U.S. patent application number 13/482542 was filed with the patent office on 2012-11-29 for methods for manipulating biomolecules.
This patent application is currently assigned to LIFE TECHNOLOGIES CORPORATION. Invention is credited to Zhoutao CHEN, Xiaoping DUAN, Kyusung PARK.
Application Number | 20120301926 13/482542 |
Document ID | / |
Family ID | 46210447 |
Filed Date | 2012-11-29 |
United States Patent
Application |
20120301926 |
Kind Code |
A1 |
CHEN; Zhoutao ; et
al. |
November 29, 2012 |
METHODS FOR MANIPULATING BIOMOLECULES
Abstract
In some embodiments, the present teachings provide compositions,
systems, methods and kits for generating a population of nucleic
acid fragments. In some embodiments, nucleic acids can be
fragmented enzymatically. For example, methods for generating a
population of nucleic acid fragments can include a nucleic acid
nicking reaction. In one embodiment, the methods can include a nick
translation reaction. A nicking reaction can introduce nicks at
random positions on either strand of a double-stranded nucleic
acid. A nick translation reaction can move the position of nicks to
a new position so that the new positions of two of the nicks are
aligned to create a double-stranded break. In some embodiments,
methods for generating a population of nucleic acid fragments can
include joining at least one end of a fragmented nucleic acid to
one or more oligonucleotide adaptors.
Inventors: |
CHEN; Zhoutao; (Carlsbad,
CA) ; DUAN; Xiaoping; (Carlsbad, CA) ; PARK;
Kyusung; (Vista, CA) |
Assignee: |
LIFE TECHNOLOGIES
CORPORATION
Carlsbad
CA
|
Family ID: |
46210447 |
Appl. No.: |
13/482542 |
Filed: |
May 29, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61490982 |
May 27, 2011 |
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61535281 |
Sep 15, 2011 |
|
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61579109 |
Dec 22, 2011 |
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61601148 |
Feb 21, 2012 |
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Current U.S.
Class: |
435/91.5 ;
536/23.1 |
Current CPC
Class: |
C12N 15/1093 20130101;
C12N 15/10 20130101; C12Q 1/6806 20130101; C12P 19/40 20130101;
C12Q 2521/501 20130101; C12Q 2525/191 20130101; C12Q 2521/101
20130101; C12Q 2521/301 20130101; C12Q 2521/307 20130101; C12Q
1/6806 20130101; C12N 15/1093 20130101 |
Class at
Publication: |
435/91.5 ;
536/23.1 |
International
Class: |
C12P 19/34 20060101
C12P019/34; C07H 21/00 20060101 C07H021/00 |
Claims
1. A method for generating a population of nucleic acid fragments,
comprising: introducing one or more nicks into a nucleic acid by
subjecting a sample including a plurality of nucleic acids to
nicking conditions; and generating at least one double stranded
break in at least one of the nucleic acids.
2. A method for generating a population of nucleic acid fragments
comprising: (a) introducing at least one nick into a double
stranded nucleic acid; and (b) forming a double stranded break in
the nucleic acid by translating at least one nick.
3. A method for generating a population of nucleic acid fragments
comprising: (a) nicking a nucleic acid at least once on each
strand; and (b) nick translating the nicks thereby generating a
double-stranded break to produce nucleic acid fragments.
4. The method of claim 2, wherein the nicking comprises enzymatic
nicking.
5. The method of claim 3, wherein the nick translating comprises a
5' to 3' DNA polymerization/degradation reaction or a 5' to 3' DNA
polymerization/strand displacement reaction.
6. The method of claim 2, wherein the translating includes
polymerizing one or more unlabeled nucleotides onto the 3' end of
at least one nick.
7. The method of claim 2, wherein at least one of the nucleic acid
fragments is not labeled.
8. The method of claim 2, wherein substantially all of the nucleic
acid fragments are not labeled.
9. The method of claim 2, further comprising: ligating at least one
oligonucleotide adapter to at least one end of one or more nucleic
acid fragments in the population of nucleic acid fragments.
10. The method of claim 2, wherein one strand of at least one end
of a fragment of the population can be joined to one strand of a
double-stranded oligonucleotide adaptor to generate a
fragment-adaptor molecule having a break.
11. The method of claim 2, wherein both strands of at least one end
of a fragment of the population can be joined to both strands of a
double-stranded oligonucleotide adaptor.
12. The method of claim 2, further comprising modulating the
nicking conditions so as to adjust the average size of the nucleic
acid fragments.
13. A population of nucleic acid fragments generated by the method
of claim 2.
Description
[0001] This application claims the filing date benefit of U.S.
Provisional Application Nos. 61/490,982, filed on May 27, 2011, and
61/535,281, filed on Sep. 15, 2011,and 61/579,109, filed on Dec.
22, 2011, and 61/601,148, filed Feb. 21, 2012.
[0002] Throughout this application various publications, patents,
and/or patent applications are referenced. The disclosures of these
publications, patents, and/or patent applications 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.
FIELD
[0003] In some embodiments, the present teachings provide methods
for manipulating double-stranded nucleic acids to produce a
population of nucleic acid fragments.
INTRODUCTION
[0004] Nucleic acid manipulations can often involve a fragmenting
step. Nucleic acid fragments can be used in a variety of processes
and methods, including in the preparation of nucleic acids for
sequencing. Sequencing of fragments of nucleic acids is common in
capillary electrophoretic, hybridization-based, ligation-based and
sequence-by-synthesis-bases sequencing processes. Fragmenting
sample nucleic acids can be useful for next-generation sequencing
processes, in which large numbers of relatively small nucleic acid
fragments can be sequenced at the same time in parallel. Many
sample and library preparation process for next-generation
sequencing include a fragmentation step as part of the
workflow.
SUMMARY
[0005] In some embodiments, the present teachings provide
compositions, systems, methods and kits for generating a population
of nucleic acid fragments.
[0006] In some embodiments, the present teachings provide
compositions, systems, methods and kits for introducing at least
one double stranded break into a sample nucleic acid.
[0007] In some embodiments, the present teachings provide
compositions, systems, methods and kits for generating a double
stranded break, resulting in the formation of at least two nucleic
acid fragments derived from an original double stranded sample
nucleic acid.
[0008] In some embodiments, a sample nucleic acid can include
single stranded or double stranded nucleic acids.
[0009] Optionally, the methods comprise subjecting the sample
nucleic acid to nicking conditions. In some embodiments, the
nicking conditions comprise introducing one or more nicks into a
sample nucleic acid. In some embodiments, the nicking conditions
comprise introducing at least one nick on each strand of a double
stranded sample nucleic acid. In some embodiments, the nicks are
introduced at random positions in the sample nucleic acid.
[0010] Optionally, the methods comprise subjecting the sample
nucleic acid to nick translating conditions. In some embodiments,
the methods comprise translating at least one nick. In some
embodiments, the nick translating conditions comprise translating
at least one nick on each strand of a double stranded sample
nucleic acid. In some embodiments, the nick translating conditions
include translating at least two nicks located on opposing nucleic
acid strands towards each other. In some embodiments, the nick
translating conditions include translating the position of a first
nick on one strand to a new position that can be aligned with a
second nick, break, or other gap in the opposing strand. In some
embodiments, alignment of nicks, breaks, or gaps can result in
double-stranded breaks or fragmentation points in the sample
nucleic acid.
[0011] In some embodiments, methods for generating a population of
nucleic acid fragments comprise: nicking a nucleic acid; and nick
translating the nicks.
[0012] Optionally, the nick translating conditions can include
labeled or unlabeled nucleotides, or a mixture of both. In some
embodiments, the nick translating conditions conducted with
unlabeled nucleotides generate a population of unlabeled nucleic
acid fragments.
[0013] In some embodiments, methods for generating a population of
nucleic acid fragments comprise introducing at least one double
stranded break into a sample nucleic acid to generate a population
of unlabeled nucleic acid fragments.
[0014] In some embodiments, methods for generating a population of
nucleic acid fragments comprise: nicking a nucleic acid at least
once on each strand; and nick translating the nicks thereby
generating a double-stranded break to produce nucleic acid
fragments.
[0015] In some embodiments, methods for generating a population of
nucleic acid fragments comprise: introducing a double stranded
break (cleave) in a sample nucleic acid by: nicking the sample
nucleic acid at least once on each strand; and nick translating the
nicks thereby generating a double-stranded break to produce nucleic
acid fragments.
[0016] In some embodiments, methods for generating a population of
nucleic acid fragments comprise: providing a double-stranded
nucleic acid having a first and a second strand; and nicking the
first and second strand. Optionally the first strand can be nicked
at least once to produce a first nick and the second strand can be
nicked at least once to produce a second nick. Optionally, the
methods further comprise nick translating the first nick and the
second nick towards each other, thereby generating a
double-stranded break to produce nucleic acid fragments.
[0017] In some embodiments, methods for generating a population of
nucleic acid fragments comprise: introducing one or more nicks on
each strand of a double-stranded nucleic acid. Optionally, the
methods include generating at least one double-stranded break by
moving the positions of at least two of the nicks along their
respective strands, thereby cleaving the double stranded nucleic
acid into at least two nucleic acid fragments.
[0018] In some embodiments, methods for generating a population of
nucleic acid fragments comprise: subjecting two or more different
double stranded nucleic acids to nicking conditions, thereby
forming at least two different nicked double stranded nucleic
acids. In some embodiments, each of at least two different nicked
double stranded nucleic acids includes at least one nick in each
strand. Optionally, the methods comprise translating the at least
one nick in each strand so as to align the nicks on opposing
strands. In some embodiments, the translating includes subjecting
the at least two different nicked double stranded nucleic acids to
nick translating conditions.
[0019] In some embodiments, methods for generating a population of
nucleic acid fragments comprise: subjecting two or more different
double stranded nucleic acids to a nicking conditions, thereby
forming at least two different nicked double stranded nucleic
acids. In some embodiments, each of the two or more different
double stranded nucleic acids includes at least one nick in each
strand. Optionally, the methods comprise cleaving the at least two
different nicked double stranded nucleic acids, wherein the
cleaving includes creating at least one double stranded break in
each of the at least two different nicked double stranded nucleic
acids. In some embodiments, the creating includes nick translating
the least one nick in each strand, thereby generating a population
of nucleic acid fragments.
[0020] In some embodiments, methods for generating a population of
nucleic acid fragments comprise: cleaving at least two different
double stranded nucleic acid molecules into nucleic acid fragments.
In some embodiments, the cleaving includes introducing at least one
nick into each strand of the at least two different double stranded
nucleic acid molecules by subjecting the at least two different
double stranded nucleic acid molecules to nicking conditions,
thereby forming nicked double stranded nucleic acid molecules.
Optionally, the method comprises generating one or more double
stranded breaks in the nicked double stranded nucleic acid
molecules by nick translating one or more nicks in a first strand
and one or more nicks in a second strand of the nicked double
stranded nucleic acid molecule until at least two nicks on opposing
strands are aligned.
[0021] In some embodiments, methods for generating a population of
nucleic acid fragments comprise: subjecting two or more different
double stranded nucleic acids to nicking conditions, thereby
forming at least two different nicked double stranded nucleic
acids, each including at least one nick in each strand; cleaving
the at least two different nicked double stranded nucleic acids,
wherein the cleaving includes creating at least one double stranded
break in each of the at least two different nicked double stranded
nucleic acids, wherein the creating includes nick translating the
least one nick in each strand, thereby generating a population of
nucleic acid fragments.
[0022] In some embodiments, methods for generating a population of
nucleic acid fragments comprise: cleaving at least two different
double stranded nucleic acid molecules into nucleic acid fragments,
wherein the cleaving includes introducing at least one nick into
each strand of the at least two different double stranded nucleic
acid molecules by subjecting the at least two different double
stranded nucleic acid molecules to nicking conditions, thereby
forming nicked double stranded nucleic acid molecules; and
generating one or more double stranded breaks in the nicked double
stranded nucleic acid molecules by nick translating one or more
nicks in a first strand and one or more nicks in the second strand
of the nicked double stranded nucleic acid molecule until at least
two nicks on opposing strands are aligned.
[0023] In some embodiments, the method comprises modulating the
nicking conditions so as to adjust the average size of the nucleic
acid fragments.
[0024] In some embodiments, the translating includes polymerizing
one or more nucleotides onto the 3' end of at least one nick. In
some embodiments, labeled or unlabeled nucleotides can be
polymerized onto the 3' end of at least one nick.
[0025] In some embodiments, at least one of the nucleic acid
fragments is not labeled.
[0026] In some embodiments, substantially all of the nucleic acid
fragments are not labeled.
[0027] In some embodiments, the method generates a population of
unlabeled nucleic acid fragments.
[0028] Optionally, the methods further comprise: ligating at least
one oligonucleotide adapter to at least one end of one or more
nucleic acid fragments in the population of nucleic acid
fragments.
[0029] Optionally, the methods further comprise: cloning one or
more of the nucleic acid fragments.
[0030] In some embodiments, the population of nucleic acid
fragments includes substantially similar-sized fragments.
[0031] In some embodiments, the population of nucleic acid
fragments includes substantially dissimilar-sized nucleic acid
fragments.
[0032] In some embodiments, in a population of nucleic acids,
substantially similar-sized fragments can differ from each other on
average by about less than 50 bp, or can differ from each other on
average by about 50-100 bp, or by about 100-200 bp, or by about
200-300 bp, or by about 300-400 bp, or by about 400-500 bp, or by
about 500-600 bp, or by about 600-700 bp.
[0033] In some embodiments, a population of nucleic acids comprises
an average size range of about 50-150 bp, or about 150-250 bp, or
about 250-500 bp, or about 500-750 bp, or about 750-1000 bp, or
about 1-2 kb, or about 2-5 kb, or about 5-8 kb, or about 8-10 kb,
or about 10-20 kb, or about 20-40 kb, or about 40-60 kb, or
longer.
[0034] In some embodiments, at least one end of a fragment in the
population comprises a blunt end.
[0035] In some embodiments, at least one end of a fragment in the
population comprises an overhang end.
[0036] In some embodiments, at least one end of a fragment in the
population comprises or lacks a 5' phosphate group.
[0037] In some embodiments, at least one end of a fragment in the
population comprises or lacks a 3' OH group.
[0038] In some embodiments, the nicking comprises enzymatic
nicking.
[0039] In some embodiments, the nick translating comprises a 5' to
3' DNA polymerization/degradation reaction or a 5' to 3' DNA
polymerization/strand displacement reaction.
[0040] Optionally, the methods comprise joining at least one
oligonucleotide adaptor to at least one end of a fragment of the
population o nucleic acid fragments.
[0041] In some embodiments, one strand of at least one end of a
fragment of the population can be joined to one strand of a
double-stranded oligonucleotide adaptor to generate a
fragment-adaptor molecule having a break or nick between the
adaptor and the fragment.
[0042] In some embodiments, both strands of at least one end of a
fragment of the population can be joined to both strands of a
double-stranded oligonucleotide adaptor.
[0043] Optionally, the nicking step comprises at least one nucleic
acid binding protein. Optionally, nick translating step comprises
at least one nucleic acid binding protein.
[0044] In some embodiments, the nucleic acid binding protein
comprises a single-stranded binding protein.
[0045] In some embodiments, the single-stranded binding protein
comprises a phage T4 gp 32 protein, a Sulfolobus solfataricus
single-stranded binding protein, a Methanococcus jannaschii
single-stranded binding protein, or an E. coli single-stranded
binding protein.
[0046] In some embodiments, the nucleic acid binding protein
comprises an amino acid sequence according to any one of SEQ ID
NOS:1, 2, 3 or 4.
[0047] A population of nucleic acid fragments generated by the
teachings provided herein.
DRAWINGS
[0048] FIG. 1 is a schematic depicting non-limiting embodiments of
a nucleic acid fragmenting method.
[0049] FIG. 2 is a schematic depicting non-limiting embodiments of
a nucleic acid fragmenting method.
[0050] FIG. 3 is a schematic depicting non-limiting embodiments of
a nucleic acid adaptor-ligation method.
[0051] FIG. 4 shows a non-limiting embodiment of an amino acid
sequence of a phage T4 gp 32 protein (SEQ ID NO:1).
[0052] FIG. 5 shows a non-limiting embodiment of an amino acid
sequence of a single-stranded binding protein from Sulfolobus
solfataricus (SEQ ID NO:2)
[0053] FIG. 6 shows a non-limiting embodiment of an amino acid
sequence of a single-stranded binding protein from E. coli (SEQ ID
NO:3).
[0054] FIG. 7 shows a non-limiting embodiment of an amino acid
sequence of a single-stranded binding protein from Methanococcus
jannaschii (SEQ ID NO:4).
[0055] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the described
subject matter in any way. All literature and similar materials
cited in this application, including but not limited to, patents,
patent applications, articles, books, treatises, and internet web
pages are expressly incorporated by reference in their entirety for
any purpose. When definitions of terms in incorporated references
appear to differ from the definitions provided in the present
teachings, the definition provided in the present teachings shall
control. 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 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. It will be
appreciated that there is an implied "about" prior to the
temperatures, concentrations, times, etc discussed in the present
teachings, such that slight and insubstantial deviations are within
the scope of the present teachings herein. In this application, the
use of the singular includes the plural unless specifically stated
otherwise. Also, the use of "comprise", "comprises", "comprising",
"contain", "contains", "containing", "include", "includes", and
"including" are not intended to be limiting. As used herein, the
terms "comprises," "comprising," "includes," "including," "has,"
"having" or any other variation thereof, are intended to cover a
non-exclusive inclusion. For example, a process, method, article,
or apparatus that comprises a list of features is not necessarily
limited only to those features but may include other features not
expressly listed or inherent to such process, method, article, or
apparatus. Further, unless expressly stated to the contrary, "or"
refers to an inclusive-or and not to an exclusive-or. For example,
a condition A or B is satisfied by any one of the following: A is
true (or present) and B is false (or not present), A is false (or
not present) and B is true (or present), and both A and B are true
(or present). It is to be understood that both the foregoing
general description and the following detailed description are
exemplary and explanatory only and are not restrictive of the
invention.
DEFINITIONS
[0056] Unless otherwise defined, scientific and technical terms
used in connection with the present teachings described herein
shall have the meanings that are commonly understood by those of
ordinary skill in the art. Further, unless otherwise required by
context, singular terms shall include pluralities and plural terms
shall include the singular. Generally, nomenclatures utilized in
connection with, and techniques of, cell and tissue culture,
molecular biology, and protein and oligo- or polynucleotide
chemistry and hybridization described herein are those well known
and commonly used in the art. Standard techniques are used, for
example, for nucleic acid purification and preparation, chemical
analysis, recombinant nucleic acid, and oligonucleotide synthesis.
Enzymatic reactions and purification techniques are performed
according to manufacturer's specifications or as commonly
accomplished in the art or as described herein. The techniques and
procedures described herein are generally performed according to
conventional methods well known in the art and as described in
various general and more specific references that are cited and
discussed throughout the instant specification. See, e.g., Sambrook
et al., Molecular Cloning: A Laboratory Manual (Third ed., Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 2000). The
nomenclatures utilized in connection with, and the laboratory
procedures and techniques described herein are those well known and
commonly used in the art.
[0057] As utilized in accordance with exemplary embodiments
provided herein, the following terms, unless otherwise indicated,
shall be understood to have the following meanings:
[0058] As used herein, when used in reference to a nucleic acid,
the term "double stranded" does not necessarily require that the
nucleic acid molecule be double stranded across its entire length;
instead, some single stranded regions (or unhybridized regions) may
still be present in the double stranded nucleic acid. Typically, at
least 50% of the nucleotides within a double stranded nucleic acid
undergo base pairing according to the Watson Crick paradigm; in
another typical example, at least some of the nucleotides will
undergo base pairing according to a different (i.e., non
Watson-Crick) model. In some embodiments, a double stranded nucleic
acid includes a pair of single stranded nucleic acids that interact
with each other so that at least a portion of one of the single
stranded molecules hybridizes with a corresponding portion of the
other single stranded nucleic acid.
[0059] As used herein, when used in reference to a nucleic acid,
the term "fragmenting", includes any process or operation whereby a
nucleic acid is physically separated to form at least two nucleic
acid fragments. In some embodiments, the nucleic acid to be
fragmented can be single stranded or double stranded. In some
embodiments, the nucleic acid fragments that are formed are single
stranded or double stranded. In some embodiments, a nucleic acid
fragment includes a segment or portion of a single-stranded or
double-stranded deoxyribonucleic acid or ribonucleic acid. The
nucleic acid fragments derived from fragmentation of a given
nucleic acid need not include, either singly or collectively, all
of the sequence of the given nucleic acid. In some embodiments,
fragmentation can include cleavage of a nucleic acid through
formation of a double stranded break.
[0060] A "double stranded break" in a nucleic acid molecule
includes any examples of double stranded nucleic acid having a
first nick in a first strand and a second nick in a second strand,
where the first and second nicks are either in complete alignment
or in sufficiently close proximity to allow the physical separation
of the double stranded nucleic acid into two double stranded
nucleic acid fragments. Introducing a break into a double stranded
nucleic acid results in the formation of two new terminal ends
(e.g., upstream and downstream terminal ends) at the break site. In
some embodiments, the upstream and downstream terminal ends of a
break can include any combination of blunt ends, 5' overhang ends
and/or 3' overhang ends. In some embodiments, one strand of a
double stranded nucleic acid can lack a phosphodiester bond between
adjacent nucleotides, while the other strand can also lack a
phosphodiester bond between adjacent nucleotides at that same
location or at nearly the same location, so as to create a double
stranded break. In some embodiments, a single stranded nucleic acid
can lack a phosphodiester bond between adjacent nucleotides, so as
to create a single stranded break. In some embodiments, a
phosphodiester bond includes analog linkages that join adjacent
nucleotides (or join nucleotide analogs).
[0061] As used herein, the term "nicking" includes any suitable
process or treatment whereby the linkage between two adjacent or
contiguous nucleotides in one nucleic acid strand of a double
stranded nucleic acid is broken or disrupted, while the two
corresponding nucleotides opposite the nick in the opposing strand
remain linked. In some embodiments, the double stranded nucleic
acid includes two strands, each having a 5' end and a 3' end (or
equivalent thereof) that are substantially complementary across at
least some portion of their respective lengths. Introducing a nick
into one strand (referred to herein as the "nicked" strand) results
in the formation of a new 5' end and a new 3' end at the position
of the nick and the formation of two new strands derived from the
nicked strand. These two new strands typically remain aligned and
attached to the opposing strand through base pairing interactions
(the opposing strand can be free of any nicks, or may include one
or more nicks at other positions). In some embodiments, in a double
stranded nucleic acid, nicking can include breaking a
phosphodiester bond between adjacent nucleotides of one of the
nucleic acid strands, while the other strand has adjacent
nucleotides joined by a phosphodiester bond at the position
opposite the break. In some embodiments, a nicking agent can break
a phosphodiester bond (or any other equivalent bond in the case of
nucleic acid molecules incorporating nucleotide analogs) at a
random or at a site-specific position on at least one strand of a
double stranded molecule. In some embodiments, a phosphodiester
bond includes analog linkages that join adjacent nucleotides (or
join nucleotide analogs). In some embodiments, double stranded
nucleic acids can be enzymatically or chemically nicked.
[0062] As used herein, the term "nicking conditions" can include
any condition that is suitable for generating a nick in a nucleic
acid. In some embodiments, nicking conditions include enzymatic or
chemical reaction conditions, where the resulting reaction breaks
or disrupts at least one covalent bond between any two or more
contiguous nucleotides in the nucleic acid strand. In some typical
embodiments, the nicking reaction can be performed on a double
stranded nucleic acid substrate having two strands that are
substantially complementary to each other over at least some
portion of their length, at least one strand including two
contiguous nucleotides linked to each other through a
phosphodiester bond that is disrupted or broken during the nicking
process. Typically, the two corresponding nucleotides in the
opposing or complementary strand remain linked to each other. For
example, the nick can be generated between adjacent nucleotides on
one strand of a double stranded nucleic acid, while the other
strand has adjacent nucleotides joined by a phosphodiester bond at
the position opposite the break. In some embodiments, the nucleic
acid substrate can include synthetic nucleotides that are not
linked to each other by phosphodiester bonds but instead by at
least one other type of bond that is disrupted or broken as a
result of the nicking process. In some embodiments, nicking
conditions can include nicking both strands of a double stranded
nucleic acid, where at least some of the nicks are located opposite
corresponding nucleotides in the opposing or complementary strand
that remain joined. In a nick translation reaction, if one or more
nicks are translated towards each other, a double-stranded break
can be formed when the two nicks come into close proximity or into
complete alignment. In some embodiments, nicking conditions include
contact or mixture with a nicking enzyme, which can optionally have
endonuclease activity. In some embodiments, a nicking enzyme can be
wild-type or mutant form. In some embodiments, nicking conditions
can include contact, treatment or mixture with a compound that
nicks nucleic acids, including: 1,2,4-benzenetriol, gallic acid,
caffeic acid or gossypol in the presence of copper; or chromium
(VI) with hydrogen peroxide.
