U.S. patent application number 12/632730 was filed with the patent office on 2010-11-25 for methods of producing and sequencing modified polynucleotides.
This patent application is currently assigned to LIFE TECHNOLOGIES CORPORATION. Invention is credited to Kevin J. McKernan, Douglas R. Smith.
Application Number | 20100298551 12/632730 |
Document ID | / |
Family ID | 37596075 |
Filed Date | 2010-11-25 |
United States Patent
Application |
20100298551 |
Kind Code |
A1 |
Smith; Douglas R. ; et
al. |
November 25, 2010 |
Methods Of Producing And Sequencing Modified Polynucleotides
Abstract
The present invention encompasses methods for producing a
modified polynucleotide sequence that comprises a (e.g., one or
more) phosphorothiolate linkage, methods for determining a
polynucleotide sequence comprising a (e.g., one or more)
phosphorothiolate linkage, and methods for separating forward and
reverse extension products that comprise a (e.g., one or more)
phosphorothiolate linkage. The invention also encompasses kits for
producing and/or determining the sequence of a modified
polynucleotide that comprises a (e.g., one or more)
phosphorothiolate linkage.
Inventors: |
Smith; Douglas R.;
(Gloucester, MA) ; McKernan; Kevin J.;
(Marblehead, MA) |
Correspondence
Address: |
LIFE TECHNOLOGIES CORPORATION;C/O INTELLEVATE
P.O. BOX 52050
MINNEAPOLIS
MN
55402
US
|
Assignee: |
LIFE TECHNOLOGIES
CORPORATION
Carlsbad
CA
|
Family ID: |
37596075 |
Appl. No.: |
12/632730 |
Filed: |
December 7, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11476423 |
Jun 28, 2006 |
7645866 |
|
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12632730 |
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60694783 |
Jun 28, 2005 |
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Current U.S.
Class: |
536/23.1 |
Current CPC
Class: |
C12Q 1/6869 20130101;
C12Q 1/6869 20130101; C12Q 2525/113 20130101 |
Class at
Publication: |
536/23.1 |
International
Class: |
C07H 21/04 20060101
C07H021/04 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] The invention was supported, in whole or in part, by a grant
HG00357 from the National Institutes of Health. The Government has
certain rights in the invention.
Foreign Application Data
Date |
Code |
Application Number |
Jun 28, 2006 |
US |
PCT/US06/25529 |
Claims
1.-94. (canceled)
95. A method of separating one or more forward extension products
from one or more reverse extension products comprising: a)
annealing a first primer and a second primer to a template
polynucleotide sequence comprising a sense nucleotide strand and an
antisense nucleotide strand, wherein the first primer anneals to
the sense strand and the second primer anneals to the antisense
strand and wherein at least one primer comprises a first tag; b)
extending the first primer and the second primer in the presence of
one or more nucleoside triphosphates wherein at least one of the
nucleoside triphosphates is modified, thereby producing at least
one forward extension product and at least one reverse extension
product, wherein the at least one forward extension product and the
at least one reverse extension product comprises a modified
nucleotide sequence having one or more phosphorothiolate linkages;
c) cleaving the one or more phosphorothiolate linkages in the at
least one forward extension product and the at least one reverse
extension product under conditions in which a plurality of
fragments are produced; d) identifying from among the fragments
produced in c), one or more fragments that comprise a first primer
and one or more fragments that comprise a second primer; and e)
separating the one or more fragments that comprise a first primer
from the one or more fragments that comprise a second primer,
thereby separating one or more forward extension products from one
or more reverse extension products.
96. The method of claim 95, wherein the one or more fragments that
comprise a first primer and the one or more fragments that comprise
a second primer are separated using the first tag.
97. A method of separating one or more forward extension products
from one or more reverse extension products comprising: a)
annealing a first primer and a second primer to a template
polynucleotide sequence comprising a sense nucleotide strand and an
antisense nucleotide strand, wherein the first primer anneals to
the sense strand and the second primer anneals to the antisense
strand and wherein the first primer and the second primer each
comprise a tag, wherein the tag on the first primer is distinct
from the tag on the second primer; b) extending the first primer
and the second primer in the presence of one or more nucleoside
triphosphates wherein at least one of the nucleoside triphosphates
is modified, thereby producing at least one forward extension
product and at least one reverse extension product, wherein the at
least one forward extension product and the at least one reverse
extension product comprises a modified nucleotide sequence having
one or more phosphorothiolate linkages; c) cleaving the one or more
phosphorothiolate linkages in the at least one forward extension
product and the at least one reverse extension product under
conditions in which a plurality of fragments are produced; d)
identifying from among the fragments produced in c), one or more
fragments that comprise a first primer and one or more fragments
that comprise a second primer; and e) separating the one or more
fragments that comprise a first primer from the one or more
fragments that comprise a second primer, thereby separating one or
more forward extension products from one or more reverse extension
products.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 11/476,423, filed Jun. 28, 2006, which claims the benefit of
U.S. Provisional Application No. 60/694,783, filed on Jun. 28,
2005. The entire teachings of the above applications are
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] Widespread efforts have been made in recent years to
determine the sequence of the human genome, as well as the genomes
of various other organisms. The advent of genomics has relied upon
accurate and efficient DNA sequencing techniques, and the ability
to determine the nucleotide sequence of a gene remains an essential
component of molecular genetic research. The widespread use of DNA
sequencing in biological research necessitates the development of
new DNA sequencing techniques that are simpler and more efficient
than traditional, commonly-used techniques.
