bath
of Tris-NaCl and agitated for 10 seconds. Slides are stored in
Tris-NaCl buffer at 4.degree. C. prior to use.
[0074] 3' bioinylated target nucleic acid templates: 5'-TCA GCT GCG
TCA GCT AGC GAC AGT AAA GGA TAA GTG CAT MG GGG TTT TT-TEG-Biotin
(SEQ ID NO: 2), are obtained from Integrated DNA Technologies
(Coral, Iowa). 5' biotinylated primers: 5'-Biotin-TEG-M AAA CCC CTT
ATG CAC TTA TCC TTT ACT (SEQ ID NO: 1), comprising a cyanine-5 dye
were hybridized to the templates and exposed to the
streptavidinated surfaces described above at a concentration of 10
pM. After incubation for 10 minutes, the surfaces are washed with
MES buffer and the surface was imaged using a Nikon TE-2000U
upright microscope equipped with a total internal reflection
objective (Nikon). The location of label represented the location
of bound hybrid and the positions of label are noted. Positional
detection can also be accomplished using unlabeled template/primer
and adding labeled first base. To determine the stability of bound
duplexes, nucleotide additions are accomplished using the Klenow
fragment (exo-) polymerase (New England Biolabs) at 10 mM in Ecopol
reaction buffer and a series of cyanine-labeled nucleotide
triphosphates. To reduce bleaching of the fluorescent dyes, an
oxygen scavenging system is used (glucose (0.36%), glucose oxidase
(8 U/ml), catalase (423 U/ml), Trolox (5 mM), Gallate (5 mM), DABCO
(10 mM), and 2,4,6-octatrienoic acid).
[0075] The positions of cyanine-5-labeled primer are recorded and
bleached. dUTP-Cy3 in polymerase is added to the slides. If dUTP is
incorporated into the primer, fluorescence resonance energy
transfer (FRET) from the cyanine-5 on the primer will caus the
cyanine-3 dye to emit and the location of the emission is detected.
The cyanine-3 dye is kept unbleached and subsequent additions are
with cyanine-5-labeled dNTPs, using FRET with cyanine-3 as the
donor for detection of incorporation. The results show that
dual-biotin duplex is a stable template for template-dependent
sequencing.
[0076] The skilled artisan understands that there are numerous
other embodiments of the invention in terms of surfaces, binding
partners and the like that can be manipulated in order to achieve
the stability results shown above.
Example 2
Locked Nucleic Acid
[0077] A primer is designed to be complementary to a known primer
attachment site of the target nucleic acid, and locked nucleic acid
bases are substituted for certain nucleotides within the selected
primer sequence. As many locked nucleic acid bases are selected as
desired depending on the temperature and length of primer, up to a
primer comprising 100% locked nucleic acids. The more locked
nucleic acid substitutions into the primer, the greater the melting
point of the primer/target nucleic acid duplex relative a primer of
the same length lacking locked nucleic acid residues.
[0078] FIG. 3 shows three primers synthesized with locked nucleic
acids. DXS17, 7G7A, and 377 primers were synthesized as
complementary strands to known regions of a template, each primer
incorporating nine locked nucleic acids. When these primers are
annealed to their respective templates, hybridization may be
carried out at temperatures between about 80.degree. C. to about
90.degree. C.
[0079] The invention may be embodied in other specific forms
without departing from the spirit or essential characteristics
thereof. The foregoing embodiments are therefore to be considered
in all respects illustrative rather than limiting on the invention
described herein. Scope of the invention is thus indicated by the
appended claims rather than by the foregoing description, and all
changes which come within the meaning and range of equivalency of
the claims are therefore intended to be embraced therein.
Sequence CWU 1
1
7 1 29 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 1 aaaaacccct tatgcactta tcctttact 29 2 50
DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 2 tcagctgcgt cagctagcga cagtaaagga
taagtgcata aggggttttt 50 3 57 DNA Artificial Sequence Description
of Artificial Sequence Synthetic oligonucleotide 3 tcagctgcgt
cagctagcga cagtaaagga taagtgcata aggggttttt ttttttt 57 4 33 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 4 aaaaaaaaaa aaccccttat gcacttatcc ttt 33 5 21 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 5 ccccttatgc acttatcctt t 21 6 22 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 6 gtctgggctt ttggtttctg gg 22 7 22 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 7 cttgcatcca tcctctgccc tg 22
* * * * *