[0063] As used herein, the term "nick translation", "nick
translating" and its variants, can include any process or treatment
whereby the position of a nick within a nucleic acid strand is
effectively moved to a new position in a nucleic acid strand. Nick
translation typically includes extension of one new strand
accompanied by digestion or erosion of the other new strand. In
some embodiments, nick translation includes polymerization of
nucleotides or nucleotide analogs onto the new 3' end as well as
digestion or erosion of nucleosides from the new 5' end. With each
successive nucleotide polymerization onto the new 3' end, the
position of the nick is effectively moved by one nucleotide
position along the nicked strand. Nick translation can optionally
continue until the nick is translated to the end of the nicked
strand, or until the translated nick comes into either complete
alignment or into sufficiently close proximity to another nick in
the opposing strand as to form a double stranded break, resulting
in the generation of two nucleic acid fragments derived from the
original double stranded nucleic acid. The double stranded break
may generate two new blunt ends or two new overhang ends (e.g.,
"sticky" ends) in the resulting nucleic acid fragments.
[0064] As used herein, the term "nick translation conditions", and
its variants, can include any suitable condition for moving the
position of a nick in one strand of a double stranded nucleic acid
to a new position within the strand. In some embodiments,
conventional nick translation conditions employ two enzymes that
couple nucleic acid nicking and nick translating activities in the
presence of nucleotides labeled with a detectable moiety (e.g.,
radioactively labeled nucleotides) to produce end-labeled nucleic
acids. Of particular interest are nick translation reactions
including nicking and nick translating activities in the presence
of unlabeled nucleotides, resulting in the production of unlabeled
nucleic acid fragments, where a sample of nucleic acids are
subjected to such nick translation within the same reaction vessel.
In some embodiments, methods for generating a population of nucleic
acid fragments conducted according to the present teachings
comprise nick translation conditions employing one or more enzymes
that couple nucleic acid nicking and nick translating activities in
the presence of nucleotides that lack a detectable moiety, or in
the presence of labeled nucleotides. In some embodiments, nick
translation conditions conducted according to the present teachings
produce unlabeled nucleic acid fragments. For example, the present
teachings can include nick translation conditions comprising a
nicking enzyme (e.g., DNase I) and a polymerase having 5'.fwdarw.3'
degradation/polymerization activity, or can include a nicking
enzyme (e.g., DNase I) and a polymerase having 5'.fwdarw.3' strand
displacing activity (e.g., Taq polymerase). A nick translation
reaction according to the present teachings can further include one
or more unlabeled nucleotides (e.g., dATP, dTTP, dCTP, dGTP, dUTP,
or analogs thereof). A nick translation reaction can include a
cation, such as magnesium, manganese or calcium.
DESCRIPTION OF VARIOUS EMBODIMENTS
[0065] In some embodiments, the present teachings provide
compositions, systems, methods and kits for generating a population
of nucleic acid fragments. Fragmentation can be at random locations
in a nucleic acid. Fragmentation can be catalyzed by one or more
enzymes. In some embodiments, the present teachings provide
fragmenting nucleic acids comprising two or more enzymatic
reactions. In some embodiments, methods for generating a population
of nucleic acid fragments can include a nucleic acid nicking
reaction, a nick translation reaction, or both. A nicking reaction
can introduce nicks at one or more positions on either strand of a
double-stranded nucleic acid. A nick translation reaction can move
the position of a first nick on one strand to a new position that
can be aligned with a second nick, break, or other gap in the other
strand. Alignment of nicks, breaks, or gaps can result in
double-stranded breaks or fragmentation points. A nicking and/or
nick translating reaction can be conducted on nucleic acids in
solution.
[0066] In some embodiments the disclosed methods for generating a
population of nucleic acid fragments can be practiced on any
suitable nucleic acid sample, including a sample comprising DNA,
cDNA, RNA, RNA/DNA hybrids, and nucleic acid analogs.
[0067] Methods for generating a population of nucleic acid
fragments can be conducted with unlabeled nucleotides.
[0068] Compositions, systems, methods and kits disclosed herein for
generating a population of nucleic acid fragments for use in
preparing nucleic acids for sequencing. Nucleic acid sequencing
techniques, platforms, and systems for which this disclosure is
useful include, among others, sequencing-by-synthesis, chemical
degradation sequencing, ligation-based sequencing, hybridization
sequencing, pyrophosphate detection sequencing, capillary
electrophoresis, gel electrophoresis, next-generation, massively
parallel sequencing platforms, sequencing platforms that detect
hydrogen ions or other sequencing by-products, and single molecule
sequencing platforms. DNA fragments can be generated to have any
desired size or size range, including sizes useful for preparing
the nucleic acid for sequencing with any of the aforementioned
sequencing techniques, platforms, and/or systems.
[0069] Many next-generation or massively parallel sequencing
systems involve the generation of nucleic acid libraries, which
often comprise numerous fragments of larger nucleic acids that are
to be sequenced. For example, many next-generation sequencing
systems use fragment libraries, which comprise a collection of
nucleic acid fragments which can be used as sequencing templates.
Other types of libraries used in or for next-generation sequencing
include mate pair libraries, RNA libraries (e.g., mRNA libraries,
RNA-Seq libraries, whole transcriptome libraries, cell-specific RNA
libraries), chromatin immunoprecipitation (ChIP) libraries, exome
libraries and methylated DNA libraries.
[0070] The compositions, systems, methods, and kits disclosed
herein can be useful for preparing nucleic acid libraries for use
with any next-generation sequencing system, including: sequencing
by oligonucleotide probe ligation and detection (e.g., SOLiD.TM.
from Life Technologies, WO 2006/084131), probe-anchor ligation
sequencing (e.g., Complete Genomics.TM. or Polonator.TM.),
sequencing- by-synthesis (e.g., Genetic Analyzer and HiSeq.TM.,
from Illumina), pyrophosphate sequencing (e.g., Genome Sequencer
FLX from 454 Life Sciences), ion-sensitive sequencing (e.g.,
Personal Genome Machine and Proton from Ion Torrent Systems, Inc.),
and single molecule sequencing platforms (e.g., HeliScope.TM. from
Helicos.TM.). The size or size range of DNA fragments can be
selected for use in preparing the nucleic acid for sequencing on
any of the aforementioned sequencing techniques and systems.
[0071] In some embodiments, compositions, systems, methods and kits
disclosed herein can be used in a workflow for constructing a
nucleic acid library for sequencing in an oligonucleotide probe
ligation and detection system (e.g., SOLiD.TM. from Life
Technologies) or for ion-sensitive sequencing (e.g., Personal
Genome Machine and Proton from Ion Torrent Systems, Inc.). Nucleic
acid starting material can be any nucleic acid (for example, DNA,
cDNA, RNA, RNA/DNA hybrids, etc.), can be chromosomal, genomic,
transcriptomic, organellar, methylated, chromatin-linked, cloned,
unamplified or amplified, natural or synthetic, and can be isolated
from any source (for example, from an organism, normal or diseased
cells or tissues, body fluids, archived tissue (e.g., tissue
archived in formalin and/or in paraffin),
[0072] Nucleic acid starting material can be randomly fragmented
according the methods disclosed herein to generate fragmented DNA
useful for preparing sequencing libraries.
[0073] Compositions, systems, methods and kits disclosed herein can
be used to generate a population of nucleic acid fragments that are
selected to have any desired size or size range, including, for
example, from about 100 to about 250 by in length for use in
preparing a SOLiD.TM. fragment library, from about 100 to about 300
by in length for use in preparing an Ion Torrent PGM.TM. fragment
library, or from about 0.8 kb to about 1.4 kb in length for
preparing a SOLiD.TM. mate pair library. Nucleic acid fragments can
also be generated with sizes or size ranges appropriate for RNA
libraries (e.g., mRNA libraries, RNA-Seq libraries, whole
transcriptome libraries, cell-specific RNA libraries), chromatin
immunoprecipitation (ChIP) libraries, and methylated DNA
libraries.
[0074] At least one molecule in a population of nucleic acid
fragments can be joined to an oligonucleotide adaptor. For example,
a fragmented DNA can be joined to an adaptor to conduct a primer
extension reaction, amplification of the fragment, or for
attachment to particles (e.g., beads), or any combination thereof.
An adaptor that is joined to a fragmented DNA can anneal to an
oligonucleotide capture primer which is attached to a particle, and
a primer extension reaction can be conducted to generate a
complementary copy of the fragmented nucleic acid attached to the
particle or surface, thereby attaching a fragmented nucleic acid to
a surface or particle. Adaptors can have one or more amplification
primer hybridization sites, sequencing primer hybridization sites,
barcode sequences, or any combinations thereof. In some
embodiments, DNA fragments can be joined to one or more
SOLiD.TM.-compatible or Ion Torrent PGM.TM.-compatible or Ion
Torrent Proton.TM.-compatible adaptors to construct a fragment
library.
[0075] Double-stranded nucleic acids can be fragmented by
enzymatically nicking either strand at one or more positions and
nick translating one or more of the nicks to move the position of
the nick to align with a nick, break or gap on the opposite strand.
Alignment of a nick on one strand with a nick, break, or gap on the
other strand can generate a double-stranded nick, break or gap,
which can release a double-stranded fragment from the source
nucleic acid. Placement of multiple nicks into either or both
strands of a nucleic acid, followed by translation of the nicks to
positions in alignment with nicks, breaks, or gaps in the opposing
strand can yield multiple fragments (e.g., a population of
fragments) from the source nucleic acid. In some embodiments,
double-stranded nucleic acids can be fragmented by enzymatically
nicking either strand at one or more positions to produce
blunt-ended nucleic acids fragments. The blunt-ended fragments can
possess a terminal 5' phosphate and at least one end can be ligated
to an adaptor to form adaptor-double stranded nucleic acid
molecules. The adaptor-double stranded nucleic acid molecules can
include a nick in each nucleic acid strand opposite the ligated 5'
phosphate. Denaturing the adaptor-double stranded nucleic acid
molecules can remove the unligated portion of the adaptor and a
nick repair enzyme, such as a nick repair polymerase, can be used
to fill-in the overhang thereby generating double stranded
fragments from a nucleic acid source (FIG. 3).
[0076] In some embodiments, a method according to this disclosure
can comprise: (a) nicking the nucleic acids; and (b) nick
translating the nicks. In some embodiments, a method according to
this disclosure can comprise (a) enzymatically nicking
double-stranded nucleic acids on either strand at random positions;
and (b) nick translating the nicks so as to move the position of
the nicks on opposite strands into alignment, thereby generating a
double-stranded break. In some embodiments, enzymatic nicking can
be conducted with an enzyme having endonuclease activity. In some
embodiments, nick translating can be a reaction that couples a 5'
to 3' DNA polymerization/degradation reaction, or a reaction that
couples a 5' to 3' DNA polymerization/strand displacement reaction.
In some embodiments, the method can further comprise contacting the
fragmented nucleic acid with a non-template-dependent terminal
transferase enzyme (e.g., tailing reaction). In some embodiments,
the method can further comprise contacting the nucleic acid with a
phosphotransferase enzyme. In some embodiments, any combination of
reactions, including nicking, nick translating, tailing and/or
phosphotransferase reactions, can be conducted on nucleic acids in
solution. In some embodiments, the method can further comprise
contacting the double stranded gap nucleic acids with adaptors. In
some embodiments, the method can further comprise contacting the
double stranded gap nucleic acids with one or more nick repair
enzymes. In some embodiments, a double stranded nucleic acid can be
double stranded DNA, double stranded RNA or double stranded DNA/RNA
hybrid.
[0077] In some embodiments, the present teachings provide methods
for randomly fragmenting double-stranded nucleic acids to generate
a population of nucleic acid fragments, comprising the steps: (a)
enzymatically nicking either strand at random positions; and (b)
nick translating the nicks so as to move the position of the nicks
on opposite strands into alignment, thereby generating a
double-stranded break. In some embodiments, the enzymatic nicking
can be conducted with an enzyme having endonuclease activity. In
some embodiments, the nick translating can be a reaction that
couples a 5' to 3' DNA polymerization/degradation reaction, or a
reaction that couples a 5' to 3' DNA polymerization/strand
displacement reaction. In some embodiments, the method can further
comprise contacting the fragmented nucleic acid with a
non-template-dependent terminal transferase enzyme. In some
embodiments, the method can further comprise contacting the nucleic
acid with a phosphotransferase enzyme. In some embodiments, any
combination of reactions, including nicking, nick translating,
tailing and/or phosphotransferase reactions, can be conducted on
nucleic acids in solution. In some embodiments, the method can
further comprise contacting the double stranded nucleic acid
fragments with oligonucleotide adaptors. In some embodiments, the
method can further comprise contacting the double stranded nucleic
acid fragments with one or more nick repair enzymes. In some
embodiments, a double stranded nucleic acid can be double stranded
DNA, double stranded RNA or double stranded DNA/RNA hybrid.
[0078] In some embodiments, the present teachings provide methods
for to generate a population of nucleic acid fragments comprising
the steps: (a) providing a double-stranded nucleic acid having a
first and second nucleic acid strand; (b) nicking the first nucleic
acid strand at a first position and nicking the second nucleic acid
strand at a second position, wherein the first and second positions
are at different locations on the double-stranded nucleic acid; and
(c) moving the position of the first nick and the position of the
second nick into alignment along the double-stranded nucleic acid,
thereby generating a double-stranded break. In some embodiments,
the nicking can be conducted with an enzyme having endonuclease
activity. In some embodiments, the moving the position of the first
nick and the position of the second nick can be conducted with a
nick translation reaction. In some embodiments, the nick
translation reaction can be a reaction that couples a 5' to 3' DNA
polymerization/degradation reaction, or a reaction that couples a
5' to 3' DNA polymerization/strand displacement reaction. In some
embodiments, the method can further comprise contacting the
fragmented nucleic acid with a non-template-dependent terminal
transferase enzyme. In some embodiments, the method can further
comprise contacting the nucleic acid with a phosphotransferase
enzyme. In some embodiments, any combination of reactions,
including nicking, nick translating, tailing and/or
phosphotransferase reactions, can be conducted on nucleic acids in
solution. In some embodiments, the method can further comprise
contacting the double stranded nucleic acid fragments with
oligonucleotide adaptors. In some embodiments, the method can
further comprise contacting the double stranded nucleic acid
fragments with one or more nick repair enzymes. In some
embodiments, a double stranded nucleic acid can be double stranded
DNA, double stranded RNA or double stranded DNA/RNA hybrid.
[0079] Methods for randomly fragmenting nucleic acids can be used
to generate nucleic acid fragments which can be used as part of a
workflow for preparing nucleic acid libraries for sequencing (e.g.,
next generation sequencing). Workflows can include fragmenting,
adaptor joining, size selection, purification, amplification and/or
attaching to a surface. It will be readily apparent to one of skill
in the art that the workflow can repeat or omit any one or more of
the above steps. It will also be apparent to one of skill in the
art that the order and combination of steps may be modified to
generate the required double-stranded nucleic acid fragments, and
is not therefore limited to the exemplary workflow provided.
[0080] For example, methods for randomly fragmenting nucleic acids
can generally include reacting a nucleic acid with a nicking
enzyme, nick translation enzymes and co-factors. In some
embodiments, randomly fragmenting nucleic acids can also include
reacting nucleic acids with a non-template-dependent terminal
transferase enzyme and/or a phosphotransferase enzyme. In some
embodiments, randomly fragmenting nucleic acids can also include
reacting nucleic acids with adaptors and a nick repair enzyme. A
reaction for randomly fragmenting nucleic acids can be practiced in
a reaction vessel. A reaction for randomly fragmenting nucleic
acids can be practiced using a thermal-control apparatus.
[0081] Nucleic acid fragments generated by such methods can be
joined to one or more oligonucleotide adaptors for library
construction to be compatible with a next generation sequencing
platform. An oligonucleotide adaptor can be used to attach a
fragmented nucleic acid to a surface for sequencing.
[0082] In some embodiments, a reaction for randomly fragmenting
nucleic acids can be practiced on a nucleic acid which can be
isolated from any source, including: an organism; normal or
diseased cells or tissues; body fluids; or archived tissue (e.g.,
tissue archived in formalin and/or in paraffin). Nucleic acids can
be in any form, including chromosomal, genomic, organellar,
methylated, cloned, amplified, DNA, cDNA, RNA, RNA/DNA or
synthesized.
[0083] In some embodiments, a reaction for randomly fragmenting
nucleic acids can include one or more nicking enzymes that catalyze
nicking one strand of a double-stranded nucleic acid. For example,
a nicking enzyme can have endonuclease activity. In some
embodiments, a nicking enzyme can be a DNase I enzyme (FIGS. 1 and
2).
[0084] In some embodiments, a reaction for randomly fragmenting
nucleic acids can include one or more enzymes that can perform a
nick translation reaction that couples a 5'.fwdarw.3'
polymerization/degradation reaction, such as E. coli DNA Pol I
(FIG. 1). In some embodiments, a reaction for randomly fragmenting
nucleic acids can include one or more enzymes that can perform a
nick translation reaction that couples a 5'.fwdarw.3'
polymerization/strand displacement reaction, such as a Taq
polymerase, Tfi polymerase, or phi29 polymerase.
[0085] In some embodiments, a non-template-dependent terminal
transferase reaction can be catalyzed by one or more enzymes in the
presence of a plurality of nucleotides (FIG. 2). In some
embodiments, a non-template-dependent terminal transferase reaction
can be catalyzed by a Taq polymerase, Tfi DNA polymerase, 3'
exonuclease minus- large (Klenow) fragment, or 3' exonuclease
minus- T4 polymerase.
[0086] In some embodiments, one enzyme can catalyze a nick
translation reaction and a non-template-dependent terminal
transferase reaction (FIG. 2).
[0087] In some embodiments, one or more oligonucleotide adaptors
can be ligated to the fragmented nucleic acids to form
adaptor-double stranded nucleic acid molecules (FIG. 3).
[0088] In some embodiments, an adaptor-double stranded nucleic acid
molecule can be denatured such that the non-ligated portion of the
adaptor is removed and one or more nick repair enzymes, such as a
nick repair polymerase, for example Taq DNA polymerase, Bst DNA
polymerase, Platinum.RTM. Pfx DNA polymerase (Invitrogen), Tfi
Exo(-) DNA polymerase (Invitrogen) or Phusion.RTM. Hot Start
High-Fidelity DNA polymerase (New England Biolabs) perform a
fill-in reaction (FIG. 3) to generate a double-stranded nucleic
acid molecule.
[0089] In some embodiments, methods for generating a population of
nucleic acid fragments can further comprise an enzyme reaction that
adds a phosphate to a 5' end and/or removes a phosphate from a 3'
end of nicked nucleic acids. These reactions can be conducted with
one or more enzymes that catalyze addition of a phosphate group to
a 5' terminus of a single-stranded or double-stranded nucleic acid
(e.g., 5' side of a nick lacking a phosphate group) and/or that
catalyze removal of 3' phosphoryl groups from a nucleic acid (e.g.,
3' side of a nick having a phosphate group). In some embodiments,
addition or removal of a phosphate group can be catalyzed by a
polynucleotide kinase. A polynucleotide kinase can be a T4
polynucleotide kinase, or can be isolated from other sources (e.g.,
human). A polynucleotide kinase reaction can be conducted in the
presence of ATP.
[0090] In some embodiments, methods for generating a population of
nucleic acid fragments can be conducted in the presence of one or
more co-factors. For example, a nucleic acid nicking reaction can
be conducted in the presence of a cation. A cation can be
magnesium, manganese or calcium.
[0091] In some embodiments, methods for generating a population of
nucleic acid fragments can be conducted in any type of reaction
vessel. For example, a reaction vessel includes any type of tube or
well (e.g., 96-well plate).
[0092] In some embodiments, methods for generating a population of
nucleic acid fragments can be practiced in any type of
thermal-control apparatus. In some embodiments, a thermal-control
apparatus can maintain a desired temperature, or can elevate and
decrease the temperature, or can elevate and decrease the
temperature for multiple cycles. In some embodiments, a
thermal-control apparatus can maintain a temperature range of about
0.degree. C.-100.degree. C., or can cycle between different
temperature ranges of about 0.degree. C.-100.degree. C. Examples of
thermal-control apparatus include: a water bath and thermal cycler
machine. Many thermal cycler machines are commercially-available,
including (but not limited to) Applied Biosystems, Agilent,
Eppendorf, Bio-Rad and Bibby Scientific.
[0093] In some embodiments, one or both ends of nucleic acid
fragments can be joined to at least one oligonucleotide adaptor to
construct a nucleic acid library. Oligonucleotide adaptors can
include amplification primer sequences, sequencing primer sites
and/or barcodes. Oligonucleotide adaptors can have any structure,
including linear, hairpin, forked, or stem-loop. Fragmented nucleic
acids can be joined to an oligonucleotide adaptor to permit
attachment to particles (e.g., beads) or to a surface. For example,
an oligonucleotide adaptor can include a nucleotide sequence that
is complementary to an oligonucleotide capture primer that is
attached to a particle or surface. An oligonucleotide capture
primer can anneal to an adaptor that is joined to a fragmented
nucleic acid, and a primer extension reaction can be conducted to
generate a complementary copy of the fragmented nucleic acid
attached to the particle or surface, thereby attaching a fragmented
nucleic acid to a surface or particle. In some embodiments,
fragmented nucleic acids can be joined at both ends to
oligonucleotide adaptors that are complementary to different
oligonucleotide capture primers which are attached to a surface for
bridge amplification. Attachment of a fragmented nucleic acid to a
particle or surface can be achieved by conducting a primer
extension reaction or an amplification reaction in an aqueous
condition. Primer extension and amplification reactions can be
conducted under isothermal or thermocyclic conditions, or can be
reacted in a tube, a well, an oil-and-water emulsion droplet or an
agarose droplet (Yang 2010 Lab Chip 10(21):2841-2843).
[0094] In some embodiments, one or both ends of nucleic acid
fragments can be modified for attachment to a surface or particles.
For example, a 5' or 3' end can be modified to include an amino
group that can bind to a carboxylic acid compound on a surface or
particles. A 5' end can include a phosphate group for reacting with
an amine-coated surface (or particles) in the presence of a
carbodiimide (e.g., water soluble carbodiimide). A nucleic acid can
be biotinylated at one end to bind with an avidin-like compound
(e.g. streptavidin) attached to a surface.
[0095] In some embodiments, a surface can be planar, convex,
concave, or any combination thereof. A surface can be porous,
semi-porous or non-porous. A surface can comprise an inorganic
material, natural polymers, synthetic polymers, or non-polymeric
material. A surface includes a flowcell, well, groove, channel,
reservoir, filter, gel or inner walls of a capillary. A surface can
be coated with an acrylamide compound. Nucleic acid fragments can
be immobilized to an acrylamide compound coating on a surface.
[0096] In some embodiments, particles can have a shape that is
spherical, hemispherical, cylindrical, barrel-shaped, toroidal,
rod-like, disc-like, conical, triangular, cubical, polygonal,
tubular, wire-like or irregular. Particles can have an iron core,
or comprise a hydrogel or agarose (e.g., Sepharose.TM.). Particles
can be paramagnetic. Particles can be spherical or irregular shape.
Particles can have cavitation or pores, or can include
three-dimensional scaffolds. Particles can be coated with a
carboxylic acid compound or an amine compound for attaching nucleic
acid fragments. Particles can be coated with an avidin-like
compound (e.g., streptavidin) for binding biotinylated nucleic acid
fragments. In some embodiments, particles can be Ion Sphere.TM.
particles. Particles can be deposited to a surface of a sequencing
instrument. Sequencing reagents can be delivered to the deposited
particles to conduct sequencing reactions.