[0004] Classical DNA sequencing techniques, such as the Sanger
chain termination method (Sanger, F., et al. Proc. Natl. Acad. Sci.
USA 74: 5463-5467 (1977); incorporated herein by reference) and the
Maxam and Gilbert chemical cleavage method (Maxam, A. M. and
Gilbert, W. Proc. Natl. Acad. Sci. USA 74: 560-564 (1977);
incorporated herein by reference), are somewhat cumbersome and
inefficient, as both of these approaches require researchers to
perform multiple reactions in order to derive a nucleotide
sequence. Attempts to simplify DNA sequencing by coupling a single
DNA amplification/synthesis reaction with sequence analysis (i.e.,
direct sequencing) have shown limited success, because these
techniques often result in DNA damage and/or degradation (Das et
al., Physiol Genomics 6: 57-80 (2001)), as well as low fidelity DNA
synthesis (Lin et al., Biochemistry 40: 8749-8755 (2001); Xia et
al., Proc Natl Acad Sci USA 99: 6597-6602 (2002); U.S. Pat. No.
5,939,292; U.S. Pat. No. 6,329,178; and U.S. Pat. No.
6,887,690).
[0005] Presently, there is a clear need to develop improved methods
of sequencing DNA. More specifically, there is a need to develop
reliable and efficient direct sequencing techniques that yield
accurate DNA sequence information.
SUMMARY OF THE INVENTION
[0006] The invention of the instant application provides new
methods of sequencing nucleic acids (e.g., DNA), as well as
improved methods for performing direct nucleic acid (e.g., DNA)
sequencing. The present invention is based, in part, on the
discovery that nucleic acid polymerases (e.g., DNA polymerases) are
capable of incorporating thiol-nucleoside triphosphates (e.g., 5'
thiol-nucleoside triphosphates, 3' thiol-nucleoside triphosphates)
into a growing polynucleotide strand to generate a polynucleotide
sequence that comprises at least one phosphorothiolate linkage.
Accordingly, the invention encompasses a method for producing a
modified polynucleotide sequence that comprises a (one or more)
phosphorothiolate linkage. The method comprises annealing at least
one primer to a template polynucleotide sequence and extending the
primer in the presence of one or more nucleoside triphosphates,
wherein at least one of the nucleoside triphosphates is modified,
such that a polynucleotide sequence that comprises a
phosphorothiolate linkage is produced.
[0007] In a particular embodiment, the invention provides a method
for producing a modified polynucleotide sequence that comprises a
3' phosphorothiolate linkage. The method comprises annealing at
least one primer to a template polynucleotide sequence and
extending said primer in the presence of one or more nucleoside
triphosphates, wherein at least one of the nucleoside triphosphates
in the mixture is a modified nucleoside triphosphate comprising the
general structure [I]:
##STR00001##
such that a polynucleotide sequence comprising at least one
modified nucleotide is produced. The modified polynucleotide
sequence comprises a 3' phosphorothiolate linkage, illustrated by
the general structure [II]:
##STR00002##
[0008] R.sub.1 in general structure [I] can be, for example,
hydrogen (--H), a substituted or non-substituted: alkyl, akenyl,
alkynyl, or aryl group, or R.sub.2, wherein R.sub.2 can be, for
example, --SH, or --SR.sub.3, and wherein R.sub.3 can be, for
example, a substituted or non-substituted: alkyl, akenyl, alkynyl,
or aryl group. In a particular embodiment, R.sub.1 is
--SCH.sub.3.