Composition
[0097] In some embodiments, the present teachings provide a
population of nucleic acid fragments prepared by nucleic acid
fragmentation methods. In some embodiments, a population of nucleic
acid fragments can include substantially similar-sized fragments or
substantially dissimilar-sized fragments. In some embodiments, a
population of nucleic acid fragments can be single-stranded or
double-stranded. In some embodiments, a population of nucleic acid
fragments can be DNA, RNA or chimeric DNA/RNA. In some embodiments,
a population of nucleic acid fragments can have a first end and a
second end. In some embodiments, a population of nucleic acid
fragments can have one or more blunt ends or overhang ends. In some
embodiments, a population of nucleic acid fragments can have one or
more tailed ends. In some embodiments, a population of nucleic acid
fragments can be chemically-modified, or joined to one or more
oligonucleotide adaptors. In some embodiments, a population of
nucleic acid fragments can be immobilized to a surface or
particles, or can be in solution.
[0098] Methods for Generating a Population of Nucleic Acid
Fragments
[0099] In some embodiments, the present teachings provide methods
for randomly fragmenting nucleic acids to generate a population of
nucleic acid fragments. In some embodiments, methods for randomly
fragmenting nucleic acids can generate a population of unlabeled
nucleic acid fragments.
[0100] Methods for generating a population of nucleic acids offer
advantages over conventional fragmentation methods. For example,
the methods provided by the present teachings employ enzymatic
reactions which produce less oxidative damage compared to
conventional shearing methods. The methods provided by the present
teachings exhibit an increase in yield of fragments that are useful
for further manipulations (e.g., nucleic acid ligation reactions).
Other advantages include, nucleic acids can be randomly fragmented,
showing little or no sequence preference, such as little or no
preference for GC-rich or GC-poor sequences. Methods for generating
a population of nucleic acids can be conducted in one or more
reaction vessels, can be performed on very small amounts of
starting material, can be performed in small reaction volume,
and/or can produce tunable size ranges. These methods can also be
performed manually or adapted for automated performance.
[0101] In some embodiments, the disclosure relates generally to
methods (and associated compositions, kits, systems and
apparatuses) for generating a population of nucleic acid fragments,
comprising: introducing at least one double stranded break into a
nucleic acid.
[0102] In some embodiments, the disclosure relates generally to
methods (and associated compositions, kits, systems and
apparatuses) for generating a population of nucleic acid fragments,
comprising: subjecting a first double stranded nucleic acid to
nicking conditions and to nick translating conditions.
[0103] In some embodiments, the disclosure relates generally to
methods (and associated compositions, kits, systems and
apparatuses) for generating a population of nucleic acid fragments,
comprising: subjecting a first double stranded nucleic acid to
nicking conditions, thereby generating a first nicked double
stranded nucleic acid having at least one nick in each strand; nick
translating at least one nick in each strand of the first nicked
double stranded nucleic acid; and generating at least one double
stranded break in the first nicked double stranded nucleic acid,
thereby forming at least two nucleic acid fragments derived from
the first double stranded nucleic acid.
[0104] In some embodiments, the disclosure relates generally to
methods (and associated compositions, kits, systems and
apparatuses) for generating a population of nucleic acid fragments,
comprising: (a) nicking the nucleic acids; and (b) nick translating
the nicks.
[0105] In some embodiments, the disclosure relates generally to
methods (and associated compositions, kits, systems and
apparatuses) for generating a population of nucleic acid fragments,
comprising: (a) nicking a nucleic acid at least once on each
strand; and (b) nick translating the nicks thereby generating a
double-stranded break to produce at least one nucleic acid
fragment.
[0106] In some embodiments, the disclosure relates generally to
methods (and associated compositions, kits, systems and
apparatuses) for generating a population of nucleic acid fragments,
comprising: (a) nicking a plurality of nucleic acids at least once
on each strand; and (b) nick translating the nicks thereby
generating double-stranded breaks in the plurality of nucleic acids
to produce a population of nucleic acid fragments.
[0107] In some embodiments, the disclosure relates generally to
methods (and associated compositions, kits, systems and
apparatuses) for generating a population of nucleic acid fragments,
comprising: cleaving a nucleic acid by (i) nicking the nucleic acid
at least once on each strand and (ii) nick translating the nicks
thereby generating a double-stranded break to produce at least one
nucleic acid fragment.
[0108] In some embodiments, the disclosure relates generally to
methods (and associated compositions, kits, systems and
apparatuses) for generating a population of nucleic acid fragments,
comprising: (a) providing a double-stranded nucleic acid having a
first and a second strand; (b) nicking the first strand at least
once to produce a first nick and nicking the second strand at least
once to produce a second nick; and (c) nick translating the first
nick and the second nick thereby generating a double-stranded break
to produce one or more nucleic acid fragments.
[0109] In some embodiments, the disclosure relates generally to
methods (and associated compositions, kits, systems and
apparatuses) for generating a population of nucleic acid fragments,
comprising: (a) introducing at least one nick into each strand of a
first and a second double stranded nucleic acid; (b) translating
one or more nicks in each strand of the first and the second double
stranded nucleic acid; and (c) generating at least one double
stranded break in the first and second double stranded nucleic acid
molecule, thereby forming a plurality (population) of nucleic acid
fragments. The first and second double stranded nucleic acids can
be subjected to the same fragmenting reaction in the same reaction
vessel. Typically, the first and second nucleic acids include
different nucleic acid sequences. In some embodiments, many
different nucleic acid molecules in a sample are fragmented to form
a population of nucleic acid fragments.
[0110] In some embodiments, one or more nicks can be introduced at
random positions on either strand of a double-stranded nucleic
acid.
[0111] In some embodiments, nick translating conditions can be
conducted with labeled or unlabeled nucleotides. In some
embodiments, at least one resulting nucleic acid fragment is
unlabeled. In some embodiments, at least one resulting nucleic acid
fragment is labeled.
[0112] In some embodiments, one, some, most or substantially all of
the nucleic acid fragments are substantially similarly sized. The
average size of the resulting nucleic acid fragments can be about
100 bp, about 200 bp, about 300 bp, about 500 bp, about 1000 bp,
about 2500 bp, about 5000 bp, about 10000 bp, about 50000 bp, about
100000 bp, about 1 Mb, about 5 Mb, about 10 Mb or greater in
length.
[0113] In some embodiments, a population of nucleic acid fragments
can include substantially similar-sized or substantially
dissimilar-sized nucleic acid fragments. For example, substantially
similar-sized fragments can differ from each other by an average of
about less than 50 bp, or differ from each other by an average of
about 50-75 bp, or by an average of about 75-100 bp, or by an
average of about 100-125 bp, or by an average of about 125-150 bp,
or by an average of about 150-175 by or more.
[0114] In some embodiments, methods for generating a population of
nucleic acid fragments comprise the steps: (a) introducing one or
more nicks on either strand of a double-stranded nucleic acid; and
(b) moving the positions of the nicks to a new position along the
double-stranded nucleic acid, under conditions suitable for
introducing one or more nicks on either strand of a double-stranded
nucleic acid and/or suitable for moving the positions of the nicks
to a new position along the double-stranded nucleic acid.
[0115] In some embodiments, methods for generating a population of
nucleic acid fragments comprise: introducing one or more nicks into
a nucleic acid by subjecting a sample including a plurality of
nucleic acids to nicking conditions; and generating at least one
double stranded break in at least one of the nucleic acids.
[0116] In some embodiments, methods for generating a population of
nucleic acid fragments comprise: (a) introducing at least one nick
into a double stranded nucleic acid; and (b) forming a double
stranded break in the nucleic acid by translating at least one
nick. In some embodiments, the introducing includes introducing at
least one nick into each strand of the double stranded nucleic
acid. Optionally, the translating includes translating at least two
nicks located on opposing nucleic acid strands towards each other.
In some embodiments, the method can include generating a double
stranded break, resulting in the formation of at least two nucleic
acid fragments derived from the original double stranded nucleic
acid.
[0117] In some embodiments, methods for generating a population of
nucleic acid fragments comprise: (a) nicking a nucleic acid at
least once on each strand; and (b) nick translating the nicks
thereby generating a double-stranded break to produce nucleic acid
fragments.
[0118] In some embodiments, methods for generating a population of
nucleic acid fragments comprise: cleaving a nucleic acid by (i)
nicking the nucleic acid at least once on each strand and (ii) nick
translating the nicks thereby generating a double-stranded break to
produce nucleic acid fragments.
[0119] In some embodiments, methods for generating a population of
nucleic acid fragments comprise: (a) providing a double-stranded
nucleic acid having a first and a second strand; (b) nicking the
first strand at least once to produce a first nick and nicking the
second strand at least once to produce a second nick; and (c) nick
translating the first nick and the second nick towards each other,
thereby generating a double-stranded break to produce nucleic acid
fragments.
[0120] In some embodiments, methods for generating a population of
nucleic acid fragments comprise: (a) introducing one or more nicks
on each strand of a double-stranded nucleic acid; and (b)
generating at least one double-stranded break by moving the
positions of at least two of the nicks along their respective
strands, thereby cleaving the double stranded nucleic acid into at
least two nucleic acid fragments.
[0121] In some embodiments, methods for generating a population of
nucleic acid fragments comprise: subjecting two or more different
double stranded nucleic acids to nicking conditions, thereby
forming at least two different nicked double stranded nucleic acids
each including at least one nick in each strand; and translating
the at least one nick in each strand so as to align the nicks on
opposing strands, wherein the translating includes subjecting the
at least two different nicked double stranded nucleic acids to nick
translating conditions.
[0122] In some embodiments, methods for generating a population of
nucleic acid fragments comprise: subjecting two or more different
double stranded nucleic acids to nicking conditions, thereby
forming at least two different nicked double stranded nucleic acids
each including at least one nick in each strand; and cleaving the
at least two different nicked double stranded nucleic acids,
wherein the cleaving includes creating at least one double stranded
break in each of the at least two different nicked double stranded
nucleic acids, wherein the creating includes nick translating the
least one nick in each strand, thereby generating a population of
nucleic acid fragments.
[0123] In some embodiments, methods for generating a population of
nucleic acid fragments comprise: cleaving at least two different
double stranded nucleic acid molecules into nucleic acid fragments,
wherein the cleaving includes introducing at least one nick into
each strand of the at least two different double stranded nucleic
acid molecules by subjecting the at least two different double
stranded nucleic acid molecules to nicking conditions, thereby
forming nicked double stranded nucleic acid molecules; and
generating one or more double stranded breaks in the nicked double
stranded nucleic acid molecules by nick translating one or more
nicks in a first strand and one or more nicks in a second strand of
the nicked double stranded nucleic acid molecule until at least two
nicks on opposing strands are aligned.
[0124] In some embodiments, methods for generating a population of
nucleic acid fragments comprise: subjecting two or more different
double stranded nucleic acids to nicking conditions, thereby
forming at least two different nicked double stranded nucleic
acids, each including at least one nick in each strand; cleaving
the at least two different nicked double stranded nucleic acids,
wherein the cleaving includes creating at least one double stranded
break in each of the at least two different nicked double stranded
nucleic acids, wherein the creating includes nick translating the
least one nick in each strand, thereby generating a population of
nucleic acid fragments.
[0125] In some embodiments, methods for generating a population of
nucleic acid fragments comprise: cleaving at least two different
double stranded nucleic acid molecules into nucleic acid fragments,
wherein the cleaving includes introducing at least one nick into
each strand of the at least two different double stranded nucleic
acid molecules by subjecting the at least two different double
stranded nucleic acid molecules to nicking conditions, thereby
forming nicked double stranded nucleic acid molecules; and
generating one or more double stranded breaks in the nicked double
stranded nucleic acid molecules by nick translating one or more
nicks in a first strand and one or more nicks in the second strand
of the nicked double stranded nucleic acid molecule until at least
two nicks on opposing strands are aligned.
[0126] In some embodiments, a nick refers to a location on a
double-stranded nucleic acid that lacks a phosphodiester bond
between adjacent nucleotides of one of the nucleic acid strands,
while the other strand has adjacent nucleotides joined by a
phosphodiester bond at that same location. In some embodiments, a
phosphodiester bond includes analog linkages that join adjacent
nucleotides (or nucleotide analogs),In some embodiments, methods
for generating a population of nucleic acid fragments can generate
a plurality of fragments having at least one blunt-end or overhang
end. In some embodiments, one or both ends of a double stranded
nucleic acid fragment can be blunt ended comprising ends that are
flush with each other. The terminal nucleosides at the blunt end
can have base pairing or can lack base pairing. In some
embodiments, one or both ends of a double stranded nucleic acid
fragment can include a 5' or 3' overhang end which comprises a
double stranded portion and a terminal single stranded portion.
[0127] In some embodiments, introducing one or more nicks can be
catalyzed by one or more enzymes. In some embodiments, an enzyme
that catalyzes nucleic acid nicking includes an enzyme having
endonuclease activity. In some embodiments, introducing one or more
nicks can be catalyzed by one or more enzymes in the presence of a
cation. In some embodiments, a cation can include magnesium,
manganese or calcium.
[0128] In some embodiments, the methods for generating a population
of nucleic acid fragments can further comprise one or more nick
repair enzymes, such as a nick repair polymerase. In some
embodiments, the nick repair polymerase can be Taq DNA polymerase,
Bst DNA polymerase, Platinum.RTM. Pfx DNA polymerase (Invitrogen),
Tfi Exo(-) DNA polymerase (Invitrogen) or Phusion.RTM. Hot Start
High-Fidelity DNA polymerase (New England Biolabs). For example, a
nick repair reaction can be conducted in the presence of a cation.
A cation can be magnesium, manganese or calcium.
[0129] In some embodiments, moving the position of the nicks can be
catalyzed by one or more enzymes in the presence of a plurality of
nucleotides. In some embodiments, moving the position of the nicks
can be catalyzed by one or more nick translation enzymes in the
presence of a plurality of nucleotides. In some embodiments, an
enzyme that catalyzes nick translation includes an enzyme that
couples a 5'.fwdarw.3' polymerization/degradation reaction, or an
enzyme that couples a 5'.fwdarw.3' polymerization/strand
displacement reaction.
[0130] In some embodiments, nicking and/or nick translating
reactions can be conducted on nucleic acids in solution or on
nucleic acids attached to a solid surface.
[0131] In some embodiments, methods for generating a population of
nucleic acid fragments can include contacting a nucleic acid with
one or more nucleic acid binding proteins at any step, or can lack
a nucleic acid binding protein. In some embodiments, methods for
generating a population of nucleic acid fragments can include
contacting a nucleic acid with one or more of nucleic acid binding
proteins in any combination with an enzyme that nicks at least one
nucleic acid strand and/or with a nick translating enzyme. In some
embodiments, a nicking and/or a nick translating reaction can
comprise at least one nucleic acid binding protein. In some
embodiments, methods for generating a population of nucleic acid
fragments can include contacting a nucleic acid with one or more of
nucleic acid binding proteins serially or simultaneously (or
essentially simultaneously) with any combination of an enzyme that
nicks at least one nucleic acid strand and/or with a nick
translating enzyme. In some embodiments, the nicking step and/or
the nick translating step can be conducted in the presence of at
least one nucleic acid binding protein. In some embodiments, a
nicking step and/or a nick translating step can be conducted in the
presence of at least one nucleic acid binding protein in solution.
Inclusion of a nucleic acid binding protein can improve the yield
of fragmented nucleic acids and/or can reduce formation of nucleic
acid fragments having rearranged portions. In some embodiments, a
nucleic acid binding protein can be a single-stranded nucleic acid
binding protein.
[0132] In some embodiments, a nucleic acid binding protein can be a
single-stranded nucleic acid binding protein. For example, a
single-stranded nucleic acid binding protein can be a phage T4 gp
32 protein, or can be from Sulfolobus solfataricus (e.g., S so SSB)
or from Methanococcus jannaschii (Mja SSB) or E. coli SSB
protein.
[0133] In some embodiments, methods for generating a population of
nucleic acid fragments can comprise a non-template-dependent
terminal transferase reaction (e.g., tailing reaction). In some
embodiments, a non-template-dependent terminal transferase reaction
can be catalyzed by one or more enzymes in the presence of a
plurality of nucleotides. In some embodiments, a
non-template-dependent terminal transferase reaction can be
catalyzed by one or more enzymes in the presence of one or more
types of nucleotides (e.g., A, G, C, or T/U).
[0134] In some embodiments, methods for generating a population of
nucleic acid fragments can further comprise an enzyme reaction that
adds a phosphate to a 5' end and/or removes a phosphate from a 3'
end. These reactions can be conducted with one or more enzymes that
catalyze addition of a phosphate group to a 5' terminus of a
single-stranded or double-stranded nucleic acid and/or that
catalyze removal of 3' phosphoryl groups from a nucleic acid. In
some embodiments, addition or removal of a phosphate group can be
catalyzed by a polynucleotide kinase. A polynucleotide kinase can
be a T4 polynucleotide kinase, or can be isolated from other
sources (e.g., human). A polynucleotide kinase reaction can be
conducted in the presence of ATP. In some embodiments, the method
can further comprise contacting the double stranded gap nucleic
acids with adaptors. In some embodiments, the method can further
comprise contacting the double stranded gap nucleic acids with one
or more nick repair enzymes.
[0135] In some embodiments, multiple reactions can be conducted in
one reaction vessel, such as a nicking reaction and nick
translation reaction, or such as a nicking reaction and nick
translation reaction and a tailing reaction, or such as a nicking
reaction and nick translation reaction and a tailing reaction and a
polynucleotide kinase reaction, or such as a nicking reaction and
ligation reaction and nick translation reaction and a nick repair
reaction. In some embodiments, different reactions (e.g., nicking
reaction, ligation reaction, nick translation reaction, tailing
reaction, nick repair reaction and/or polynucleotide kinase
reaction) can be conducted in separate reaction vessels or these
different reactions can be conducted at different times in the same
reaction vessel. A nucleic acid binding protein can be present in
any reaction vessel during a nicking and/or nick translating step.
In some embodiments, a reaction vessel can be any type of tube or
well (e.g., 96-well plate). In some embodiments, different
reactions (e.g., nicking reaction, ligation reaction, nick
translation reaction, tailing reaction, nick repair reaction and/or
polynucleotide kinase reaction) can be conducted in an
oil-and-water emulsion droplet or an agarose droplet (Yang 2010 Lab
Chip 10(21):2841-2843).
[0136] In some embodiments, the size range of fragments resulting
from conducting a nucleic acid fragmentation reaction can be about
50-150 bp, or about 150-250 bp, or about 250-500 bp, or about
500-750 bp, or about 750-1000 bp, or about 1-2 kb, or about 2-5 kb,
or about 5-8 kb, or about 8-10 kb, or about 10-20 kb, or about
20-40 kb, or about 40-60 kb, or longer. In some embodiments, the
resulting average fragment size (or average size range of nucleic
acid fragments) can be modulated by: adjusting the nicking
conditions and/or the nick translating conditions. For example, the
nicking conditions and/or the nick translating conditions can be
adjusted by increasing or decreasing an enzyme concentration (e.g.,
nicking or nick translating enzyme); by increasing or decreasing
the cation concentration; by increasing or decreasing the
nucleotide concentration; by increasing or decreasing a reaction
temperature, time and/or pH.
[0137] In some embodiments, the resulting average fragment size (or
average size range) can be modulated by: increasing or decreasing
an enzyme concentration (e.g., nicking or nick translating enzyme);
by increasing or decreasing the cation concentration; by increasing
or decreasing the nucleotide concentration; by increasing or
decreasing a reaction temperature, time and/or pH.
[0138] In some embodiments, the number of nicks introduced on
either strand of a double-stranded nucleic acid can be modulated
by: increasing or decreasing an enzyme concentration; by increasing
or decreasing the cation concentration; by increasing or decreasing
the nucleotide concentration; by increasing or decreasing a
reaction temperature, time and/or pH. In some embodiments, the nick
translation reaction can be modulated by: increasing or decreasing
the enzyme concentration; by increasing or decreasing the
nucleotide concentration; by increasing or decreasing the cation
concentration; by increasing or decreasing a reaction temperature,
time and/or pH.
[0139] In some embodiments, the average number of nicks introduced
into nucleic acid molecules within a mixed population of different
nucleic acid molecules on either strand of a double-stranded
nucleic acid can be modulated by: increasing or decreasing an
enzyme concentration (e.g., DNase I); by increasing or decreasing
the cation concentration (e.g., magnesium); by increasing or
decreasing the nucleotide concentration; by increasing or
decreasing a reaction temperature, time and/or pH.
[0140] In some embodiments, the nick repair reaction can be
modulated by: increasing or decreasing the nick repair enzyme
concentration; by increasing or decreasing the nucleotide
concentration; by increasing or decreasing the cation
concentration; by increasing or decreasing a reaction temperature,
time and/or pH.
[0141] In some embodiments, methods for generating a population of
nucleic acid fragments, can include the steps: (a) providing a
double-stranded nucleic acid having a first and second nucleic acid
strand; (b) nicking the first nucleic acid strand at a first
position and nicking the second nucleic acid strand at a second
position; and (c) moving the position of the first nick and the
position of the second nick to a new position, under conditions
suitable for nicking the first nucleic acid strand, suitable for
nicking the second nucleic acid strand, suitable for moving the
position of first nick, and/or suitable for moving the position of
the second nick.
[0142] In some embodiments, methods for generating a population of
nucleic acid fragments, can include the steps: (a) providing a
double-stranded nucleic acid having a first and second nucleic acid
strand; (b) nicking the first nucleic acid strand at a first
position and nicking the second nucleic acid strand at a second
position, wherein the first and second positions are at different
locations on the double-stranded nucleic acid; and (c) moving the
position of the first nick and the position of the second nick in a
direction towards each other until the position of the first nick
and the second nick are aligned so as to generate a double-stranded
gap, thereby fragmenting the nucleic acid, under conditions
suitable for nicking the first nucleic acid strand, suitable for
nicking the second nucleic acid strand, suitable for moving the
position of first nick, and/or suitable for moving the position of
the second nick. In some embodiments, the positions of the first
and second nick can be proximal to each other to cause
fragmentation of the nucleic acids.
[0143] In some embodiments, methods for generating a population of
nucleic acid fragments can include additional enzyme steps to
improve the yield of fragments useful for further manipulations.
For example, a 5' end of a nicked nucleic acid can lack a phosphate
group which can inhibit ligation to another nucleic acid fragment.
In another example, a 3' end of a nicked nucleic acid can have a
phosphate group which can inhibit nick translation. In some
embodiments, the methods can include an enzyme reaction that adds a
phosphate to a 5' end and/or removes a phosphate from a 3' end of
nicked nucleic acids. These reactions can be conducted with one or
more enzymes that catalyze addition of a phosphate group to a 5'
terminus of a single-stranded or double-stranded nucleic acid
and/or that catalyze removal of 3' phosphoryl groups from a nucleic
acid. In some embodiments, addition or removal of a phosphate group
can be catalyzed by a polynucleotide kinase. A polynucleotide
kinase can be a T4 polynucleotide kinase, or can be isolated from
other sources (e.g., human). A polynucleotide kinase reaction can
be conducted in the presence of ATP. In some embodiments, the can
further comprise contacting the double stranded gap nucleic acids
with adaptors. In some embodiments, the method can further comprise
contacting the double stranded gap nucleic acids with one or more
nick repair enzymes. In some embodiments, the methods can include
the steps: (a) providing a double-stranded nucleic acid having a
first and second nucleic acid strand; (b) nicking the first nucleic
acid strand at a first position to generate a first nick and
nicking the second nucleic acid strand at a second position to
generate a second nick, wherein the first and second positions are
at different locations on the double-stranded nucleic acid; (c)
moving the position of the first nick and the position of the
second nick in a direction towards each other until the position of
the first nick and the second nick are aligned to generate a
double-stranded gap, thereby fragmenting the nucleic acid; and (d)
adding a phosphate group to a 5' terminus of the first or second
nick or removing a phosphate group from a 3' end of the first or
second nick, under conditions suitable for nicking the first
nucleic acid strand, suitable for nicking the second nucleic acid
strand, suitable for moving the position of first nick, suitable
for moving the position of the second nick and/or suitable for
adding a phosphate group to a 5' terminus of the first or second
nick or removing a phosphate group from a 3' end of the first or
second nick. In some embodiments, the positions of the first and
second nick can be proximal to each other to cause fragmentation of
the nucleic acids.
[0144] In some embodiments, methods for generating a population of
nucleic acid fragments can generate fragments having at least one
blunt-end, or can generate fragments having both ends blunt-ended.
In some embodiments, nicking the first nucleic acid strand can be
catalyzed by one or more enzymes.
[0145] In some embodiments, an enzyme that catalyzes nucleic acid
nicking includes an enzyme having endonuclease activity. In some
embodiments, nicking the first nucleic acid strand can be catalyzed
by one or more enzymes in the presence of a cation. In some
embodiments, nicking the second nucleic acid strand can be
catalyzed by one or more enzymes. In some embodiments, nicking the
second nucleic acid strand can be catalyzed by one or more enzymes
in the presence of a cation. In some embodiments, a cation can
include magnesium, manganese or calcium. In some embodiments, the
position of the first nick and the position of the second nick can
be at the same or different location on the double-stranded nucleic
acid.
[0146] In some embodiments, moving the position of the first nick
can be catalyzed by one or more enzymes in the presence of a
plurality of nucleotides (labeled and/or unlabeled). For example, a
nucleotide can be joined to a label such as a fluorescent,
luminescent or radioactive moiety. In some embodiments, moving the
position of the second nick can be catalyzed by one or more enzymes
in the presence of a plurality of nucleotides (labeled and/or
unlabeled). In some embodiments, an enzyme that catalyzes nick
translation includes an enzyme that couples a 5'.fwdarw.3'
polymerization/degradation reaction, or an enzyme that couples a
5'.fwdarw.3' polymerization/strand displacement reaction.
[0147] In some embodiments, methods for generating a population of
nucleic acid fragments further comprise a non-template-dependent
terminal transferase reaction (e.g., tailing reaction). In some
embodiments, a non-template-dependent terminal transferase reaction
can be catalyzed by one or more enzymes in the presence of a
plurality of nucleotides (labeled or unlabeled). In some
embodiments, a non-template-dependent terminal transferase reaction
can be catalyzed by one or more enzymes in the presence of one or
more types of nucleotides (e.g., A, G, C, T, U or analogs
thereof).
[0148] In some embodiments, methods for generating a population of
nucleic acid fragments can further include joining an
oligonucleotide adaptor to at least one end of a fragment. For
example, method for generating a population of nucleic acid
fragments can include the steps: (a) generating a population of
nucleic acid fragments by (i) nicking the nucleic acid at least
once on each strand and (ii) nick translating the nicks thereby
generating a double-stranded break to produce a population of
nucleic acid fragments; and (b) joining at least one end of each of
the fragments in the population to an oligonucleotide adaptor,
thereby generating a nucleic acid library.
[0149] In some embodiments, the method comprises: (a) providing a
double-stranded nucleic acid having a first and second nucleic acid
strand; (b) nicking the first nucleic acid strand at a first
position to generate a first nick and nicking the second nucleic
acid strand at a second position to generate a second nick, wherein
the first and second positions are at different locations on the
double-stranded nucleic acid; (c) moving the position of the first
nick and the position of the second nick in a direction towards
each other until the position of the first nick and the second nick
are aligned to generate a double-stranded gap, thereby fragmenting
the nucleic acid; (d) joining an oligonucleotide adaptor to the
fragmented nucleic acid; denaturing the adaptor-nucleic acid
fragment; and nick repairing the nucleic acid strand opposite the
site of ligation, under conditions suitable for nicking the first
nucleic acid strand, suitable for nicking the second nucleic acid
strand, suitable for moving the position of first nick, suitable
for moving the position of the second nick, suitable for ligating
the adaptor to the nucleic acid fragment and/or suitable for nick
repairing the nucleic acid strand opposite the site of
ligation.
[0150] In some embodiments, multiple reactions can be conducted
(with or without binding to nucleic acid binding proteins) in one
reaction vessel, such as a nicking reaction and nick translation
reaction, or a nicking reaction and a ligation reaction, or a
nicking reaction and a ligation reaction and a nick repair
reaction, or such as a nicking reaction and nick translation
reaction and a tailing reaction. In some embodiments, different
reactions (e.g., nicking reaction, nick translation reaction,
ligation reaction, nick repair reaction and/or tailing reaction)
can be conducted (with or without binding to nucleic acid binding
proteins) in separate reaction vessels or these different reactions
can be conducted at different times in the same reaction
vessel.
[0151] Additional Steps
[0152] In some embodiments, additional nucleic acid manipulations
can be conducted following a fragmentation reaction(s). In some
embodiments, any combination of additional reactions can be
conducted in any order, and can include: chemical modification,
size-selection, end repairing, tailing, adaptor-joining, ligation,
nick repairing, purification, nick translation, amplification,
surface attachment and/or sequencing. In some embodiments, any of
these reactions can be omitted or can be repeated.
[0153] In some embodiments, nucleic acid fragmentation reactions
and additional reactions can be conducted to prepare nucleic acid
fragments to be used for insertion into a vector, as probes, as a
source of double-stranded and single-stranded fragments, as
amplification templates, or for preparing nucleic acid
libraries.
[0154] In some embodiments, a population of nucleic acid fragments
can be modified to attach to a surface. For example, a population
of nucleic acid fragments can be amino-modified for attachment to a
surface (e.g., particles or a planar surface). In some embodiments,
an amino-modified nucleic acid fragment can be attached to a
surface that is coated with a carboxylic acid. In some embodiments,
an amino-modified nucleic acid can be reacted with EDC (or EDAC)
for attachment to a carboxylic acid coated surface (with or without
NHS). In some embodiments, nucleic acid fragments can be attached
to particles, such as Ion Sphere.TM. particles (Life
Technologies).
[0155] In some embodiments, a surface can be an outer or top-most
layer or boundary of an object. In some embodiments, a surface can
be a solid surface or semi-solid surface. In some embodiments, a
surface can be porous or non-porous. In some embodiments, a surface
can be a planar surface, as well as concave, convex, or any
combination thereof. In some embodiments, a surface can be a bead,
particle, sphere, filter, flowcell, or gel. In some embodiments, a
surface includes the inner walls of a capillary, a channel, a well,
groove, channel, reservoir. In some embodiments, a surface can
include texture (e.g., etched, cavitated, pores, three-dimensional
scaffolds or bumps). In some embodiments, a surface can 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). In some embodiments, nucleic
acid fragments can be arranged on a surface in a random pattern,
organized pattern, rectilinear pattern, hexagonal pattern, or
addressable array pattern.
[0156] In some embodiments, a population of nucleic acid fragments
can be modified to attach to one member of a binding partner (e.g.,
biotin). In some embodiments, a biotinylated nucleic acid fragment
can be attached to another member of a binding partner (e.g.,
avidin-like, such as streptavidin) which is attached to a
surface.
[0157] In some embodiments, molecules that function as binding
partners include: biotin (and its derivatives) and their binding
partners avidin, streptavidin (and their derivatives); His-tags
which bind with nickel, cobalt or copper; cysteine, histidine, or
histidine patch which bind Ni-NTA; maltose which binds with maltose
binding protein (MBP); lectin-carbohydrate binding partners;
calcium-calcium binding protein (CBP); acetylcholine and
receptor-acetylcholine; protein A and binding partner anti-FLAG
antibody; GST and binding partner glutathione; uracil DNA
glycosylase (UDG) and ugi (uracil-DNA glycosylase inhibitor)
protein; antigen or epitope tags which bind to antibody or antibody
fragments, particularly antigens such as digoxigenin, fluorescein,
dinitrophenol or bromodeoxyuridine and their respective antibodies;
mouse immunoglobulin and goat anti-mouse immunoglobulin; IgG bound
and protein A; receptor-receptor agonist or receptor antagonist;
enzyme-enzyme cofactors; enzyme-enzyme inhibitors; and
thyroxine-cortisol. Another binding partner for biotin can be a
biotin-binding protein from chicken (Hytonen, et al., BMC
Structural Biology 7:8).
[0158] In some embodiments, a population of nucleic acid fragments
can be used to generate DNA fragments that are selected to have any
desired size or size range, including, for example, from about 100
to about 250 by in length for use in preparing a SOLiD.TM. fragment
library, from about 100 to about 300 by in length for use in
preparing an Ion Torrent PGM.TM. fragment library, or from about
0.8 kb to about 1.4 kb in length for preparing a SOLiD.TM. mate
pair library, or about 100 to about 60 kb in length for any type of
nucleic acid library. DNA fragments can also be generated with
sizes or size ranges appropriate for RNA libraries (e.g., mRNA
libraries, RNA-Seq libraries, whole transcriptome libraries,
cell-specific RNA libraries), chromatin immunoprecipitation (ChIP)
libraries, and methylated DNA libraries.
[0159] Library Preparation Methods
[0160] In some embodiments, a population of nucleic acid fragments
produced by the present teachings can be used to prepare any type
of nucleic acid library that is compatible with any type of
sequencing platform including chemical degradation,
chain-termination, sequence-by-synthesis, pyrophosphate, massively
parallel, ion-sensitive, and single molecule platforms. For
example, a sequencing platform can include any type of sequencing
reaction, including: Maxam Gilbert, Sanger, capillary
electrophoresis (e.g., Applied Biosystems, now part of Life
Technologies), or any type of next generation sequence platform,
including oligonucleotide probe ligation sequencing (e.g., SOLiD),
probe-anchor ligation sequencing (e.g., Complete Genomics or
Polonator), sequence by synthesis (e.g., Illumina, Helicos),
pyrophosphate sequencing (e.g., 454/Roche), and ion-sensitive
sequencing (e.g., Ion Personal Genome Machine.TM., produced by Ion
Torrent Systems, Inc., a subsidiary of Life Technologies Corp,
Carlsbad, Calif.).
[0161] In some embodiments, additional nucleic acid manipulations
can be conducted following a fragmentation reaction(s), including
any combination of additional reactions can be conducted in any
order, and can include: chemical modification, size-selection, end
repairing, tailing, ligation, nick repairing, adaptor-joining,
purification, nick translation, amplification surface attachment
and/or sequencing. In some embodiments, any of these reactions can
be omitted or can be repeated.
[0162] In some embodiments, a nucleic acid fragmentation reaction
can include: size-selection, adaptor-joining, and nick translation.
In some embodiments, a nucleic acid fragmentation reaction can
include: size-selection, adaptor-joining, nick translation and
amplification. In some embodiments, a nucleic acid fragmentation
reaction can include: ligation, nick repair reaction and size
selection. In some embodiments, a nucleic acid fragmentation
reaction can include: ligation, nick repair reaction, size
selection and amplification. In some embodiments, a nucleic acid
fragmentation reaction can include: purification, ligation, nick
repair reaction, purification and size selection. In some
embodiments, a nucleic acid fragmentation reaction can include:
size selection, ligation, nick repair reaction, purification and
size selection.
[0163] In some embodiments, amplification can include
thermo-cycling amplification or isothermal amplification reactions.
In some embodiments, amplification can be conducted with polymerase
that are thermo-stable or thermo-labile. In some embodiments,
amplification can be conducted as a PCR reaction.
[0164] In some embodiments, nucleic acid fragments produced by the
present teachings can result in advantages over the teachings of
the prior art. For example, nucleic acid fragments produced by the
present teachings can result in increased yield. In some
embodiments, the nucleic acid fragments produced by the present
teachings are generated in a more efficient manner and therefore
decrease the amount of time required to produce the fragmented
nucleic acid library. In some embodiments, the nucleic acid
fragments produced by the teachings of the present disclosure are
sufficient in yield to be used in a downstream application without
an amplification step. For example, nucleic acid fragments produced
by the present teachings can be directly incorporated into a
downstream template preparation step, such as the Ion Xpress.TM.
Template Kit using an Ion Torrent.TM. PGM system (e.g.,
PCR-mediated addition of the nucleic acid fragment library onto Ion
Sphere.TM. Particles)(Life Technologies, Part No. 4467389). For
example, instructions to prepare a template library from the
nucleic acid fragment library can be found in the Ion Xpress
Template Kit User Guide (Life Technologies, Part No. 4465884),
hereby incorporated by reference in its entirety. Instructions for
loading the template library onto the Ion Torrent.TM. Chip for
sequencing are described in the Ion Sequencing User Guide (Part No.
4467391), hereby incorporated by reference in its entirety.
[0165] Size-Selection:
[0166] In some embodiments, a population of nucleic acid fragments
can be subjected to any size-selection procedure to obtain any
desired size range. In some embodiments, a population of nucleic
acid fragments is not size-selected. In some embodiments, nucleic
acid fragments generated by practicing the present teachings can be
size-selected to produce a population of nucleic acid
fragments.
[0167] In some embodiments, nucleic acid size selection method
includes without limitation: solid phase adherence or
immobilization; electrophoresis, such as gel electrophoresis; and
chromatography, such as HPLC and size exclusion chromatography. In
some embodiments, a solid phase adherence/immobilization methods
involves paramagnetic beads coated with a chemical functional group
that interacts with nucleic acids under certain ionic strength
conditions with or without polyethylene glycol or polyalkylene
glycol.
[0168] Examples of solid phase adherence/immobilization methods
include but are not limited to: SPRI (Solid Phase Reversible
Immobilization) beads from Agencourt (see Hawkins 1995 Nucleic
Acids Research 23:22) which are carboxylate-modified paramagnetic
beads; MAGNA PURE magnetic glass particles (Roche Diagnostics,
Hoffmann-La Roche Ltd.); MAGNESIL magnetic bead kit from Promega;
BILATEST magnetic bead kit from Bilatec AG; MAGTRATION paramagnetic
system from Precision System Science, Inc.; MAG BIND from Omega
Bio-Tek; MAGPREP silica from Merck/Estapor; SNARe DNA purification
system from Bangs; CHEMAGEN M-PVA beads from CHEMAGEN; and magnetic
beads from Aline Bioscience (DNA Purification Kit).
[0169] In some embodiments, size-selected nucleic acid fragments
can be about 50-250 bp, or about 250-500 bp, or about 500-750 bp,
or about 750-1000 bp, or about 1-5 kb, or about 5-10 kb, or about
10-25 kb, or about 25-50 kb, or about 50-60 kb or longer.
[0170] Repairing Nucleic Acid Fragments:
[0171] In some embodiments, repairing a population of nucleic acid
fragments may be desirable. In some embodiments, a nucleic acid
fragment from a population can have a first end, a second end, or
an internal portion, having undesirable features, such as nicks,
overhang ends, ends lacking a phosphorylated end, ends having a
phosphorylated end, or nucleic acid fragments having apurinic or
apyrimidinic residues. In some embodiments, enzymatic reactions can
be conducted to repair one or more ends or internal portions. In
some embodiments, nucleic acid fragments can be subjected to
enzymatic reactions to convert overhang ends to blunt ends, or to
phosphorylate or de-phosphorylate the 5' end of a strand, or to
close nicks, to repair oxidized purines or pyrimidines, to repair
deaminated cytosines, or to hydrolyze the apurinic or apyrimidinic
residues. In some embodiments, repairing or end-repairing nucleic
acid fragments includes contacting nucleic acid fragments with: an
enzyme to close single-stranded nicks in duplex DNA (e.g., T4 DNA
ligase); an enzyme to phosphorylate the 5' end of at least one
strand of a duplex DNA (e.g., T4 polynucleotide kinase); an enzyme
to remove a 5' or 3'phosphate (e.g., any phosphatase enzyme, such
as calf intestinal alkaline phosphatase, bacterial alkaline
phosphatase, shrimp alkaline phosphatase, Antarctic phosphatase,
and placental alkaline phosphatase); an enzyme to remove 3'
overhang ends (e.g., DNA polymerase I, Large (Klenow) fragment, T4
DNA polymerase, mung bean nuclease); an enzyme to fill-in 5'
overhang ends (e.g., T4 DNA polymerase, Tfi DNA polymerase, Tli DNA
polymerase, Taq DNA polymerase, Large (Klenow) fragment, phi29 DNA
polymerase, Mako DNA polymerase (Enyzmatics, Beverly, Mass.), or
any heat-stable or heat-labile DNA polymerase); an enzyme to remove
5' overhang ends (e.g., S1 nuclease); an enzyme to remove 5' or 3'
overhang ends (e.g., mung bean nuclease); an enzyme to hydrolyze
single-stranded DNA (e.g., nuclease P1); an enzyme to remove both
strands of double-stranded DNA (e.g., nuclease Bal-31); and/or an
enzyme to remove an apurinic or apyrimidinic residue (e.g.,
endonuclease IV). In some embodiments, the polymerases can have
exonuclease activity, or have a reduced or lack exonuclease
activity.
[0172] In some embodiments, a repairing or end-repairing reaction
can be supplemented with additional repairing enzymes in any
combination and in any amount, including: endonuclease IV
(apurinic- apyrimidinic removal), Bst DNA polymerase (5'>3'
exonuclease for nick translation), formamidopyrimidine DNA
glycosylase (FPG) (e.g., base excision repair for oxidize purines),
uracil DNA glycosylase (uracil removal), T4 endonuclease V
(pyrimidine removal) and/or endonuclease VIII (removes oxidized
pyrimidines). In some embodiments, a repairing or end-repairing
reaction can be conducted in the presence of appropriate
co-factors, including dNTPs, NAD, (NH.sub.4).sub.2SO.sub.4, KCl,
and/or MgSO.sub.4.
[0173] Adaptor-Joining
[0174] In some embodiments, a population of nucleic acid fragments
(e.g., generated by any method disclosed herein) can be joined to
at least one type of nucleic acid adaptor (e.g., oligonucleotide
adaptor). In some embodiments, at least one end of nucleic acid
fragments in a population, so generated by the disclosed methods,
can be joined to one or more oligonucleotide adaptors to generate a
nucleic acid library.
[0175] In some embodiments, methods for generating a population of
nucleic acid fragments comprise: introducing at least one double
stranded break into a sample nucleic acid so as to generate at
least one nucleic acid fragment; and joining at least one end of
the at least one nucleic acid fragment to one or more
oligonucleotide adaptors, thereby generating a fragment-adaptor
construct. In some embodiments, a fragment-adaptor construct can be
part of a nucleic acid library.
[0176] In some embodiments, methods for generating a population of
nucleic acid fragments comprise: subjecting a sample nucleic acid
to nicking conditions; subjecting the sample nucleic acid to nick
translation conditions so as to generate nucleic acid fragments;
and joining at least one end of the nucleic acid fragments to one
or more oligonucleotide adaptors, thereby generating
fragment-adaptor constructs. In some embodiments, the
fragment-adaptor constructs can be part of a nucleic acid
library.
[0177] In some embodiments, methods for generating a population of
nucleic acid fragments comprise: nicking a nucleic acid; nick
translating the nicks so as to generate a nucleic acid fragment;
and joining at least one end of the nucleic acid fragment to one or
more oligonucleotide adaptors, thereby generating a
fragment-adaptor construct. In some embodiments, a fragment-adaptor
construct can be part of a nucleic acid library.
[0178] In some embodiments, methods for generating a population of
nucleic acid fragments comprise: (a) generating a population of
nucleic acid fragments by (i) nicking the nucleic acid at least
once on each strand and (ii) nick translating the nicks thereby
generating a double-stranded break to produce a population of
nucleic acid fragments; and (b) joining at least one end of each of
the fragments in the population to an oligonucleotide adaptor,
thereby generating a nucleic acid library.
[0179] In some embodiments, nucleic acid fragments can be joined at
one or both ends to at least one nucleic acid adaptor. In some
embodiments, methods for generating nucleic acid library constructs
comprise the steps: (a) cleaving a nucleic acid by (i) nicking the
nucleic acid at least once on each strand and (ii) nick translating
the nicks thereby generating a double-stranded break to produce at
least one nucleic acid fragment, wherein the nicking and/or the
nick translating steps comprise a nucleic acid binding protein; and
(b) joining at least one end of the at least one nucleic acid
fragment to a oligonucleotide adaptor, thereby generating a
fragment-adaptor molecule.
[0180] In some embodiments, methods for generating a nucleic acid
library construct comprises the steps: (a) nicking a nucleic acid
at least once on each strand; (b) nick translating the nicks
thereby generating a double-stranded break to produce at least one
nucleic acid fragment; (c) joining at least one end of the at least
one nucleic acid fragment to an oligonucleotide adaptor, thereby
generating a fragment-adaptor molecule.
[0181] In some embodiments, methods for generating a nucleic acid
library construct comprises the steps: (a) providing a
double-stranded nucleic acid having a first and a second nucleic
acid strand; (b) nicking the first nucleic acid strand at least
once to produce a first nick and nicking the second nucleic acid
strand at least once to produce a second nick; (c) nick translating
the first nick and the second nick thereby generating a
double-stranded break to produce at least one nucleic acid
fragment; and (d) joining at least one end of the nucleic acid
fragment to an oligonucleotide adaptor, thereby generating a
fragment-adaptor molecule. In some embodiments, fragment-adaptor
molecules can be generated for preparing nucleic acid library
constructs.
[0182] In some embodiments, any step of a method for generating a
nucleic acid library construct can include a nucleic acid binding
protein or can lack a nucleic acid binding protein.
[0183] In some embodiments, a nucleic acid fragment in a population
comprises a first end and a second end. In some embodiments, a
nucleic acid fragment can be joined at its first end to a first
oligonucleotide adaptor. In some embodiments, a nucleic acid
fragment can be joined at its second end to a second
oligonucleotide adaptor. In some embodiments, on at least one end
of a double-stranded nucleic acid fragment, one strand of the
nucleic acid fragment can be joined to one strand of a
double-stranded oligonucleotide adaptor to generate a
fragment-adaptor molecule having a break (e.g., a nick or gap). In
some embodiments, on at least one end of a double-stranded nucleic
acid fragment, both strands of the nucleic acid fragment can be
joined to both strand of a double-stranded oligonucleotide adaptor
to generate a fragment-adaptor molecule. In some embodiments, the
first and second oligonucleotide adaptors can be the same or
different adaptors. In some embodiments, a nucleic acid fragment
can be circularized by joining one or more oligonucleotide adaptors
to both ends of a nucleic acid fragment. In some embodiments,
nucleic acid fragments can be joined to at least one adaptor with a
ligase enzyme, PCR amplification, nucleotide polymerization or any
combination thereof. In some embodiments, one or both ends of a
nucleic acid fragment can be joined to one or more adaptors, and
the joining reaction can be conducted in solution. In some
embodiments, one end or both ends of nucleic acid fragments can be
joined to at least one type of oligonucleotide adaptor. In some
embodiments, nucleic acid fragments and adaptors can be joined by
ligation or annealing.
[0184] In some embodiments, an oligonucleotide adaptor can be DNA,
RNA or chimeric RNA/DNA molecules. In some embodiments, an adaptor
can include one or more ribonucleoside residues. In some
embodiments, an adaptor can be single-stranded or double-stranded
nucleic acids, or can include single-stranded or double-stranded
portions. In some embodiments, an adaptor can have any structure,
including linear, hairpin, forked, or stem-loop.
[0185] In some embodiments, an oligonucleotide adaptor can be a
blocking oligonucleotide adaptor which comprises a double-stranded
oligonucleotide adaptor (duplex) having an overhang cohesive
portion that anneals with a blocking oligonucleotide which is a
separate single-stranded oligonucleotide (PCT/US2011/054053, filed
Sep. 29, 2011).
[0186] In some embodiments, an oligonucleotide adaptor can have any
length, including fewer than 10 bases in length, or about 10-20
bases in length, or about 20-50 bases in length, or about 50-100
bases in length, or longer.
[0187] In some embodiments, an oligonucleotide adaptor can have any
combination of blunt end(s) and/or sticky end(s). In some
embodiments, at least one end of an adaptor can be compatible with
at least one end of a nucleic acid fragment. In some embodiments, a
compatible end of an adaptor can be joined to a compatible end of a
nucleic acid fragment. In some embodiments, an adaptor can have a
5' or 3' overhang end.
[0188] In some embodiments, an oligonucleotide adaptor can include
a monomeric sequence (e.g., AAA, TTT, CCC, or GGG) of any length,
or an adaptor can include a complex sequence (e.g., non- monomeric
sequence), or can include both monomeric and complex sequences.
[0189] In some embodiments, an oligonucleotide adaptor can have a
5' or 3' tail. In some embodiments, the tail can be one, two,
three, or more nucleotides in length. In some embodiments, an
adaptor can have a tail comprising A, T, C, G and/or U. In some
embodiments, an adaptor can have a monomeric tail sequence of any
length. In some embodiments, at least one end of an adaptor can
have a tail that is compatible with a tail on one end of a nucleic
acid fragment.