[0009] In another embodiment, the invention provides a method for
producing a modified polynucleotide sequence that comprises a 5'
phosphorothiolate linkage. The method comprises annealing at least
one primer to a template polynucleotide sequence and extending said
primer in the presence of one or more nucleoside triphosphates,
wherein at least one of the nucleoside triphosphates is a modified
nucleoside triphosphate comprising the general structure [III]:
##STR00003##
such that a modified polynucleotide sequence is produced. The
modified polynucleotide sequence comprises a 5' phosphorothiolate
linkage, illustrated by the general structure [IV]:
##STR00004##
[0010] The present invention also encompasses a method for
determining (e.g., sequencing) a polynucleotide sequence comprising
annealing a plurality of primers to a plurality of template
polynucleotide sequences and extending the plurality primers in the
presence of nucleoside triphosphates, wherein at least one of the
nucleoside triphosphates is modified, thereby producing a plurality
of extension products that comprise a modified nucleotide sequence
having one or more phosphorothiolate linkages. The
phosphorothiolate linkages in the extension products are cleaved
under conditions in which a plurality of fragments are produced.
The fragments that comprise the primer are identified, and the
nucleotide at the 3' end of each fragment that comprises the primer
is identified, such that the polynucleotide sequence can be
determined.
[0011] For example, the fragments that comprise the primer can be
identified (e.g., using a tag on the primer) and resolved (e.g., on
a solid support, such as a gel), and the sequence of the
polynucleotide can be determined (e.g., by detecting the length of
the fragment for which the nucleotide at its 3' end is known; by
detecting a tag present on the nucleotide at the 3' end, thereby
identifying the nucleotide at the 3' end), as will be understood by
one of skill in the art. The fragments attached to a primer can be
identified either directly or indirectly, using one or more of a
variety of previously-described techniques in the art, such as, for
example, by using an isolating means that binds to and/or
recognizes a tag on each primer. In the methods for determining a
polynucleotide sequence, the phosphorothiolate linkage can be
cleaved using, for example, Ag.sup.+, Hg.sup.++ and/or Cu.sup.++.
In particular embodiments, the methods comprise cleaving the
phosphorothiolate linkage using Ag.sup.+ ions at a pH of about 7.0
and at a temperature of about 22.degree. C. to about 37.degree.
C.
[0012] In a particular embodiment, the one or more modified
nucleoside triphosphates comprises a general structure [I]:
##STR00005##
such that the modified nucleotide sequences that are produced
comprise a general structure [II]:
##STR00006##
wherein the general structure [II] comprises at least one 3'
phosphorothiolate linkage.
[0013] R.sub.1 in general structure [I] can be, for example,
hydrogen (--H), a substituted or non-substituted: alkyl, akenyl,
alkynyl or aryl group, or R.sub.2, wherein R.sub.2 can be, for
example, --SH, or --SR.sub.3, and wherein R.sub.3 can be, for
example, a substituted or non-substituted: alkyl, akenyl, alkynyl,
or aryl group. In a particular embodiment, R.sub.1 is
--SCH.sub.3.
[0014] In another embodiment, the one or more nucleoside
triphosphates comprise the general structure [III]:
##STR00007##
such that the modified nucleotide sequences that are produced
comprise a general structure [IV]:
##STR00008##
wherein the general structure [IV] comprises at least one 5'
phosphorothiolate linkage.
[0015] In a further embodiment, the methods for determining a
polynucleotide sequence also comprise isolating the cleaved
fragments that comprise a primer, prior to identifying the length
of the polynucleotide, thereby identifying the nucleotide at the 3'
end of the fragments by virtue of the modified nucleotide used in
the extension reaction, or identifying the nucleotide at the 3' end
of the fragments by detecting a tag on the nucleotide. In a
particular embodiment, the fragments that comprise a primer are
isolated using an isolating means that specifically recognizes a
tag on the fragments (e.g., by binding of the isolating means to a
tag on each primer). As used herein, the term "isolated fragment"
refers to a preparation of fragments that is purified from, or
otherwise substantially free of, other components from the
extension and/or cleavage reactions, including, but not limited to,
cleavage fragments that are not attached to a primer, buffers,
salts, metal ions, unincorporated nucleotides, nucleic acid
templates and enzymes. The term "isolating means", as used herein,
refers to a means, such as a solid support, which comprises a
moiety that specifically recognizes, and binds to, a partner moiety
on a substance to be isolated.
[0016] The invention is also directed to methods for separating one
or more forward extension products from one or more reverse
extension products, comprising annealing a plurality of first
primers and a plurality of second primers to a plurality of
template polynucleotide sequences that comprise a sense nucleotide
strand and an antisense nucleotide strand, wherein the first primer
anneals to the sense strand and the second primer anneals to the
antisense strand and wherein at least one primer (e.g., the first
primer, the second primer) comprises a tag. The first and second
primers are extended in the presence of one or more nucleoside
triphosphates wherein at least one of the nucleoside triphosphates
is modified, thereby producing a plurality of extension products
that comprise a modified nucleotide sequence having one or more
phosphorothiolate linkages. In particular, as will be understood by
a person of skill in the art, extension of a first primer annealed
to a sense nucleotide strand produces a reverse extension product
and extension of a second primer annealed to an antisense
nucleotide strand produces a forward extension product. The
phosphorothiolate linkages in the modified nucleotide sequences are
cleaved under conditions in which a plurality of fragments of the
reverse extension product and a plurality of fragments of the
forward extension product are produced. The fragments of the
reverse extension product that comprise the first primer are then
separated from the fragments of the forward extension product that
comprise the second primer (e.g., using a tag on the first and/or
second primer), thereby separating forward and reverse extension
products. For example, one or more reverse extension products that
comprise a first primer, which comprises a biotin tag, are
separated from fragments of the forward extension product that
comprise the second primer, which do not comprise a biotin tag by
binding to, for example, streptavidin.