[0190] In some embodiments, an oligonucleotide adaptor can include
an internal nick. In some embodiments, an adaptor can have at least
one strand that lacks a terminal 5' phosphate residue. In some
embodiments, an adaptor lacking a terminal 5' phosphate residue or
lacking a terminal 3' OH can be joined to a nucleic acid fragment
to introduce a nick at the junction between the adaptor and the
nucleic acid fragment. In some embodiments, an adaptor can be
ligated to a fragmented nucleic acid. In some embodiments, ligation
of the adaptor to a fragmented nucleic acid results in the
formation of a nick in the nucleic acid strand opposite the site of
ligation. In some embodiments, the nick opposite the site of
ligation can be repaired by denaturing the adaptor (thereby
releasing the nucleotides of the adaptor adjacent to the nick to
the termini of the adaptor), and extending the nucleic acid strand
from the site of the nick to the termini of the adaptor using a
nick repair enzyme. In some embodiments, the nick repair enzyme
used to repair the nucleic acid strand can be Taq DNA polymerase,
Bst DNA polymerase, Platinum.RTM. Pfx DNA polymerase (Invitrogen),
Tfi Exo(-) DNA polymerase (Invitrogen) or Phusion.RTM. Hot Start
High-Fidelity DNA polymerase (New England Biolabs).
[0191] In some embodiments, an oligonucleotide adaptor can include
nucleotide sequences that are complementary to sequencing primers
or amplification primers. In some embodiments, an adaptor can
include a universal sequence that includes a nucleotide sequence
that is part of, or is complementary to, a universal adaptor, a P1
adaptor, P2 adaptor, A (Ion-compatible adaptor), IA (internal
adaptor), barcode sequence, amplification primer, or sequencing
primer.
[0192] In some embodiments, an oligonucleotide adaptor can include
degenerate sequences. In some embodiments, an adaptor can include
one or more inosine residues.
[0193] In some embodiments, an oligonucleotide adaptor can include
at least one scissile linkage. In some embodiments, a scissile
linkage can be susceptible to cleavage or degradation by an enzyme
or chemical compound. In some embodiments, an adaptor can include
at least one phosphorothiolate, phosphorothioate, and/or
phosphoramidate linkage.
[0194] In some embodiments, an oligonucleotide adaptor can include
identification sequences. In some embodiments, an identification
sequences can be used for sorting or tracking. In some embodiments,
an identification sequences can be a unique sequence (e.g., barcode
sequence). In some embodiments, a barcode sequence can allow
identification of a particular adaptor among a mixture of different
adaptors having different barcodes sequences. For example, a
mixture can include 2, 3, 4, 5, 6, 7-10, 10-50, 50-100, 100-200,
200-500, 500-1000, or more different adaptors having unique barcode
sequences.
[0195] In some embodiments, an oligonucleotide adaptor can include
any type of restriction enzyme recognition sequence, including type
I, type II, type IIs, type IIB, type III or type IV restriction
enzyme recognition sequences.
[0196] In some embodiments, an oligonucleotide adaptor can include
a cell regulation sequences, including a promoter (inducible or
constitutive), enhancers, transcription or translation initiation
sequence, transcription or translation termination sequence,
secretion signals, Kozak sequence, cellular protein binding
sequence, and the like.
[0197] Purification Steps:
[0198] In some embodiments, a population of nucleic acid fragments
can be subjected to any purification procedure to remove
non-desirable materials (buffers, salts, enzymes, primer-dimers, or
excess adaptors or primers). In some embodiments, a purification
procedure can be conducted between any two steps to remove buffers,
salts, enzymes, adaptors, non-reacted nucleic acid fragments, and
the like. Purification procedures include without limitation: bead
purification, column purification, gel electrophoresis, dialysis,
alcohol precipitation, and size-selective PEG precipitation.
[0199] Nucleic Acids
[0200] In some embodiments, a suitable nucleic acid sample to be
fragmented can include single-stranded and double-stranded nucleic
acids. In some embodiments, nucleic acids can include polymers of
deoxyribonucleotides, ribonucleotides, and/or analogs thereof. In
some embodiments, nucleic acids can include naturally-occurring and
synthetic forms. In some embodiments, nucleic acids include
single-stranded and double-stranded molecules. In some embodiments,
nucleic acids can include DNA, cDNA RNA or chimeric RNA/DNA.
[0201] In some embodiments, a sample of nucleic acids to be
fragmented can include single-or double-stranded DNA. In some
embodiments, nucleic acids to be fragmented can be isolated in any
form including chromosomal, genomic, organellar (e.g.,
mitochondrial, chloroplast or ribosomal), recombinant molecules,
cloned, amplified (e.g., PCR amplified), cDNA, RNA such as
precursor mRNA or mRNA, oligonucleotide, or any type of nucleic
acid library such as an amplicon library. In some embodiments,
nucleic acids to be fragmented can be isolated from any source
including from organisms such as prokaryotes, eukaryotes (e.g.,
humans, plants and animals), fungus, and viruses; cells; tissues;
normal or diseased cells or tissues or organs, 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. In some embodiments, nucleic acids to be
fragmented can be chemically synthesized to include any type of
nucleic acid analog. In some embodiments, nucleic acids to be
fragmented can be isolated from a formalin-fixed tissue, or from a
paraffin-embedded tissue, or from a formalin-fix paraffin-embedded
(FFPE) tissue.
[0202] In some embodiments, nucleic acids to be fragmented can be
about 100 bp-1000 bp, or about 1 kb -50 kb, or about 50 kb-100 kb,
or longer.
[0203] In some embodiments, nucleic acids to be fragmented can
include a GC% content of about 0-10%, or about 10-25%, or about
25-40%, or about 40-55%, or about 55-70%, or about 70-85%, or about
85-100%.
[0204] In some embodiments, nucleic acid fragmentation reaction can
be conducted with about 0.01-0.1 ng, or about 0.1-1 ng, or about
1-5 ng, or about 5-10 ng, or about 10-50 ng, or about 50-100 ng, or
about 100-500 ng, or about 500-1000 ng, or about 1-2 ug, or about
2-5 ug, or about 5-10 ug, or about 10-50 ug, or about 50-100 ug, or
about 100-500 ug, or about 500-1000 ug, or more.
[0205] Polymerases
[0206] In some embodiments, methods for generating a population of
nucleic acid fragments can include one or more different
polymerases. In some embodiments, a polymerase includes any enzyme,
or fragment or subunit of thereof, that can catalyze polymerization
of nucleotides and/or nucleotide analogs. In some embodiments, a
polymerase requires the terminal 3' OH of a nucleic acid primer to
initiate nucleotide polymerization. In some embodiments, a linker
nucleic acid provides a terminal 3'OH for the polymerase to
polymerize the nucleotides.
[0207] A polymerase comprises 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. In some
embodiments, a polymerase can be a high fidelity polymerase. 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 from the polymerase, or the linkage of
parts of two or more 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. Typically,
the polymerase comprises one or more active sites at which
nucleotide binding and/or catalysis of nucleotide polymerization
can occur. In some embodiments, a polymerase includes other
enzymatic activities, such as for example, 3' to 5' exonuclease
activity or 5' to 3' exonuclease activity. In some embodiments, a
polymerase can be isolated from a cell, or generated using
recombinant DNA technology or chemical synthesis methods. In some
embodiments, a polymerase can be expressed in prokaryote,
eukaryote, viral, or phage organisms. In some embodiments, a
polymerase can be post-translationally modified proteins or
fragments thereof.
[0208] In some embodiments, a 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.
[0209] In some embodiments, a polymerase can be a replicase,
DNA-dependent polymerase, primases, RNA-dependent polymerase
(including RNA-dependent DNA polymerases such as, for example,
reverse transcriptases), a strand-displacement polymerase, a
thermo-labile polymerase, or a thermo-stable polymerase. In some
embodiments, a 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. In some embodiments, a polymerase can be a
T3, T5, T7, or SP6 RNA polymerase.
[0210] 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.
[0211] In some embodiments, an archaeal DNA polymerase can be,
without limitation, a thermostable or thermophilic DNA polymerase
such as, for example: a 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; or
heterodimeric DNA polymerase DP1/DP2. In some embodiments, a
polymerase can be a commercially-available polymerase, such as
AmpliTaq.TM. or AmpliTaq Gold.TM. (both from Applied
Biosystems).
[0212] These and other polymerases are described by Rothwell and
Watsman (2005 Advances in Protein Chemistry 71:401-440). One
skilled in the art will know which polymerase(s) to select to
conduct a cleaving, nick translating, and/or tailing reaction.
[0213] Nicking Reaction
[0214] In some embodiments, methods for generating a population of
nucleic acid fragments can include nicking a nucleic acid with an
enzyme. In some embodiments, nucleic acid nicking enzymes include
any enzyme having endonuclease activity, with or without
exonuclease activity. In some embodiments, nucleic acid nicking
enzymes include any enzyme that can catalyze nicking one or both
strands of a double-stranded nucleic acid. In some embodiments,
nucleic acid nicking enzymes include any enzyme that can catalyze
introducing a nick at random positions in one or both strands of a
double-stranded nucleic acid. In some embodiments, nucleic acid
nicking enzymes include any enzyme that can introduce one or more
nicks at random (or nearly random) positions in either strand of a
double-stranded nucleic acid. In some embodiments, nucleic acid
nicking enzymes include any enzyme that can introduce one or more
nicks in a non-specific sequence manner at any position in either
strand of a double-stranded nucleic acid. In some embodiments,
nucleic acid nicking enzymes include any wild-type or mutant
deoxyribonucleases I (DNase I) enzyme isolated from any organism or
tissue, or isolated as a recombinant enzyme. In some embodiments, a
DNase I can be isolated from bovine. In some embodiments, a DNase I
can be isolated from pancreas.
[0215] In some embodiments, a nucleic acid nicking enzyme can be a
DNase from a family Virionaceae, such as genus Vibrio, which
includes Vibrio vulnificus. In some embodiments, a nucleic acid
nicking enzyme can be a Vvn nuclease. In some embodiments, a
nucleic acid nicking enzyme can be a nuclease from Vibrio cholera
(Focareta and Manning 1987 Gene 53(1):31-400, or an NucM polymerase
from Erwinia chrysanthemi (Moulard 1993 Mol. Microbiol.
8)4):685-695, or an Endo I nuclease from E. coli (Jekel 1995 Gene
154(1):55-59, or a Dns or DnsH nuclease from Aeromonas hydrophila
(Chang 1992 Gene 122(1):175-180, Dodd 1999 FEMS Microbiol. Lett.
173:41-46, and Wang 2007 Nucleic Acids Research 35:584-594). In
some embodiments, a nucleic acid nicking enzyme can be a DNase from
a family Enterobacteriaceae, such as a genus Serratia, which
includes Serratia marcescens (Benzonase.TM., U.S. Pat. No.
5,173,418).
[0216] In some embodiments, a nucleic acid nicking enzyme exhibits
little or no preference for nicking nucleic acids at sequences
having a high or low GC % content, including nucleic acids having
about 0-10%, or about 10-25%, or about 25-40%, or about 40-55%, or
about 55-70%, or about 70-85%, or about 85-100% GC % content.
[0217] Nick Translating
[0218] In some embodiments, methods for generating a population of
nucleic acid fragments can include a nick translation reaction. A
nick translation can include any process or treatment whereby the
position of a nick within a nucleic acid strand is effectively
moved to a new position in a nucleic acid strand. Nick translation
typically includes extension of one new strand accompanied by
digestion or erosion of the other new strand. In some embodiments,
nick translation includes polymerization of nucleotides or
nucleotide analogs onto the new 3' end as well as digestion or
erosion of nucleosides from the new 5' end. With each successive
nucleotide polymerization onto the new 3' end, the position of the
nick is effectively moved by one nucleotide position along the
nicked strand. Nick translation can optionally continue until the
nick is translated to the end of the nicked strand, or until the
translated nick comes into either complete alignment or into
sufficiently close proximity to another nick in the opposing strand
as to form a double stranded break, resulting in the generation of
two nucleic acid fragments derived from the original double
stranded nucleic acid. The double stranded break may generate two
new blunt ends or two new "sticky" ends in the resulting nucleic
acid fragments.
[0219] In some embodiments, nick translating can include two or
more enzymatic activities that act on double stranded nucleic acids
to: (1) nick a double stranded nucleic acid and (2) translate the
nick. For example, a nicking enzyme can introduce a nick on at
least one nucleic acid strand, and a polymerase can act at the nick
to remove nucleotides in a 5'.fwdarw.3' direction (exonuclease
activity) while incorporating nucleotides in a 5'.fwdarw.3'
direction (polymerization activity). Alternatively, a nicking
enzyme can introduce a nick on at least one nucleic acid strand,
and a polymerase can move in a 5'.fwdarw.3' direction to displace
one strand (strand displacing activity) while incorporating
nucleotides in a 5'.fwdarw.3' direction (polymerization
activity).
[0220] In some embodiments, the position of a nick can be moved to
a new position by reacting a nick on a double stranded nucleic acid
with an polymerase that moves in a 5'.fwdarw.3' direction to
degrade nucleotides or nucleosides (exonuclease activity) while
polymerizing nucleotides onto the free 3' end of the nick in a
5'.fwdarw.3' direction (polymerization activity).
[0221] In some embodiments, a nick on a double stranded nucleic
acid can be reacted with a polymerase that moves in a 5'.fwdarw.3'
direction to displace one strand (strand displacing activity) while
polymerizing nucleotides onto the free 3' end of the nick in a
5'.fwdarw.3' direction (polymerization activity).
[0222] In some embodiments, a nick translation reaction can be
catalyzed by one or more enzymes that couples a 5'.fwdarw.3'
nucleic acid polymerization and degradation reaction. In some
embodiments, a nick translation reaction can be catalyzed by any
nucleic acid polymerase having a 5'.fwdarw.3' nucleotide
polymerization activity and a 5'.fwdarw.3' exonuclease activity. In
some embodiments, a nick translation reaction can be catalyzed by
any nucleic acid polymerase lacking a 3'.fwdarw.5' exonuclease
activity. In some embodiments, a nick translation reaction can be
catalyzed by any DNA polymerase. In some embodiments, a nick
translation reaction can be catalyzed by any Family A DNA
polymerase (also known as pol I family). In some embodiments, a
nick translation reaction can be catalyzed by Klenow fragment.
[0223] In some embodiments, a nick translation reaction can be
catalyzed by E. coli DNA Pol I. In some embodiments, a nick
translation reaction can be catalyzed by one or more thermostable
enzymes having 5'.fwdarw.3' nucleotide polymerization activity and
a 5'.fwdarw.3' exonuclease activity. In some embodiments, a
thermostable enzyme includes Taq polymerase (from Thermus
aquaticus), Tfi polymerase (from Thermus filiformis), Pfu
polymerase (from Pyrococcus furiosus), Tth (from Thermus
thermophilus), Pow polymerase (from Pyrococcus woesei), Tli
polymerase (from Thermococcus litoralis), Pol I and II polymerases
(from Pyrococcus abyssi), and Pab (from Pyrococcus abyssi).
[0224] In some embodiments, a nick translation reaction can be
catalyzed by one or more enzymes that couples a 5' to 3' DNA
polymerization and strand displacement reaction. In some
embodiments, a strand displacing polymerase includes Taq
polymerase, Tfi polymerase, Bst polymerase (from Bacillus
stearothermophilus), Tli polymerase, 9.degree. N polymerase, and
phi29 polymerase.
[0225] In some embodiments, a nick translation reaction can be
catalyzed by a combination of a helicase and a DNA polymerase.
[0226] In some embodiments, a nick translation reaction includes a
nick translation enzyme and at least one type of nucleotide. In
some embodiments, the nick translation enzyme catalyzes
polymerization of one or more nucleotides onto the new 3' end at
the nick site. In some embodiments, the nucleotides that are
polymerized onto the new 3' end can be unlabeled to or labeled with
a detectable moiety, or a combination of unlabeled and labeled
nucleotides. In some embodiments, a nick translation reaction can
generate unlabeled or labeled ends.
[0227] Tailing
[0228] In some embodiments, methods for generating a population of
nucleic acid fragments can include a non-template-dependent
terminal transferase reaction (e.g., tailing reaction). In some
embodiments, a non-template-dependent terminal transferase reaction
can be catalyzed by a Taq polymerase, Tfi DNA polymerase, 3'
exonuclease minus- large (Klenow) fragment, or 3' exonuclease
minus- T4 polymerase.
[0229] Nick Repair
[0230] In some embodiments, methods for generating a population of
nucleic acid fragments can include a nick repair enzyme (e.g., nick
repairing or nick repair reaction). In some embodiments, a nick
repair reaction can be catalyzed by a nick repair polymerase such
as Taq DNA polymerase, Bst DNA polymerase, Platinum.RTM. Pfx DNA
polymerase (Invitrogen), Tfi Exo(-) DNA polymerase (Invitrogen) or
Phusion.RTM. Hot Start High-Fidelity DNA polymerase (New England
Biolabs). In some embodiments, the nick repair enzyme can be used
to extend the nucleic acid strand from the site of the nick to the
original termini of the adaptor sequence.
[0231] Nucleotides
[0232] In some embodiments, methods for generating a population of
nucleic acid fragments can be conducted with one or more types of
nucleotides. A nucleotide 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 O
can be a substituted phosphate group. In the phosphorus chain,
phosphorus atoms with an intervening atom other than O can be a
substituted phosphate group. Some examples of nucleotide analogs
are described in Xu, U.S. Pat. No. 7,405,281.
[0233] 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, modified 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.
[0234] In some embodiments, the nucleotide comprises a label 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 any portion of a nucleotide including a base, sugar
or any intervening phosphate group or a terminal phosphate group,
i.e., the phosphate group most distal from the sugar.
[0235] Labels
[0236] In some embodiments, a nucleotide (or analog thereof) can be
attached to a label. In some embodiments, a label comprises a
detectable moiety. In some embodiments, a label can generate, or
cause to generate, a detectable signal. A detectable signal can be
generated from a chemical or physical change (e.g., heat, light,
electrical, pH, salt concentration, enzymatic activity, or
proximity events). For example, a proximity event can include two
reporter moieties approaching each other, or associating with each
other, or binding each other. A detectable signal can be detected
optically, electrically, chemically, enzymatically, thermally, or
via mass spectroscopy or Raman spectroscopy. A label can include
compounds that are luminescent, photoluminescent,
electroluminescent, bioluminescent, chemiluminescent, fluorescent,
phosphorescent or electrochemical. A label can include compounds
that are fluorophores, chromophores, radioisotopes, haptens,
affinity tags, atoms or enzymes. In some embodiments, the label
comprises a moiety not typically present in naturally occurring
nucleotides. For example, the label can include fluorescent,
luminescent or radioactive moieties.
[0237] Nucleic Acid Binding Proteins
[0238] In some embodiments, the methods for generating a population
of nucleic acid fragments can include binding a nucleic acid with a
protein that binds nucleic acids (e.g., nucleic acid binding
protein), or can lack this step. In some embodiments, the methods
can include binding a nucleic acid with a nucleic acid binding
protein at any step, which can be conducted once or can be
repeated.
[0239] In some embodiments, the methods can include binding a
nucleic acid with a nucleic acid binding protein before, during
and/or after a nicking reaction. In some embodiments, the methods
can include binding a nucleic acid with a nucleic acid binding
protein before, during and/or after a nick translation reaction.
For example, a nucleic acid can be subjected to a nicking reaction
and/or a nick translation reaction in the presence of a nucleic
acid binding protein. In some embodiments, a nucleic acid binding
protein can be added after conducting nicking reaction and/or a
nick translation reaction.
[0240] In some embodiments, a nucleic acid binding protein can be a
protein or at least a portion thereof. In some embodiments, a
nucleic acid binding protein can be a multimeric protein complex
(e.g., dimers, trimers, tetramers or higher order multimers), that
binds a nucleic acid. In some embodiments, a multimeric protein
complex can be hetero-polymeric or homo-polymeric. In some
embodiments, a nucleic acid binding protein can bind DNA, RNA, or
any analog or derivative thereof. In some embodiments, a nucleic
acid binding protein can bind single-stranded nucleic acids with
higher affinity compared to binding double-stranded or
triple-stranded nucleic acids. In some embodiments, a nucleic acid
binding protein can be a single-stranded nucleic acid binding
protein (Chase and Williams 1986 Ann. Rev. Biochem. 55:103-136). In
some embodiments, a nucleic acid binding protein can bind a double
strand and a single strand (e.g., recA can bind three nucleic acid
strands). In some embodiments, a nucleic acid binding protein can
bind a folded or non-folded nucleic acid. In some embodiments, a
nucleic acid binding protein can exhibit little or no sequence
specificity for binding nucleic acids. In some embodiments, one or
more nucleic acid binding proteins can bind a nucleic acid strand.
In some embodiments, multiple nucleic acid binding proteins can
cooperatively bind a nucleic acid strand (Lohman and Ferrari 1994
Ann. Rev. Biochem. 63:527-570) or can bind non-cooperatively. In
some embodiments, a nucleic acid binding protein can be wild-type,
mutant or truncated. In some embodiments, at nucleic acid binding
protein can be a naturally-occurring or can be a recombinant
protein prepared using recombinant DNA methods (Haseltine 2002 Mol.
Microbiol. 43:1505-1515).
[0241] In some embodiments, a nucleic acid binding protein can be a
mesophilic or thermostable protein. A thermostable nucleic acid
binding protein can be resistant to inactivation by heat, such as a
temperature range of about 50-95.degree. C. for about 15 seconds to
10 minutes or longer. For example, a thermostable nucleic acid
binding protein can retain about 50-95% activity at a temperature
range of about 50-95.degree. C.
[0242] In some embodiments, a nucleic acid binding protein can be
from any type of organism, including prokaryotic, eukaryotic, virus
or phage. A nucleic acid binding protein can originate from any
type of cell, tissue or cell culture. A nucleic acid binding
protein from a eukaryotic organism can originate from any
organelle, including nuclear, mitochondria, or chloroplast, or can
originate from cytoplasm. A nucleic acid binding protein from a
eukaryotic organism can originate from any organ, including thymus.
A nucleic acid binding protein from a prokaryotic organism can be
episomally-encoded.
[0243] In some embodiments, a nucleic acid binding protein can
mediate in vivo and/or in vitro reactions, including: DNA
replication, repair and/or recombination (Kowalczykowski 1994
Microbiol Rev. 58:401-465; Lohman and Ferrari 1994 Ann. Rev.
Biochem. 63:527-570; Wold 1997 Annu. Rev. Biochem. 66:61-92; Chedin
1998 Trends Biochem. Sci. 23:273-277; Kelly 1998 Proc. Natl. Acad.
Sci. USA 95:14634-14639; Iftody 1999 Crit. Rev. Biochem. Mol. Biol
34:141-180); helix destabilization; reduction of DNA secondary
structures; renaturation of complementary sequences; protection of
nucleic acids from nucleases; and/or repair via homologous
recombination (e.g., RecA, Zhumabayeva 1990 Biotechniques
27:834-845; LexA, Radman, 1974 "Phenomenology of an inducible
mutagenic DNA repair pathway in Escherichia coli: SOS repair
hypothesis" in Sherman(ed) in: Molecular and Environmental Aspects
of Mutagenesis, Springfield, Ill., Charles C. Thomas publisher, pp.
128-142; and Bridges 2005, in: DNA Repair, (Amst) vol 4(6), pp.
725-739).
[0244] In some embodiments, a nucleic acid binding protein can
include one or more OB folds or OB fold-like structures
(oligonucleotide/oligosaccharide binding fold) having a
five-stranded antiparallel beta-barrel terminating in an
alpha-helix (Murzin 1993 EMBO J. 12:861-867; Philipova 1996 Genes
Dev. 10:2222-2233).
[0245] In some embodiments, a nucleic acid binding protein can be a
phage T4 gp 32 protein (Williams 1981 J. Biol. Chem. 256:1754-1762;
Topal and Sinha 1983 J. Biol. Chem. 258:12274-12279; GenBank
accession BAG54790; FIG. 4), or a T7 gp 2.5 protein or phi29
protein p5 protein.
[0246] In some embodiments, a nucleic acid binding protein can be
from Sulfolobus solfataricus (Sso SSB) (Haseltine and
Kowalczykowski 2002 Mol. Microbiol. 43:1505-1515; FIG. 5).
[0247] In some embodiments, a nucleic acid binding protein can be
from E. coli (Skyberg 2006 Infect. Immun. 74:6287-6292; Sigal 1972
Proc. Natl. Acad. Sci. USA 69:3537-3541; Weiner 1975 J. Biol. Chem.
250:1972-1980; GenBank accession ABC42252; FIG. 6).
[0248] In some embodiments, a nucleic acid binding protein can be
from Methanococcus jannaschii (Mja SSB) (Kelly 1998 PNAS
95:14634-14639; GenBank accession NP.sub.--248153 ; FIG. 7).
[0249] In some embodiments, a nucleic acid binding protein can be
a: phage T7 SSB; T4 gene 44/62 protein; coliphage N4 SSB;
adenovirus DNA binding protein (Ad DBP or Ad SSB); calf thymus
unwinding protein (UP1); episomal encoded SSB (Kolodkin 1983 Proc.
Natl. Acad. Sci. USA 80:4422-4426); mitochondrial (rim-1); yeast
(e.g., rpa-1, SSB I, SSB II, or SSB III); HeLa A.sub.1 protein;
Bacillus subtilis SSB; Saccharomyces cerevisiae RPA70
single-stranded DNA-binding region 1; eukaryotic replication
protein A (RPA) (Smith 1997 J. Bacteriol. 179:7135-7155; Wold 1997
Annu. Rev. Biochem. 66:61-92); or from Homo sapiens.
[0250] In some embodiments, a nucleic acid binding protein can be a
RecA or RecA-like protein including RecA (bacteria), Rad51
(eukaryotes), and RadA (archaeal) (Kowalczykowski 1994 Annu. Rev.
Biochem. 63:991-1043; Kuzminov 1999 Microbiol. Mol. Biol. Rev.
63:751-813; Bianco 2005 in "RecA protein" John Wiley and Sons, Ltd.
Chichester, UK).
[0251] In some embodiments, nucleic acid binding proteins can
originate from thermophilic organisms, including Methanococcus
(e.g., Methanococcus jannachii), Methanobacterium (e.g.,
Methanobacterium thermoautrophicum), Archaeoglobus (e.g.,
Archaeoglobus fulgidus), Sulfolobus (e.g., Sulfolobus
sulfataricus), Aeropyrum (e.g., Aeropyrum pernix) (see e.g.,
Chedin, 1998 Trends Biochem. Sci. 23:273-277; Haseltine 2002 Mol.
Microbiol. 43:1505-1515; Kelly 1998 Proc. Natl. Acad. Sci. USA
95:14634-14639; Klenk 1997 Nature 390:364-370; Smith 1997 J.
Bacteriol. 179:7135-55; Wadsworth and White 2001 Nucl. Acids Res.
29:914-920).
[0252] For example, a thermostable nucleic acid binding protein can
be a: Thermus thermophilus SSB (e.g., GenBank AJ564626); Thermus
aquaticus SSB (e.g., GenBank AF276705); Methanococcus
thermoautotrophicum SSB; Methanococcus jannaschii RPA protein;
Aeropyrum pernix (ApeSSB); Archaeoglobus fulgidus SSB; Pyrococcus
abyssii SSB; or Pyrococcus horikoshii SSB.
[0253] In some embodiments, a single stranded binding protein can
be any SSB found in Table 1 at pages 9-16 of published patent
application No. U.S. 2007/0178491 (Park and Lee).
[0254] Suitable Conditions
[0255] In some embodiments, methods for generating a population of
nucleic acid fragments can be conducted under conditions that are
suitable for introducing one or more nicks on either strand of a
double-stranded nucleic acid and/or suitable for moving the
positions of the nicks to a new position along the double-stranded
nucleic acid and/or suitable for binding nucleic acids to one or
more nucleic acid binding proteins and/or suitable conditions for
joining ends of nucleic acid fragments to oligonucleotide
adaptors.
[0256] In some embodiments, methods for generating a population of
nucleic acid fragments can be conducted under conditions that are
suitable for nicking the first nucleic acid strand and/or suitable
for nicking the second nucleic acid strand and/or suitable for
moving the position of first nick and/or suitable for moving the
position of the second nick and/or suitable for joining at least
one end of a nucleic acid fragment to an oligonucleotide
adaptor.
[0257] In some embodiments, suitable conditions include well known
parameters, such as: time, temperature, pH, buffers, reagents,
cations, salts, co-factors, nucleotides, nucleic acids, and
enzymes. In some embodiments, a reagent or buffer can include a
source of ions, such as KCl, K-acetate, NH.sub.4-acetate,
K-glutamate, NH.sub.4Cl, or ammonium sulfate. In some embodiments,
a reagent or buffer can include a source of divalent ions, such as
Mg.sup.2+ or Mn.sup.2+, MgCl.sub.2, MnCl.sub.2, or Mg-acetate. In
some embodiments, a reagent or buffer can include magnesium,
manganese and/or calcium. In some embodiments, a buffer can include
Tris, Tricine, HEPES, MOPS, ACES, MES, or inorganic buffers such as
phosphate or acetate-based buffers which can provide a pH range of
about 4-12. In some embodiments, a buffer can include chelating
agents such as EDTA or EGTA. In some embodiments, a buffer can
include dithiothreitol (DTT), glycerol, spermidine, and/or BSA
(bovine serum albumin). In some embodiments, a buffer can include
ATP.
[0258] In some embodiments, suitable conditions include conducting
a nick translation reaction with an enzyme and one or more types of
nucleotides. In some embodiments, suitable conditions include
conducting a non-template-dependent terminal transferase reaction
with an enzyme and one or more types of nucleotides. In some
embodiments, a suitable condition includes joining at least one end
of a nucleic acid fragment to an oligonucleotide adaptor with a
ligase enzyme (e.g., T4 DNA ligase, Taq DNA ligase or a derivative
thereof).
[0259] In some embodiments, suitable conditions include cyclical
temperature changes, or isothermal temperature conditions, or a
combination of both. In some embodiments, a reaction can be
conducted at a temperature range of about 0-10.degree. C., or about
10-20.degree. C., or about 20-30.degree. C., or about 30-40.degree.
C., or about 40-50.degree. C., or about 50-60.degree. C., or about
60-70.degree. C., or about 70-80.degree. C., or about 80-90.degree.
C., or about 90-100.degree. C., or high temperatures.
[0260] In some embodiments, suitable conditions include conducting
a reaction for a time, such as about 10-30 seconds, or about 30-60
seconds, or about 1-3 minutes, or about 3-5 minutes, or about 5-6
minutes, or about 6-7 minutes, or about 7-8 minutes, or about 8-9
minutes, or about 9-10 minutes, or about 10-11 minutes, or about
11-12 minutes, or about 12-13 minutes, or about 13-14 minutes, or
about 14-15 minutes, or about 15-20 minutes, or about 20-30
minutes, or about 30-45 minutes, or about 45-60 minutes, or about
1-3 hours, or about 3-6 hours, or about 6-10 hours, or longer.
[0261] In some embodiments, suitable conditions include conducting
a reaction in a volume of about 1-10 uL, or about 10-25 uL, or
about 25-50 uL, or about 50-75 uL, or about 75-100 uL, or about
100-125 uL, or about 125-150 uL, or about 150-200 uL, or more.
[0262] In some embodiments, suitable conditions include conducting
a reaction in a tube or well. In some embodiments, the well can be
a part of a 96-well plate.
[0263] In some embodiments, the number of nicks introduced on
either strand of a double-stranded nucleic acid and/or a nick
translation reaction can be adjusted by varying any parameters,
including varying: the time; temperature; pH; amount of template;
enzyme concentration; nucleotide concentration; type of salts,
cations and/or ions; amount of salts, cations, and/or ions;
reaction volume; or any combination thereof.
[0264] Sequencing Methods
[0265] In some embodiments, one or more nucleic acid fragments
produced according to the present teachings can be sequenced using
methods that detect one or more byproducts of nucleotide
incorporation. The detection of polymerase extension by detecting
physicochemical byproducts of the extension reaction, can include
pyrophosphate, hydrogen ion, charge transfer, heat, and the like,
as disclosed, for example, in Pourmand et al, Proc. Natl. Acad.
Sci., 103: 6466-6470 (2006); Purushothaman et al., IEEE ISCAS,
IV-169-172; Rothberg et al, U.S. Patent Publication No.
2009/0026082; Anderson et al, Sensors and Actuators B Chem., 129:
79-86 (2008); Sakata et al., Angew. Chem. 118:2283-2286 (2006);
Esfandyapour et al., U.S. Patent Publication No. 2008/01666727; and
Sakurai et al., Anal. Chem. 64: 1996-1997 (1992).
[0266] Reactions involving the generation and detection of ions are
widely performed. The use of direct ion detection methods to
monitor the progress of such reactions can simplify many current
biological assays. For example, template-dependent nucleic acid
synthesis by a polymerase can be monitored by detecting hydrogen
ions that are generated as natural byproducts of nucleotide
incorporations catalyzed by the polymerase. Ion-sensitive
sequencing (also referred to as "pH-based" or "ion-based" nucleic
acid sequencing) exploits the direct detection of ionic byproducts,
such as hydrogen ions, that are produced as a byproduct of
nucleotide incorporation. In one exemplary system for ion-based
sequencing, the nucleic acid to be sequenced can be captured in a
microwell, and nucleotides can be floated across the well, one at a
time, under nucleotide incorporation conditions. The polymerase
incorporates the appropriate nucleotide into the growing strand,
and the hydrogen ion that is released can change the pH in the
solution, which can be detected by an ion sensor. This technique
does not require labeling of the nucleotides or expensive optical
components, and allows for far more rapid completion of sequencing
runs. Examples of such ion-based nucleic acid sequencing methods
and platforms include the Ion Torrent PGM.TM. or Proton.TM.
sequencer (Ion Torrent.TM. Systems, Life Technologies
Corporation).
[0267] In some embodiments, one or more nucleic acid fragments
produced using the methods, systems and kits of the present
teachings can be used as a substrate for a biological or chemical
reaction that is detected and/or monitored by a sensor including a
field-effect transistor (FET). In various embodiments the FET is a
chemFET or an ISFET. A "chemFET" or chemical field-effect
transistor, is a type of field effect transistor that acts as a
chemical sensor. It is the structural analog of a MOSFET
transistor, where the charge on the gate electrode is applied by a
chemical process. An "ISFET" or ion-sensitive field-effect
transistor, is used for measuring ion concentrations in solution;
when the ion concentration (such as H+) changes, the current
through the transistor will change accordingly. A detailed theory
of operation of an ISFET is given in "Thirty years of ISFETOLOGY:
what happened in the past 30 years and what may happen in the next
30 years," P. Bergveld, Sens. Actuators, 88 (2003), pp. 1-20.
[0268] In some embodiments, the FET may be a FET array. As used
herein, an "array" is a planar arrangement of elements such as
sensors or wells. The array may be one or two dimensional. A one
dimensional array can be an array having one column (or row) of
elements in the first dimension and a plurality of columns (or
rows) in the second dimension. The number of columns (or rows) in
the first and second dimensions may or may not be the same. The FET
or array can comprise 102, 103, 104, 105, 106, 107 or more
FETs.
[0269] In some embodiments, one or more microfluidic structures can
be fabricated above the FET sensor array to provide for containment
and/or confinement of a biological or chemical reaction. For
example, in one implementation, the microfluidic structure(s) can
be configured as one or more wells (or microwells, or reaction
chambers, or reaction wells, as the terms are used interchangeably
herein) disposed above one or more sensors of the array, such that
the one or more sensors over which a given well is disposed detect
and measure analyte presence, level, and/or concentration in the
given well. In some embodiments, there can be a 1:1 correspondence
of FET sensors and reaction wells.
[0270] Microwells or reaction chambers are typically hollows or
wells having well-defined shapes and volumes which can be
manufactured into a substrate and can be fabricated using
conventional microfabrication techniques, e.g. as disclosed in the
following references: Doering and Nishi, Editors, Handbook of
Semiconductor Manufacturing Technology, Second Edition (CRC Press,
2007); Saliterman, Fundamentals of BioMEMS and Medical Microdevices
(SPIE Publications, 2006); Elwenspoek et al, Silicon Micromachining
(Cambridge University Press, 2004); and the like. Examples of
configurations (e.g. spacing, shape and volumes) of microwells or
reaction chambers are disclosed in Rothberg et al, U.S. patent
publication 2009/0127589; Rothberg et al, U.K. patent application
GB24611127.
[0271] In some embodiments, the biological or chemical reaction can
be performed in a solution or a reaction chamber that is in contact
with or capacitively coupled to a FET such as a chemFET or an
ISFET. The FET (or chemFET or ISFET) and/or reaction chamber can be
an array of FETs or reaction chambers, respectively.
[0272] In some embodiments, a biological or chemical reaction can
be carried out in a two-dimensional array of reaction chambers,
wherein each reaction chamber can be coupled to a FET, and each
reaction chamber is no greater than 10 .mu.m.sup.3 (i.e., 1 pL) in
volume. In some embodiments each reaction chamber is no greater
than 0.34 pL, 0.096 pL or even 0.012 pL in volume. A reaction
chamber can optionally be 22, 32, 42, 52, 62, 72, 82, 92, or 102
square microns in cross-sectional area at the top. Preferably, the
array has at least 102, 103, 104, 105, 106, 107,108, 109, or more
reaction chambers. In some embodiments, the reaction chambers can
be capacitively coupled to the FETs.
[0273] FET arrays as used in various embodiments according to the
disclosure can be fabricated according to conventional CMOS
fabrications techniques, as well as modified CMOS fabrication
techniques and other semiconductor fabrication techniques beyond
those conventionally employed in CMOS fabrication. Additionally,
various lithography techniques can be employed as part of an array
fabrication process.
[0274] Exemplary FET arrays suitable for use in the disclosed
methods, as well as microwells and attendant fluidics, and methods
for manufacturing them, are disclosed, for example, in U.S. Patent
Publication No. 20100301398; U.S. Patent Publication No.
20100300895; U.S. Patent Publication No. 20100300559; U.S. Patent
Publication No. 20100197507, U.S. Patent Publication No.
20100137143; U.S. Patent Publication No. 20090127589; and U.S.
Patent Publication No. 20090026082, which are incorporated by
reference in their entireties.
[0275] In one aspect, the disclosed methods, compositions, systems,
apparatuses and kits can be used for carrying out label-free
nucleic acid sequencing, and in particular, ion-based nucleic acid
sequencing. The concept of label-free detection of nucleotide
incorporation has been described in the literature, including the
following references that are incorporated by reference: Rothberg
et al, U.S. patent publication 2009/0026082; Anderson et al,
Sensors and Actuators B Chem., 129: 79-86 (2008); and Pourmand et
al, Proc. Natl. Acad. Sci., 103: 6466-6470 (2006). Briefly, in
nucleic acid sequencing applications, nucleotide incorporations are
determined by measuring natural byproducts of polymerase-catalyzed
extension reactions, including hydrogen ions, polyphosphates, PPi,
and Pi (e.g., in the presence of pyrophosphatase). Examples of such
ion-based nucleic acid sequencing methods and platforms include the
Ion Torrent PGM.TM. or Proton.TM. sequencer (Ion Torrent.TM.
Systems, Life Technologies Corporation).
[0276] In some embodiments, the disclosure relates generally to
methods for sequencing a nucleic acid using the nucleic acid
fragment library produced by the teachings provided herein. In some
embodiments, the nucleic acid fragment library can be used to
generate a template library and the template library can be used to
obtain sequence information. In one exemplary embodiment, the
disclosure relates generally to a method for obtaining sequence
information from a nucleic acid template, comprising:
[0277] (a) fragmenting a nucleic acid molecule into two or more
fragments;
[0278] (b) performing template-dependent nucleic acid synthesis
using at least one of the fragments produced during step (a) as a
template.
[0279] In some embodiments, the fragmenting can include: (a)
introducing one or more nicks on either strand of a double-stranded
nucleic acid; and (b) moving the position of at least two of the
nicks into alignment along the double-stranded nucleic acid.
Alignment of nicks can result in double-stranded breaks or
fragmentation points.
[0280] In some embodiments, the introducing can include introducing
a nick on either strand of the double-stranded nucleic acid using
an endonuclease. In some embodiments, the moving can include moving
the positions of a nick to a new position along the double-stranded
nucleic acid using one or more nick translating enzymes. In some
embodiments, the fragmenting can further include enzymatically
adding a 3' tail to a nick at a new position. In some embodiments,
the fragmenting can further include joining an oligonucleotide
adaptor to the fragmented nucleic acid, denaturing the adaptor and
nick repairing the fragmented nucleic acid strand.
[0281] In some embodiments, the template-dependent synthesis
includes incorporating one or more nucleotides in a
template-dependent fashion into a newly synthesized nucleic acid
strand.
[0282] Optionally, the methods can further include producing one or
more ionic byproducts of such nucleotide incorporation.
[0283] In some embodiments, the methods can further include
detecting the incorporation of the one or more nucleotides into the
sequencing primer. Optionally, the detecting can include detecting
the release of hydrogen ions.
[0284] In another embodiment, the disclosure relates generally to a
method for sequencing a nucleic acid, comprising: (a) producing a
plurality of nucleic acid fragments by fragmenting a nucleic acid
molecule according to the methods disclosed herein; (b) disposing a
plurality of nucleic acid fragments into a plurality of reaction
chambers, wherein one or more of the reaction chambers are in
contact with a field effect transistor (FET). Optionally, the
method further includes contacting at least one of the nucleic acid
fragments disposed into one of the reaction chambers with a
polymerase, thereby synthesizing a new nucleic acid strand by
sequentially incorporating one or more nucleotides into a nucleic
acid molecule. Optionally, the method further includes generating
one or more hydrogen ions as a byproduct of such nucleotide
incorporation. Optionally, the method further includes detecting
the incorporation of the one or more nucleotides by detecting the
generation of the one or more hydrogen ions using the FET.
[0285] In some embodiments, the detecting includes detecting a
change in voltage and/or current at the at least one FET within the
array in response to the generation of the one or more hydrogen
ions.
[0286] In some embodiments, the FET can be selected from the group
consisting of: ion-sensitive FET (isFET) and chemically-sensitive
FET (chemFET).
[0287] One exemplary system involving sequencing via detection of
ionic byproducts of nucleotide incorporation is the Ion Torrent
PGM.TM. or Proton.TM. sequencer (Life Technologies), which is an
ion-based sequencing system that sequences nucleic acid templates
by detecting hydrogen ions produced as a byproduct of nucleotide
incorporation. Typically, hydrogen ions are released as byproducts
of nucleotide incorporations occurring during template-dependent
nucleic acid synthesis by a polymerase. The Ion Torrent PGM.TM. or
Proton.TM. sequencer detects the nucleotide incorporations by
detecting the hydrogen ion byproducts of the nucleotide
incorporations. The Ion Torrent PGM.TM. or Proton.TM. sequencer can
include a plurality of nucleic acid templates to be sequenced, each
template disposed within a respective sequencing reaction well in
an array. The wells of the array can each be coupled to at least
one ion sensor that can detect the release of H.sup.+ ions or
changes in solution pH produced as a byproduct of nucleotide
incorporation. The ion sensor comprises a field effect transistor
(FET) coupled to an ion-sensitive detection layer that can sense
the presence of H.sup.+ ions or changes in solution pH. The ion
sensor can provide output signals indicative of nucleotide
incorporation which can be represented as voltage changes whose
magnitude correlates with the H.sup.+ ion concentration in a
respective well or reaction chamber. Different nucleotide types can
be flowed serially into the reaction chamber, and can be
incorporated by the polymerase into an extending primer (or
polymerization site) in an order determined by the sequence of the
template. Each nucleotide incorporation can be accompanied by the
release of H.sup.+ ions in the reaction well, along with a
concomitant change in the localized pH. The release of H.sup.+ ions
can be registered by the FET of the sensor, which produces signals
indicating the occurrence of the nucleotide incorporation.
Nucleotides that are not incorporated during a particular
nucleotide flow may not produce signals. The amplitude of the
signals from the FET can also be correlated with the number of
nucleotides of a particular type incorporated into the extending
nucleic acid molecule thereby permitting homopolymer regions to be
resolved. Thus, during a run of the sequencer multiple nucleotide
flows into the reaction chamber along with incorporation monitoring
across a multiplicity of wells or reaction chambers can permit the
instrument to resolve the sequence of many nucleic acid templates
simultaneously. Further details regarding the compositions, design
and operation of the Ion Torrent PGM.TM. or Proton.TM. sequencer
can be found, for example, in U.S. patent application Ser. No.
12/002781, now published as U.S. Patent Publication No.
2009/0026082; U.S. patent application Ser. No. 12/474897, now
published as U.S. Patent Publication No. 2010/0137143; and U.S.
patent application Ser. No. 12/492844, now published as U.S. Patent
Publication No. 2010/0282617, all of which applications are
incorporated by reference herein in their entireties.
[0288] In some embodiments, the disclosure relates generally to use
of nucleic acid fragments produced using any of the methods,
systems and kits of the present disclosure in methods of ion-based
sequencing. Use of such nucleic acid fragments in ion-based
sequencing reactions can be advantageous because the fragmenting
methods of the disclosure permit isolation of fragments of a
desired size that can be selected to match the read length capacity
of the ion-based sequencing system.
[0289] In a typical embodiment of ion-based nucleic acid
sequencing, nucleotide incorporations can be detected by detecting
the presence and/or concentration of hydrogen ions generated by
polymerase-catalyzed extension reactions. In one embodiment,
templates each having a primer and polymerase operably bound can be
loaded into reaction chambers (such as the microwells disclosed in
Rothberg et al, cited herein), after which repeated cycles of
nucleotide addition and washing can be carried out. In some
embodiments, such templates can be attached as clonal populations
to a solid support, such as particles, bead, or the like, and said
clonal populations are loaded into reaction chambers. As used
herein, "operably bound" means that a primer is annealed to a
template so that the primer's 3' end may be extended by a
polymerase and that a polymerase is bound to such primer-template
duplex, or in close proximity thereof so that binding and/or
extension takes place whenever nucleotides are added.
[0290] In each addition step of the cycle, the polymerase can
extend the primer by incorporating added nucleotide only if the
next base in the template is the complement of the added
nucleotide. If there is one complementary base, there is one
incorporation, if two, there are two incorporations, if three,
there are three incorporations, and so on. With each such
incorporation there is a hydrogen ion released, and collectively a
population of templates releasing hydrogen ions changes the local
pH of the reaction chamber. The production of hydrogen ions is
monotonically related to the number of contiguous complementary
bases in the template (as well as the total number of template
molecules with primer and polymerase that participate in an
extension reaction). Thus, when there are a number of contiguous
identical complementary bases in the template (i.e. a homopolymer
region), the number of hydrogen ions generated, and therefore the
magnitude of the local pH change, can be proportional to the number
of contiguous identical complementary bases. If the next base in
the template is not complementary to the added nucleotide, then no
incorporation occurs and no hydrogen ion is released. In some
embodiments, after each step of adding a nucleotide, an additional
step can be performed, in which an unbuffered wash solution at a
predetermined pH is used to remove the nucleotide of the previous
step in order to prevent misincorporations in later cycles. In some
embodiments, the after each step of adding a nucleotide, an
additional step can be performed wherein the reaction chambers are
treated with a nucleotide-destroying agent, such as apyrase, to
eliminate any residual nucleotides remaining in the chamber, which
may result in spurious extensions in subsequent cycles.
[0291] In one exemplary embodiment, different kinds of nucleotides
are added sequentially to the reaction chambers, so that each
reaction can be exposed to the different nucleotides one at a time.
For example, nucleotides can be added in the following sequence:
dATP, dCTP, dGTP, dTTP, dATP, dCTP, dGTP, dTTP, and so on; with
each exposure followed by a wash step. The cycles may be repeated
for 50 times, 100 times, 200 times, 300 times, 400 times, 500
times, 750 times, or more, depending on the length of sequence
information desired.
[0292] In some embodiments, sequencing can be performed according
to the user protocols supplied with the PGM.TM. or Proton.TM.
sequencer. Example 3 provides one exemplary protocol for ion-based
sequencing using the Ion Torrent PGM.TM. sequencer (Ion Torrent.TM.
Systems, Life Technologies, CA).
[0293] Systems
[0294] In some embodiments, the present teachings provide systems
for generating a population of nucleic acid fragments, comprising:
nucleic acids, one or more nicking enzymes, one or more nick
translation enzymes and nucleotides. In some embodiments, systems
for preparing fragmented nucleic acids can further comprise at
least one nucleic acid binding protein (e.g., a single-stranded
binding protein). In some embodiments, systems for preparing
fragmented nucleic acids further comprise one or more tailing
enzymes. In some embodiments, systems for preparing fragmented
nucleic acids can further comprise at least one oligonucleotide
adaptor. In some embodiments, systems for preparing fragmented
nucleic acids further comprise any combination of: buffers;
cations; size-selection reagents; one or more end-repairing
enzyme(s); one or more repairing enzyme(s); one or more nick repair
enzymes, one or more types of adaptor(s); one or more ligation
enzyme(s); reagents for nucleic acid purification; reagents for
nucleic acid amplification; endonuclease(s); polymerase(s);
kinase(s); phosphatase(s); and/or nuclease(s).
[0295] Kits
[0296] In some embodiments, the present teachings provide kits for
generating a population of nucleic acid fragments. In some
embodiments, kits include any reagent that can be used to conduct
nucleic acid fragmentation method. In some embodiments, kits
include any combination of: buffers; cations; one or more nucleic
acid nicking enzyme(s); one or more nick translation enzyme(s); one
or more nucleotides; one or more nucleic acid tailing enzyme(s);
size-selection reagents; one or more end-repairing enzyme(s); one
or more repairing enzyme(s); one or more nick repair enzymes, one
or more types of adaptor(s); one or more ligation enzyme(s);
reagents for nucleic acid purification; and/or reagents for nucleic
acid amplification. In some embodiments, kits include any
combination of: endonuclease(s); polymerase(s); ligase(s);
kinase(s); phosphatase(s); and/or nuclease(s).
EXAMPLES
[0297] Embodiments of the present teachings can be further
understood in light of the following examples, which should not be
construed as limiting the scope of the present teachings in any
way.
Example 1
A. Enzymatic Nucleic Acid Fragmentation
[0298] Genomic DNA (0.5 ug) from DH10B, Rhodo or Vibrio was mixed
with 5 uL of 10.times. Buffer with dNTP, 10 uL of enzyme mix from
Nick Translation System (Invitrogen, catalog 18160-010), 40 mU
DNase I, and water to make a total reaction volume of 50 uL.
[0299] The mixture was incubated at 37.degree. C. for 15 minutes,
and the reaction was stopped with 5 uL of Stop Buffer (0.5 M EDTA,
pH 8). The fragmented DNA was purified with a SOLiD.TM. Library
Micro Column Purification kit, using B2-S buffer and eluting with
20 uL of E1 buffer.
B. Library Preparation
[0300] The fragmented DNA from step (A) above (20 uL) was ligated
to barcoded adaptors. The fragmented DNA was mixed in 10 uL of
5.times. Reaction buffer, barcoded adaptor P1 (50 uM stock),
barcoded adaptor P2 (50 uM stock), 5 uL of 5U/uL T4 DNA ligase, and
water to make a total reaction volume of 50 uL.
[0301] The mixture was incubated at room temperature for 30
minutes. The DNA was purified with a SOLiD.TM. Library Micro Column
Purification kit, using B2-S buffer and eluting with 20 uL of El
buffer. The DNA was reacted in a nick translation reaction to get
rid of the nick between the ligated barcoded adaptors and the DNA.
The DNA was amplified with a Platinum.TM. PCR Amplification Mix in
a total volume of 120 uL, and amplified in a thermocycler at
72.degree. C. for 20 minute, 4.degree. C. hold. The amplified DNA
was purified with a SOLiD.TM. Library Micro Column Purification
kit, using B2-S buffer and eluting with 20 uL of E1 buffer.
Example 2
A. Enzymatic Nucleic Acid Fragmentation
[0302] DNA (10 ng-1 ug) can be mixed in 1.times.iShear Buffer with
dNTP, 1.times. iShear Enzyme Mix, in a total volume of 50 uL. The
mixture can be incubated at 37.degree. C. for 15 minutes, and the
reaction can be stopped with 5 uL of Stop Buffer (0.5 M EDTA, pH
8). The fragmented DNA can be purified with AMPure XP beads
(1.8.times.)(Agencourt), and the fragmented DNA can be retrieved
with 22 uL of E1 buffer.
[0303] 10.times. iShear Buffer can include: 500 mM Tris-HCl (pH
7.5), 3 mM dNTP and 55 mM MgCl.sub.2 in water.
[0304] iShear Enzyme Mix can include: 50 mM Tris-HCl (pH 7.5), DNA
polymerase I, DNase I, MgCl.sub.2, 50% glycerol, and 100 ug/ml
BSA.
B. Library Preparation
[0305] The fragmented DNA from step (A) above can be ligated to
adaptors. The fragmented DNA can be mixed in 1.times. Reaction
buffer, with adaptor P1, adaptor P2, 25 U T4 DNA ligase, 25 U Tfi
(exo-) polymerase, 0.2 mM dNTPs, in a total volume of 50 uL. The
mixture can be incubated in a thermocycler at 20.degree. C. for 30
minutes, and 72.degree. C. for 20 minutes. The DNA can be purified
with AMPure XP beads (0.6.times.)(Agencourt) and washed with 200 uL
of 70% ethanol three times. The next steps are optional. The DNA
can be amplified with a Platinum.TM. PCR Amplification Mix using
the library PCR primer 1 and 2 in a total volume of 125 uL, and
amplified in a thermocycler at 95.degree. C. for 5 minutes
(95.degree. C. for 15 seconds, 62.degree. C. for 15 seconds,
70.degree. C. for 1 minute).times. # of cycles; 70.degree. C. for 5
minutes, 4.degree. C. hold. The amplified DNA can be purified with
AMPure XP beads (1.5.times.)(Agencourt).
Example 3
[0306] Nucleic acid molecules were fragmented and ligated to
adapters as described below and then amplified and sequenced in an
Ion Torrent.TM. PGM.TM. sequencer (Ion Torrent.TM. Systems, Life
Technologies) according to the manufacturer's supplied
protocols.
[0307] Reagents
[0308] 10.times. Shearing Buffer: 500 mM Tris HCl, pH 7.5; 55 mM
MgCl.sub.2; 3 mM dNTP in water.
[0309] Shearing Enzyme: 0.8 Units E. coli DNA Polymerase I; 0.0025
Units DNase I in storage buffer.
[0310] Stop Buffer: 0.5M EDTA (pH 8.0).
[0311] 5.times. Ligase Buffer: 250 mM Tris-HCl, pH 7.6; 50 mM
MgCl.sub.2, 5 mM ATP, 5 mM DTT, 25% PEG-8000.
[0312] E1 Buffer: 10 mM Tris-HCl, pH 8.5.
[0313] Adapters: Ion Torrent Part No: 602-1067-01, available as
part of the Ion Fragment Library Kit.
[0314] Fragmentation of Nucleic Acid Molecules; Purification of
Nucleic Acid Fragments
[0315] The following reagents were combined in a 1.5 ml LoBind tube
(Eppendorf # 022431021):
TABLE-US-00001 DNA to be fragmented: 2 .mu.g 10X Shearing Buffer 5
.mu.L Shearing Enzyme Mix 10 .mu.L Deionized water to final volume
of 50 .mu.L
[0316] The contents of the LoBind tube were mixed and incubated at
37.degree. C. for 15 minutes.
[0317] 5 .mu.L of Stop Buffer was added to the reaction, the
mixture was vortexed and placed on ice.
[0318] The fragmented nucleic acid molecules were purified using
the Agencourt.TM. AMPure.TM. XP kit (Beckman # A63880) according to
the following protocol: 99 .mu.l Agencourt.RTM. AMPure.RTM. beads
(1.8 volumes) were added to the sample, which was then vortexed and
pulse-spin. The mixture was incubated at room temperature
(20-25.degree. C.) for 5 minutes. The tube was placed in a
DynaMag.TM.-2 magnetic rack for at least 1 minute until the
solution was clear of brown tint when viewed at an angle; then the
supernatant was carefully removed and discarded. The beads were
washed three times with 300 .mu.L of 70% ethanol, then dried at
room temperature for 5 minutes. The DNA was eluted from the beads
using 50 .mu.L E1 buffer (supplied with the AMPure Kit). The
supernatant containing the eluted DNA was transferred to a new
1.5-mL LoBind tube.
[0319] Ligation Of Adapters To Sheared DNA
[0320] In a 1.5 ml LoBind tube, add 5.times. reaction buffer, 50 uM
adapters (Ion Torrent cat no: 602-1067-01), 5 U/ul T4 DNA ligase
were combined to a final volume of 53 .mu.L as follows:
TABLE-US-00002 DNA 50 ul 5x Ligase buffer 20 ul 50 uM adapters 20
ul 5U/ul T4 DNA ligase 10 ul Total Volume 100 ul
[0321] The ligation mixture was incubated at room temperature for
30 minutes.
[0322] The ligated DNA was purified using the Agencourt.TM.
AMPure.TM. XP kit (Beckman # A63880) according to the following
protocol: 180 .mu.l Agencourt.RTM. AMPure.RTM. beads (1.8 volumes)
were added to the sample, which was then vortexed and pulse-spin.
The mixture was incubated at room temperature (20-25.degree. C.)
for 5 minutes. The tube was placed in a DynaMag.TM.. 2 magnetic
rack for at least 1 minute until the solution was clear of brown
tint when viewed at an angle; then the supernatant was carefully
removed and discarded. The beads were washed three times with 300
.mu..mu.L of 70% ethanol, then dried at room temperature for 5
minutes. The DNA was eluted from the beads using 30 .mu..mu.L E1
buffer (supplied with the AMPure Kit). The supernatant containing
the eluted DNA was transferred to a new 1.5-mL LoBind tube.
[0323] The DNA was then size-selected using a Pippin Prep.TM.
instrument (Sage Sciences) to achieve a size distribution of 50-80
bp, essentially according to the manufacturer's provided
protocol.
[0324] The size-selected DNA was diluted to a total volume of 60
.mu.L, and then purified using the Agencourt.TM. AMPure.TM. XP kit
(Beckman # A63880) according to the following protocol: 108 .mu.l
Agencourt.RTM. AMPure.RTM. beads (1.8 volumes) were added to the
sample, which was then vortexed and pulse-spin. The mixture was
incubated at room temperature (20-25.degree. C.) for 5 minutes. The
tube was placed in a DynaMag.TM.-2 magnetic rack for at least 1
minute until the solution was clear of brown tint when viewed at an
angle; then the supernatant was carefully removed and discarded.
The beads were washed twice with 500 .mu.L of freshly prepared 70%
ethanol, then dried at room temperature for 5 minutes. The DNA was
eluted from the beads using 40 .mu.L E1 buffer (supplied with the
AMPure Kit). The supernatant containing the eluted DNA was
transferred to a new 1.5-mL LoBind tube.
[0325] Nick Translation and Library Amplification
[0326] The following reaction mixture was prepared:
TABLE-US-00003 Platinum PCR SuperMix High 200 .mu.L Fidelity Primer
Mix 10 .mu.L Size selected DNA 40 .mu.L Total 250
[0327] The mixture was aliquoted in batches of 125 .mu.L into each
of two PCR tubes.
[0328] The PCR tubes were subjected to the following cycle:
TABLE-US-00004 Stage Step Temp Time Holding Nick translation
72.degree. C. 20 min Holding Denature 95.degree. C. 5 min Cycling
(4 cycles) Denature 95.degree. C. 15 sec Anneal 58.degree. C. 15
sec Extend 72.degree. C. 1 min Holding Extend 72.degree. C. 3 min
Holding -- 4.degree. C. .infin.
[0329] The PCR samples were pooled into a new 1.5 ml LoBind
tube.
[0330] The mixture was then purified using the Agencourt.TM.
AMPure.TM. XP kit (Beckman # A63880) according to the following
protocol: 375 .mu.l Agencourt.RTM. AMPure.RTM. beads (1.5 volumes)
were added to the sample, which was then vortexed. The mixture was
incubated at room temperature (20-25.degree. C.) for 10 minutes on
a rotator. The tube was placed in a DynaMag.TM.. 2 magnetic rack
for at least 1 minute until the solution was clear of brown tint
when viewed at an angle; then the supernatant was carefully removed
and discarded. The beads were washed three with 500 .mu.L of
freshly prepared 70% ethanol, then dried at room temperature for 5
minutes. The DNA was eluted from the beads using 50 .mu.L E1 buffer
(supplied with the AMPure Kit). The supernatant containing the
eluted DNA was transferred to a new 1.5-mL LoBind tube.
[0331] The concentration of the eluted DNA was measured using the
Agilent Bioanalyzer.TM. High Sensitivity DNA Kit (Agilent, Catalog
No. 5067-4626), and also separately using the Invitrogen Qubit.TM.
dsDNA HS Assay Kit (Invitrogen Part no. Q32851 or Q32854).
[0332] The purified, adapter-ligated and size selected DNA
fragments were then amplified onto Ion Sphere.TM. particles (Ion
Torrent Systems/Life Technologies, Part No. 602-1075-01)
essentially according to the protocols provided in the Ion
Xpress.TM. Template Kit User Guide (Ion Torrent Systems/Life
Technologies, Part No. 4467389) , hereby incorporated by reference
in its entirety, and using the reagents provided in the Ion
Template Preparation Kit (Ion Torrent Systems/Life Technologies,
Part No. 4466461), the Ion Template Reagents Kit (Ion Torrent
Systems/Life Technologies, Part No.4466462) and the Ion Template
Solutions Kit (Ion Torrent Systems/Life Technologies, Part No.
4466463). The amplified DNA was then sequenced on an Ion PGM.TM.
sequencer (Ion Torrent Systems/Life Technologies, Part No. 4462917)
essentially according to the protocols provided in the Ion
Sequencing Kit User Guide (Ion Torrent Systems/Life Technologies,
Part No. 4467391) , hereby incorporated by reference in its
entirety, using the reagents provided in the Ion Sequencing Kit
(Ion Torrent Systems/Life Technologies, Part No. 4466456) and the
Ion Chip Kit (Ion Torrent Systems/Life Technologies, Part No.
4462923). Ion Torrent Systems is a subsidiary of Life Technologies
Corp., Carlsbad, Calif.).
Example 4
[0333] Nucleic acid molecules (Amplicon or genomic DNA) were
fragmented and ligated to adapters as described below and sequenced
using an Ion Torrent.TM. PGM.TM. sequencer (Ion Torrent.TM.
Systems, Life Technologies) according to the manufacturers supplied
protocols.
[0334] Reagents
[0335] 10.times. Ion Shear.TM. Plus Reaction Buffer can include:
500 mM Tris-HCl (pH 7.5), 55 mM MgCl.sub.2 and 3 mM dNTP in
water.
[0336] Ion Shear.TM. Plus Enzyme Mix can include: DNA polymerase I,
DNase I, 50 mM Tris-HCl (pH 7.5), MgCl.sub.2, 0.75 mg/ml BSA and
50% glycerol.
[0337] Ion Shear.TM. Plus Stop Buffer can include: 0.5M EDTA (pH
8).
[0338] Nick Repair Enzyme can include: Taq DNA polymerase, Bst DNA
polymerase, Platinum.RTM. Pfx DNA polymerase (Invitrogen), Tfi
Exo(-) DNA polymerase (Invitrogen) or Phusion.RTM. Hot Start
High-Fidelity DNA polymerase (New England Biolabs).
[0339] 10 mM dNTP: 10 mM dNTPs in water.
[0340] DNA Ligase: Life Technologies Part. No. 602-1060-01.
[0341] 10.times. Ligase Buffer can include: 250 mM Tris-HCl (pH
7.6), 50 mM MgCl.sub.2, 5 mM ATP, 5 mM DTT and 25% PEG-8000 (Life
Technologies Part. No. 602-1060-01).
[0342] Low TE can include: Tris-EDTA (Ion Torrent.TM. Part No:
602-1066-01, available as part of the Ion Fragment Library Kit
(Part No. 4466464).
[0343] Adapters: Ion Torrent.TM. Part No: 602-1067-01, available as
part of the Ion Fragment Library Kit (Part No. 4466464).
[0344] Fragmentation of Nucleic Acid Molecules
[0345] The following method fragments double stranded DNA into
blunt-ended fragments in the 200-300 bp size range. The fragmented
DNA is ready for adaptor ligation, followed by nick repair to
complete linkage between adaptors and DNA. The adaptor-ligated
fragments are then size-selected for optimum fragment length, for
example using E-Gel Size Select 2% agarose gel or Pippin Prep.TM.
instrument for automated size selection.
[0346] For inputs of 100 ng or greater, the resulting amplification
free library is sufficient for most downstream processing, for
example for use with Ion Sphere.TM. particles (Ion Torrent.TM.,
Life Technologies, Part No. 602-1075-01) essentially according to
the protocols provided in the Ion Xpress.TM. Template Kit User
Guide (Ion Torrent.TM., Life Technologies, Part No. 4467389),
hereby incorporated by reference in its entirety, using the
reagents provided in the Ion Template Preparation Kit (Ion
Torrent.TM., Life Technologies, Part No. 4466461), the Ion Template
Reagents Kit (Ion Torrent.TM., Life Technologies, Part No.4466462)
and the Ion Template Solutions Kit (Ion Torrent.TM., Life
Technologies, Part No. 4466463). Template preparations can then be
sequenced on an Ion PGM.TM. sequencer (Ion Torrent.TM., Life
Technologies, Part No. 4462917) essentially according to the
protocols provided in the Ion Sequencing Kit User Guide (Ion
Torrent.TM. Life Technologies, Part No. 4467391), hereby
incorporated by reference in its entirety.
[0347] In this example, the following reagents were combined in a
1.5 ml LoBind tube (Eppendorf, Cat. No. 022431021):
TABLE-US-00005 Nucleic acids to be fragmented at 100 ng/.mu.L 1
.mu.g 10X Ion Shear .TM. Plus Reaction Buffer 5 .mu.L Deionized
water 25 .mu.L
[0348] The contents of the tube were mixed vigorously by vortexing
for 5 seconds, pulse-spin and the following amount of Ion Shear.TM.
Plus Enzyme Mix added to the tube:
TABLE-US-00006 Ion Shear .TM. Plus Enzyme Mix 10 .mu.L
[0349] The contents of the LoBind tube were mixed and incubated at
37.degree. C. for 15 minutes.
[0350] 5 .mu.L of Ion Shear.TM. Plus Stop Buffer was added to the
reaction; the mixture was vortexed and placed on ice.
[0351] The fragmented nucleic acid molecules were purified using
the Agencourt.TM. AMPure.TM. XP kit (Beckman Part. No. A63880)
according to the following protocol:
[0352] 99 .mu.l Agencourt.RTM. AMPure.RTM. beads (1.8 volumes) were
added to the sample, which was then vortexed and pulse-spin. The
mixture was incubated at room temperature (20-25.degree. C.) for 5
minutes. The tube was placed in a DynaMag.TM.-2 magnetic rack for 3
minutes until the solution was clear of brown tint when viewed at
an angle; then the supernatant was carefully removed and discarded.
The beads were washed twice with 500 .mu.L of 70% ethanol, then
dried at room temperature for 5 minutes. The DNA was eluted from
the beads using 25 .mu.L Low TE, which was then vortexed and
pulse-spin. The sample was placed in a DynaMag.TM.-2 magnetic rack
for at least one minute until the solution was clear. The
supernatant containing the eluted DNA was transferred to a new 0.2
mL PCR tube.
[0353] Ligation Of Adapters To DNA and Nick Repair
[0354] In a 0.2 ml PCR tube containing the eluted DNA (.about.1
.mu.g) , add 10.times. Ligase Buffer, Adapters (Ion Torrent.TM.
Cat. no: 602-1067-01), dNTP mix, DNA ligase, and Nick repair enzyme
were combined to a final volume of 100 .mu.L as follows:
TABLE-US-00007 DNA 25 ul 10x Ligase buffer 10 ul Adapters 10 ul
dNTP Mix 2 ul DNA ligase 2 ul Nick repair enzyme 8 ul Nuclease-free
water 43 ul Total Volume 100 ul
[0355] The ligation mixture was placed into a thermocycler
programmed as follows:
[0356] 25.degree. C. for 15 minutes; and then 72.degree. C. for 5
minutes; followed by holding at 4.degree. C.
[0357] The adaptor-ligated and nick-repaired DNA was purified using
the Agencourt.TM. AMPure.TM. XP kit (Beckman Part. No. A63880)
according to the following protocol:
[0358] 150 .mu.l Agencourt.RTM. AMPure.RTM. beads (1.5 volumes)
were added to the sample, which was then vortexed and pulse-spin.
The mixture was incubated at room temperature (20-25.degree. C.)
for 5 minutes. The tube was placed in a DynaMag.TM.-2 magnetic rack
for 3 minutes until the solution was clear of brown tint when
viewed at an angle; then the supernatant was carefully removed and
discarded. The beads were washed twice with 500 .mu.L of 70%
ethanol, then dried at room temperature for 5 minutes. The DNA was
eluted from the beads using 20 .mu.L Low TE, which was then
vortexed and pulse-spin. The sample was placed in a DynaMag.TM.-2
magnetic rack for at least one minute until the solution was clear.
The supernatant containing the eluted DNA was transferred to a new
1.5-mL LoBind tube.
[0359] Size Selection
[0360] The DNA was then size-selected using a E-Gel SizeSelect
Agarose Gel (2%) (Life Technologies, G6610-02) to achieve 200
nucleotide sequencing reads, a DNA library with a peak size of
.about.330bp was selected, essentially according to the
manufacturer's provided protocol. The DNA solution (.about.40
.mu.l) recovered from the SizeSelect Gel did not require additional
purification for downstream processing (e.g., qPCR quantification
or emulsion PCR). In an alternative method, a Pippin Prep.TM.
instrument can be used to automate size-selection.
[0361] The concentration of the eluted DNA from the SizeSelect gel
was measured using the Agilent Bioanalyzer.TM. High Sensitivity DNA
Kit (Agilent, Catalog No. 5067-4626), and also separately using the
Invitrogen Qubit.TM. dsDNA HS Assay Kit (Invitrogen Part no. Q32851
or Q32854).
[0362] The size selected DNA fragments were then amplified onto Ion
Sphere.TM. particles (Ion Torrent.TM., Life Technologies, Part No.
602-1075-01) essentially according to the protocols provided in the
Ion Xpress.TM. Template Kit User Guide (Ion Torrent.TM., Life
Technologies, Part No. 4467389), hereby incorporated by reference
in its entirety, and using the reagents provided in the Ion
Template Preparation Kit (Ion Torrent.TM., Life Technologies, Part
No. 4466461), the Ion Template Reagents Kit (Ion Torrent.TM., Life
Technologies, Part No.4466462) and the Ion Template Solutions Kit
(Ion Torrent.TM., Life Technologies, Part No. 4466463). The
amplified DNA was then sequenced on an Ion PGM.TM.sequencer (Ion
Torrent.TM. Systems, Life Technologies, Part No. 4462917)
essentially according to the protocols provided in the Ion
Sequencing Kit User Guide (Ion Torrent.TM., Life Technologies, Part
No. 4467391), hereby incorporated by reference in its entirety, and
using the reagents provided in the Ion Sequencing Kit (Ion
Torrent.TM., Life Technologies, Part No. 4466456) and the Ion Chip
Kit (Ion Torrent.TM., Life Technologies, Part No. 4462923).
Example 5
[0363] Nucleic acid molecules (Amplicon or genomic DNA) were
fragmented and ligated to adapters as described below and sequenced
on an Ion Torrent.TM. PGM.TM. sequencer (Ion Torrent.TM. Systems,
Life Technologies) according to the manufacturers supplied
protocols.
[0364] Reagents
[0365] 10.times. Ion Shear.TM. Plus Reaction Buffer can include:
500 mM Tris-HCl (pH 7.5), 55 mM MgCl.sub.2 and 3 mM dNTP in
water.
[0366] Ion Shear.TM. Plus Enzyme Mix can include: DNA polymerase I;
DNase I; 50 mM Tris-HCl (pH 7.5), MgCl.sub.2, 0.75 mg/ml BSA and
50% glycerol. Ion Shear.TM. Plus Stop Buffer can include: 0.5M EDTA
(pH 8.0).
[0367] Nick Repair Enzyme can include: Taq DNA polymerase, Bst DNA
polymerase, Platinum.RTM. Pfx DNA polymerase (Invitrogen), Tfi
Exo(-) DNA polymerase (Invitrogen) or Phusion.RTM. Hot Start
High-Fidelity DNA polymerase (New England Biolabs).
[0368] 10 mm dNTP: 10 mm dNTPs in water.
[0369] DNA Ligase: Life Technologies Part. No. 602-1060-01.
[0370] 10.times. Ligase Buffer can include: 250 mM Tris-HCl (pH
7.6), 50 mM MgCl.sub.2, 5mM ATP, 5 mM DTT and 25% PEG-8000 (Life
Technologies Part. No. 602-1060-01). Low TE can include: Tris-EDTA
(Ion Torrent.TM. Part No: 602-1066-01, available as part of the Ion
Fragment Library Kit (Part No. 4466464).
[0371] Adapters: Ion Torrent.TM. Part No: 602-1067-01, available as
part of the Ion Fragment Library Kit (Part No. 4466464).
[0372] Fragmentation of Nucleic Acid Molecules
[0373] The following reagents were combined in a 1.5 ml LoBind tube
(Eppendorf, Cat. No. 022431021):
TABLE-US-00008 Nucleic acids to be fragmented at 100 ng/.mu.L 1
.mu.g 10X Ion Shear .TM. Plus Reaction Buffer 5 .mu.L Deionized
water 25 .mu.L
[0374] The contents of the tube were mixed vigorously by vortexing
for 5 seconds, pulse-spin and the following amount of Ion Shear.TM.
Plus Enzyme Mix added to the tube:
TABLE-US-00009 Ion Shear .TM. Plus Enzyme Mix 10 .mu.L
[0375] The contents of the LoBind tube were mixed and incubated at
37.degree. C. for 15 minutes.
[0376] 5 .mu.L of Ion Shear.TM. Plus Stop Buffer was added to the
reaction; the mixture was vortexed and placed on ice.
[0377] The fragmented nucleic acid molecules were purified using
the Agencourt.TM. AMPure.TM. XP kit (Beckman Part. No. A63880)
according to the following protocol to produce double stranded DNA
fragments in the range of 180-250 bp:
[0378] 99 .mu.l Agencourt.RTM. AMPure.RTM. beads (1.8 volumes) were
added to the sample, which was then vortexed and pulse-spin. The
mixture was incubated at room temperature (20-25.degree. C.) for 5
minutes. The tube was placed in a DynaMag.TM.-2 magnetic rack for 3
minutes until the solution was clear of brown tint when viewed at
an angle; then the supernatant was carefully removed and discarded.
The beads were washed twice with 500 .mu.L of 70% ethanol, then
dried at room temperature for 5 minutes. The DNA was eluted from
the beads using 25 .mu.L Low TE, which was then vortexed and
pulse-spin. The sample was placed in a DynaMag.TM.-2 magnetic rack
for at least one minute until the solution was clear. The
supernatant containing the eluted DNA was transferred to a new
1.5-mL LoBind tube.
[0379] AMPure.TM. XP beads
[0380] 49.5 .mu.l Agencourt.RTM. AMPure.RTM. beads (0.9 volume)
were added to the sample, which was then vortexed and pulse-spin.
The mixture was incubated at room temperature (20-25.degree. C.)
for 5 minutes. The tube was placed in a DynaMag.TM.-2 magnetic rack
for 3 minutes until the solution was clear of brown tint when
viewed at an angle; then the supernatant was carefully removed to a
new 1.5-mL LoBind tube. To the supernatant, 11 .mu.l of
Agencourt.RTM. AMPure.RTM. beads (0.2.times. original sample
volume) were added to the sample, which was then vortexed and
pulse-spin. The mixture was incubated at room temperature
(20-25.degree. C.) for 5 minutes. The tube was placed in a
DynaMag.TM.-2 magnetic rack for 3 minutes until the solution was
clear of brown tint when viewed at an angle; then the supernatant
was carefully removed and discarded. The beads were washed twice
with 500 .mu.L of 70% ethanol, then dried at room temperature for 5
minutes. The DNA was eluted from the beads using 25 .mu.L Low TE,
which was then vortexed and pulse-spin. The sample was placed in a
DynaMag.TM.-2 magnetic rack for at least one minute until the
solution was clear. The supernatant containing the eluted DNA was
transferred to a new 0.2-mL PCR tube.
[0381] Ligation Of Adapters To DNA and Nick Repair
[0382] To the 0.2-mL PCR tube containing the eluted DNA (.about.1
.mu.g) , add 10.times. Ligase Buffer, Adapters (Ion Torrent.TM.
Cat. no: 602-1067-01), dNTP mix, DNA ligase, and Nick Repair Enzyme
were combined to a final volume of 100 .mu.L as follows:
TABLE-US-00010 DNA 25 ul 10x Ligase buffer 10 ul Adapters 10 ul
dNTP Mix 2 ul DNA ligase 2 ul Nick Repair Enzyme 8 ul Nuclease-free
water 43 ul Total Volume 100 ul
[0383] The mixture was placed into a thermocycler programmed as
follows: 25.degree. C. for 15 minutes; and then 72.degree. C. for 5
minutes; followed by holding at 4.degree. C.
[0384] The adaptor-ligated and nick-repaired DNA was purified using
the Agencourt.TM. AMPure.TM. XP kit (Beckman Part. No. A63880)
according to the following protocol:
[0385] 140 .mu.l Agencourt.RTM. AMPure.RTM. beads (1.4 volumes)
were added to the sample, which was then vortexed and pulse-spin.
The mixture was incubated at room temperature (20-25.degree. C.)
for 5 minutes. The tube was placed in a DynaMag.TM.-2 magnetic rack
for 3 minutes until the solution was clear of brown tint when
viewed at an angle; then the supernatant was carefully removed and
discarded. The beads were washed twice with 500 .mu.L of 70%
ethanol, then dried at room temperature for 5 minutes. The DNA was
eluted from the beads using 20 .mu.L Low TE, which was then
vortexed and pulse-spin. The sample was placed in a DynaMag.TM.-2
magnetic rack for at least one minute until the solution was clear.
The supernatant containing the eluted DNA was transferred to a new
1.5-mL LoBind tube.
[0386] The DNA solution (.about.20 .mu.l) recovered did not require
additional purification for downstream processing (e.g., qPCR
quantification or emulsion PCR) and generated library with a mean
size of 280-310 bp.
[0387] The concentration of the eluted DNA was measured using the
Agilent Bioanalyzer.TM. High Sensitivity DNA Kit (Agilent, Catalog
No. 5067-4626), and also separately using the Invitrogen Qubit.TM.
dsDNA HS Assay Kit (Invitrogen Part no. Q32851 or Q32854).
[0388] The size selected DNA fragments were then amplified onto Ion
Sphere.TM. particles (Ion Torrent.TM., Life Technologies, Part No.
602-1075-01) essentially according to the protocols provided in the
Ion Xpress.TM. Template Kit User Guide (Ion Torrent.TM., Life
Technologies, Part No. 4467389), hereby incorporated by reference
in its entirety, and using the reagents provided in the Ion
Template Preparation Kit (Ion Torrent.TM., Life Technologies, Part
No. 4466461), the Ion Template Reagents Kit (Ion Torrent.TM., Life
Technologies, Part No.4466462) and the Ion Template Solutions Kit
(Ion Torrent.TM., Life Technologies, Part No. 4466463). The
amplified DNA was then sequenced on an Ion PGM.TM. sequencer (Ion
Torrent.TM., Life Technologies, Part No. 4462917) essentially
according to the protocols provided in the Ion Sequencing Kit User
Guide (Ion Torrent.TM., Life Technologies, Part No. 4467391),
hereby incorporated by reference in its entirety, and using the
reagents provided in the Ion Sequencing Kit (Ion Torrent.TM., Life
Technologies, Part No. 4466456) and the Ion Chip Kit (Ion
Torrent.TM., Life Technologies, Part No. 4462923).
Example 6
[0389] Nucleic acids (gDNA or Amplicons) were fragmented and
ligated to adapters as described in Examples 4 and 5. In some
instances, DNA input is less than required for downstream template
preparation, and as such the fragmented nucleic acid library (from
Examples 4 and 5) can be optionally amplified as described below to
generate a library ready for downstream Template preparation, for
example for use in the Ion XpresS.TM. Template Kit (Ion
Torrent.TM., Life Technologies, Part No. 4467389). The DNA can then
be sequenced on an Ion PGMTM sequencer (Ion Torrent.TM., Life
Technologies, Part No. 4462917) essentially according to the
protocols provided in the Ion Sequencing Kit User Guide (Ion
Torrent.TM., Life Technologies, Part No. 4467391), hereby
incorporated by reference in its entirety, using the reagents
provided in the Ion Sequencing Kit (Ion Torrent.TM., Life
Technologies, Part No. 4466456) and the Ion Chip Kit (Ion
Torrent.TM., Life Technologies, Part No. 4462923).
[0390] Library Amplification
[0391] The following reaction mixture was prepared in a PCR tube
containing 100 ng genomic DNA or amplicon prepared as described in
Examples 4 and 5:
TABLE-US-00011 Platinum PCR SuperMix High 100 .mu.L Fidelity Primer
Mix 5 .mu.L Size-selected amplification-free 25 .mu.L DNA Total
130
[0392] The PCR tube was placed into a thermal cycler and run the
following PCR cycling program:
TABLE-US-00012 Stage Step Temp Time Holding Denature 95.degree. C.
5 min Cycling (5-9 Denature 95.degree. C. 15 sec cycles) Anneal
58.degree. C. 15 sec Extend 70.degree. C. 1 min Holding --
4.degree. C. .infin.
[0393] The mixture was then purified using the Agencourt.TM.
AMPure.TM. XP kit (Beckman Cat. No. A63880) according to the
following protocol: 195 .mu.l Agencourt.RTM. AMPure.RTM. beads (1.5
volumes) were added to the sample, which was then vortexed. The
mixture was incubated at room temperature (20-25.degree. C.) for 5
minutes. The tube was placed in a DynaMag.TM.-2 magnetic rack for
at least 3 minutes until the solution was clear of brown tint when
viewed at an angle; then the supernatant was carefully removed and
discarded. The beads were washed twice with 500 .mu.L of freshly
prepared 70% ethanol, then dried at room temperature for 5 minutes.
The DNA was eluted from the beads using 20 .mu.L Low TE. The
supernatant containing the eluted DNA was transferred to a new
1.5-mL LoBind tube.
[0394] The concentration of the eluted DNA was measured using the
Agilent Bioanalyzer.TM. High Sensitivity DNA Kit (Agilent, Catalog
No. 5067-4626), and also separately using the Invitrogen Qubit.TM.
dsDNA HS Assay Kit (Invitrogen Part no. Q32851 or Q32854).
[0395] The size selected DNA fragments were then amplified onto Ion
Sphere.TM. particles (Ion Torrent.TM., Life Technologies, Part No.
602-1075-01) essentially according to the protocols provided in the
Ion Xpress.TM. Template Kit User Guide (Ion Torrent.TM., Life
Technologies, Part No. 4467389), hereby incorporated by reference
in its entirety, and using the reagents provided in the Ion
Template Preparation Kit (Ion Torrent.TM., Life Technologies, Part
No. 4466461), the Ion Template Reagents Kit (Ion Torrent.TM., Life
Technologies, Part No.4466462) and the Ion Template Solutions Kit
(Ion Torrent.TM., Life Technologies, Part No. 4466463). The
amplified DNA was then sequenced on an Ion PGM.TM.sequencer (Ion
Torrent.TM., Life Technologies, Part No. 4462917) essentially
according to the protocols provided in the Ion Sequencing Kit User
Guide (Ion Torrent.TM., Life Technologies, Part No. 4467391),
hereby incorporated by reference in its entirety, and using the
reagents provided in the Ion Sequencing Kit (Ion Torrent.TM., Life
Technologies, Part No. 4466456) and the Ion Chip Kit (Ion
Torrent.TM., Life Technologies, Part No. 4462923).
Example 7
[0396] Nucleic acid fragmentation reactions were conducted in a
LoBind tube: 1 ug DNA (e.g., genomic or amplicon) was mixed with 5
uL of 10.times. Ion Shear Plus Reaction buffer, 10 uL of Ion Shear
Plus Enzyme Mix, between about 5-50 ug of gp 32 protein (e.g., 40
ug of gp32 protein), and water to a final volume of 50 uL. The
reaction was incubated at 37 .degree. C. for 15 minutes, and 5 uL
of Stop buffer was added. The reaction was vortexed and placed on
ice.
[0397] The fragmented nucleic acid molecules were purified using
the Agencourt.TM. AMPure.TM. XP kit (Beckman Part. No. A63880)
according to the following protocol : 99 .mu.l Agencourt.RTM.
AMPure.RTM. beads (1.8 volumes) were added to the sample, which was
then vortexed and pulse-spin. The mixture was incubated at room
temperature (20-25.degree. C.) for 5 minutes. The tube was placed
in a DynaMag.TM.-2 magnetic rack for 3 minutes until the solution
was clear of brown tint when viewed at an angle; then the
supernatant was carefully removed and discarded. The beads were
washed twice with 500 .mu.L of 70% ethanol, then dried at room
temperature for 5 minutes. The DNA was eluted from the beads using
25 .mu.L Low TE, which was then vortexed and pulse-spin. The sample
was placed in a DynaMag.TM.-2 magnetic rack for at least one minute
until the solution was clear. The supernatant containing the eluted
DNA was transferred to a new 1.5-mL LoBind tube.
[0398] DNA fragmented in the presence of gp32 protein was ligated
to adapters as described in Examples 4 and 5 above. In some
instances, DNA input is less than required for downstream template
preparation, and as such the fragmented nucleic acid library can be
optionally amplified as described in Example 6 above to generate a
library ready for downstream Template preparation, for example for
use in the Ion Xpress.TM. Template Kit (Ion Torrent.TM., Life
Technologies, Part No. 4467389). The DNA can then be sequenced on
an Ion PGMTM sequencer (Ion Torrent.TM., Life Technologies, Part
No. 4462917) essentially according to the protocols provided in the
Ion Sequencing Kit User Guide (Ion Torrent.TM., Life Technologies,
Part No. 4467391), hereby incorporated by reference in its
entirety, using the reagents provided in the Ion Sequencing Kit
(Ion Torrent.TM., Life Technologies, Part No. 4466456) and the Ion
Chip Kit (Ion Torrent.TM., Life Technologies, Part No.
4462923).
[0399] While the principles of the present teachings have been
described in connection with specific embodiments, it should be
understood clearly that these descriptions are made only by way of
example and are not intended to limit the scope of the present
teachings or claims. What has been disclosed herein has been
provided for the purposes of illustration and description. It is
not intended to be exhaustive or to limit what is disclosed to the
precise forms described. Many modifications and variations will be
apparent to the practitioner skilled in the art. What is disclosed
was chosen and described in order to best explain the principles
and practical application of the disclosed embodiments of the art
described, thereby enabling others skilled in the art to understand
the various embodiments and various modifications that are suited
to the particular use contemplated. It is intended that the scope
of what is disclosed be defined by the following claims and their
equivalents.
Sequence CWU 1
1
41301PRTphage T4MISC_FEATUREgp 32 protein 1Met Phe Lys Arg Lys Ser
Thr Ala Glu Leu Ala Ala Gln Met Ala Lys1 5 10 15Leu Asn Gly Asn Lys
Gly Phe Ser Ser Glu Asp Lys Gly Glu Trp Lys 20 25 30Leu Lys Leu Asp
Asn Ala Gly Asn Gly Gln Ala Val Ile Arg Phe Leu 35 40 45Pro Ser Lys
Asn Asp Glu Gln Ala Pro Phe Ala Ile Leu Val Asn His 50 55 60Gly Phe
Lys Lys Asn Gly Lys Trp Tyr Ile Glu Thr Cys Ser Ser Thr65 70 75
80His Gly Asp Tyr Asp Ser Cys Pro Val Cys Gln Tyr Ile Ser Lys Asn
85 90 95Asp Leu Tyr Asn Thr Asp Asn Lys Glu Tyr Ser Leu Val Lys Arg
Lys 100 105 110Thr Ser Tyr Trp Ala Asn Ile Leu Val Val Lys Asp Pro
Ala Ala Pro 115 120 125Glu Asn Glu Gly Lys Val Phe Lys Tyr Arg Phe
Gly Lys Lys Ile Trp 130 135 140Asp Lys Ile Asn Ala Met Ile Ala Val
Asp Val Glu Met Gly Glu Thr145 150 155 160Pro Val Asp Val Thr Cys
Pro Trp Glu Gly Ala Asn Phe Val Leu Lys 165 170 175Val Lys Gln Val
Ser Gly Phe Ser Asn Tyr Asp Glu Ser Lys Phe Leu 180 185 190Asn Gln
Ser Ala Ile Pro Asn Ile Asp Asp Glu Ser Phe Gln Lys Glu 195 200
205Leu Phe Glu Gln Met Val Asp Leu Ser Glu Met Thr Ser Lys Asp Lys
210 215 220Phe Lys Ser Phe Glu Glu Leu Asn Thr Lys Phe Gly Gln Val
Met Gly225 230 235 240Thr Ala Val Met Gly Gly Ala Ala Ala Thr Ala
Ala Lys Lys Ala Asp 245 250 255Lys Val Ala Asp Asp Leu Asp Ala Phe
Asn Val Asp Asp Phe Asn Thr 260 265 270Lys Thr Glu Asp Asp Phe Met
Ser Ser Ser Ser Gly Ser Ser Ser Ser 275 280 285Ala Asp Asp Thr Asp
Leu Asp Asp Leu Leu Asn Asp Leu 290 295 3002148PRTSulfolobus
solfataricus 2Met Glu Glu Lys Val Gly Asn Leu Lys Pro Asn Met Glu
Ser Val Asn1 5 10 15Val Thr Val Arg Val Leu Glu Ala Ser Glu Ala Arg
Gln Ile Gln Thr 20 25 30Lys Asn Gly Val Arg Thr Ile Ser Glu Ala Ile
Val Gly Asp Glu Thr 35 40 45Gly Arg Val Lys Leu Thr Leu Trp Gly Lys
His Ala Gly Ser Ile Lys 50 55 60Glu Gly Gln Val Val Lys Ile Glu Asn
Ala Trp Thr Thr Ala Phe Lys65 70 75 80Gly Gln Val Gln Leu Asn Ala
Gly Ser Lys Thr Lys Ile Ala Glu Ala 85 90 95Ser Glu Asp Gly Phe Pro
Glu Ser Ser Gln Ile Pro Glu Asn Thr Pro 100 105 110Thr Ala Pro Gln
Gln Met Arg Gly Gly Gly Arg Gly Phe Arg Gly Gly 115 120 125Gly Arg
Arg Tyr Gly Arg Arg Gly Gly Arg Arg Gln Glu Asn Glu Glu 130 135
140Gly Glu Glu Glu1453180PRTEscherichia coli 3Met Ala Asn Leu Gln
Val Ala Thr Ser Glu Thr Trp Arg Asp Lys Gln1 5 10 15Thr Gly Glu Met
Arg Glu Gln Thr Glu Trp His Arg Val Val Leu Phe 20 25 30Gly Lys Leu
Ala Glu Val Ala Gly Glu Tyr Leu Arg Lys Gly Val Gln 35 40 45Val Tyr
Ile Glu Gly Gln Leu Arg Thr Arg Ser Trp Glu Asp Asn Gly 50 55 60Ile
Thr Arg Tyr Val His Pro Lys Phe Leu Leu Arg Pro Gln Gly Thr65 70 75
80Asn Ala Arg Cys Trp Asp Val Pro Gln Val Leu Arg Leu Lys Leu Glu
85 90 95Arg Gly Ala Asn Ser Phe Lys Thr Ala Gln Pro Phe Lys Pro Gly
Asn 100 105 110Pro Thr Arg Pro Gly Gly Pro Gly Leu Arg Lys Lys Arg
Val Ala Pro 115 120 125Lys Arg Lys Ala Val Asp Val Arg Pro Arg Ser
Arg Ser Leu Ser Cys 130 135 140Asn Arg Arg Arg Val Thr Ile Thr Gly
Phe Gln Thr Ile Ser Arg Ser145 150 155 160Glu Arg Ala Asp Cys Asp
Asn Arg Pro Ala Pro Val Leu Cys Gly Ala 165 170 175Ser Pro Glu Arg
1804645PRTMethanococcus jannaschii 4Met Ile Gly Asp Tyr Glu Arg Phe
Lys Gln Leu Lys Lys Lys Val Ala1 5 10 15Glu Ala Leu Asn Ile Ser Glu
Glu Glu Leu Asp Arg Met Ile Asp Lys 20 25 30Lys Ile Glu Glu Asn Gly
Gly Ile Ile Leu Lys Asp Ala Ala Leu Met 35 40 45Met Ile Ala Lys Glu
His Gly Val Tyr Gly Glu Glu Lys Asn Asp Glu 50 55 60Glu Phe Leu Ile
Ser Asp Ile Glu Glu Gly Gln Ile Gly Val Glu Ile65 70 75 80Thr Gly
Val Ile Thr Asp Ile Ser Glu Ile Lys Thr Phe Lys Arg Arg 85 90 95Asp
Gly Ser Leu Gly Lys Tyr Lys Arg Ile Thr Ile Ala Asp Lys Ser 100 105
110Gly Thr Ile Arg Met Thr Leu Trp Asp Asp Leu Ala Glu Leu Asp Val
115 120 125Lys Val Gly Asp Val Ile Lys Ile Glu Arg Ala Arg Ala Arg
Lys Trp 130 135 140Arg Asn Asn Leu Glu Leu Ser Ser Thr Ser Glu Thr
Lys Ile Lys Lys145 150 155 160Leu Glu Asn Tyr Glu Gly Glu Leu Pro
Glu Ile Lys Asp Thr Tyr Asn 165 170 175Ile Gly Glu Leu Ser Pro Gly
Met Thr Ala Thr Phe Glu Gly Glu Val 180 185 190Ile Ser Ala Leu Pro
Ile Lys Glu Phe Lys Arg Ala Asp Gly Ser Ile 195 200 205Gly Lys Leu
Lys Ser Phe Ile Val Arg Asp Glu Thr Gly Ser Ile Arg 210 215 220Val
Thr Leu Trp Asp Asn Leu Thr Asp Ile Asp Val Gly Arg Gly Asp225 230
235 240Tyr Val Arg Val Arg Gly Tyr Ile Arg Glu Gly Tyr Tyr Gly Gly
Leu 245 250 255Glu Cys Thr Ala Asn Tyr Val Glu Ile Leu Lys Lys Gly
Glu Lys Ile 260 265 270Glu Ser Glu Glu Val Asn Ile Glu Asp Leu Thr
Lys Tyr Glu Asp Gly 275 280 285Glu Leu Val Ser Val Lys Gly Arg Val
Ile Ala Ile Ser Asn Lys Lys 290 295 300Ser Val Asp Leu Asp Gly Glu
Ile Ala Lys Val Gln Asp Ile Ile Leu305 310 315 320Asp Asn Gly Thr
Gly Arg Val Arg Val Ser Phe Trp Arg Gly Lys Thr 325 330 335Ala Leu
Leu Glu Asn Ile Lys Glu Gly Asp Leu Val Arg Ile Thr Asn 340 345
350Cys Arg Val Lys Thr Phe Tyr Asp Arg Glu Gly Asn Lys Arg Thr Asp
355 360 365Leu Val Ala Thr Leu Glu Thr Glu Val Ile Lys Asp Glu Asn
Ile Glu 370 375 380Ala Pro Glu Tyr Glu Leu Lys Tyr Cys Lys Ile Glu
Asp Ile Tyr Asn385 390 395 400Arg Asp Val Asp Trp Asn Asp Ile Asn
Leu Ile Ala Gln Val Val Glu 405 410 415Asp Tyr Gly Val Asn Glu Ile
Glu Phe Glu Asp Lys Val Arg Lys Val 420 425 430Arg Asn Leu Leu Leu
Glu Asp Gly Thr Gly Arg Ile Arg Leu Ser Leu 435 440 445Trp Asp Asp
Leu Ala Glu Ile Glu Ile Lys Glu Gly Asp Ile Val Glu 450 455 460Ile
Leu His Ala Tyr Ala Lys Glu Arg Gly Asp Tyr Ile Asp Leu Val465 470
475 480Ile Gly Lys Tyr Gly Arg Ile Ile Ile Asn Pro Glu Gly Val Glu
Ile 485 490 495Lys Thr Asn Arg Lys Phe Ile Ala Asp Ile Glu Asp Gly
Glu Thr Val 500 505 510Glu Val Arg Gly Ala Val Val Lys Ile Leu Ser
Asp Thr Leu Phe Leu 515 520 525Tyr Leu Cys Pro Asn Cys Arg Lys Arg
Val Val Glu Ile Asp Gly Ile 530 535 540Tyr Asn Cys Pro Ile Cys Gly
Asp Val Glu Pro Glu Glu Ile Leu Arg545 550 555 560Leu Asn Phe Val
Val Asp Asp Gly Thr Gly Thr Leu Leu Cys Arg Ala 565 570 575Tyr Asp
Arg Arg Val Glu Lys Met Leu Lys Met Asn Arg Glu Glu Leu 580 585
590Lys Asn Leu Thr Ile Glu Met Val Glu Asp Glu Ile Leu Gly Glu Glu
595 600 605Phe Val Leu Tyr Gly Asn Val Arg Val Glu Asn Asp Glu Leu
Ile Met 610 615 620Val Val Arg Arg Val Asn Asp Val Asp Val Glu Lys
Glu Ile Arg Ile625 630 635 640Leu Glu Glu Met Glu 645
* * * * *