[0017] In a particular embodiment, the first primer and the second
primer each comprise a tag, wherein the tag on the first primer is
distinct from the tag on the second primer. Accordingly, the
fragments of the reverse extension product that comprise the first
primer can be separated from the fragments of the forward extension
product that comprise the second primer using the distinct tags on
the first and second primers.
[0018] The instant invention also encompasses kits that comprise
one or more nucleoside triphosphates, wherein at least one of the
nucleoside triphosphates is a modified thiol-nucleoside
triphosphate, and a nucleic acid polymerase.
[0019] In a particular embodiment, the at least one modified
thiol-nucleoside triphosphate comprises the general structure
[I]:
##STR00009##
[0020] R.sub.1 in general structure [I] can be, for example,
hydrogen (--H), a substituted or non-substituted: alkyl, akenyl,
alkynyl or aryl group, or R.sub.2, wherein R.sub.2 can be, for
example, --SH, or --SR.sub.3, and wherein R.sub.3 can be, for
example, a substituted or non-substituted: alkyl, akenyl, alkynyl
or aryl group. In a particular embodiment, R.sub.1 is
--SCH.sub.3.
[0021] In another embodiment, the at least one modified
thiol-nucleoside triphosphate comprises the general structure
[III]:
##STR00010##
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawings will be provided by the Office upon
request and payment of the necessary fee.
[0023] FIG. 1A-FIG. 1D are schematics illustrating the steps
involved in producing, cleaving and isolating primer extension
products, each of which contains a labeled 3' thiol nucleotide at
its 3' end.
[0024] FIG. 1A is a schematic depicting primer extension of a DNA
template using a mixture of unmodified nucleoside triphosphates
(pppN, also referred to herein as dNTP) and modified
3'-thiol-nucleoside triphosphates (pppAs, pppTs, pppGs, and pppCs,
also referred to herein as pppA-s, pppT-s, pppG-s, pppC-s, sdATP,
sdTTP, sdGTP, sdCTP, dAsTP, dTsTP, dGsTP, dCsTP, As, Ts, Gs and
Cs). Each of the four 3'-thiol-nucleoside triphosphates is labeled
with a distinct fluorophore, indicated by a colored star. The DNA
primer (red) is attached to an affinity probe or tag, shown as a
gray pentagonal structure at the 5' end of the primer.
[0025] FIG. 1B is a schematic depicting a DNA strand (top) which
has incorporated multiple labeled 3'-thiol nucleotides (As, Ts, Gs,
Cs) following primer extension by DNA polymerase on a DNA template
(bottom strand).
[0026] FIG. 1C is a schematic depicting the selective cleavage of
DNA strands containing 3'-thiol nucleotides in the presence of
AgNO.sub.3. Cleavage occurs at the 3' end of each modified 3'-thiol
nucleotide and results in the generation of several labeled
cleavage products.
[0027] FIG. 1D is a schematic depicting the purification of several
different 5' extension products following AgNO.sub.3-induced
cleavage of DNA strands containing 3'-thiol nucleotides. DNA
cleavage products are isolated using a solid support (red circle),
which contains a molecule (blue cross) that binds to the affinity
tag on the primer (gray pentagon). Therefore, only the 5'-most DNA
fragments, which comprise the primer sequence, are recovered. All
other labeled fragments are washed away.
[0028] FIG. 2A-FIG. 2C are schematics illustrating the steps
involved in producing, cleaving and separating forward and reverse
primer extension products, each of which contains a labeled 3'
thiol nucleotide at its 3' end.
[0029] FIG. 2A is a schematic depicting bidirectional PCR
amplification of DNA using a mixture of unmodified nucleoside
triphosphates (pppN) and modified 3'-thiol-nucleoside triphosphates
(pppA-s, pppT-s, pppG-s, and pppC-s). The four 3'-thiol-nucleoside
triphosphates are differentially labeled with distinct
fluorophores, indicated by stars of different colors. The forward
DNA primer (red) and reverse DNA primer (green) are attached to
different affinity probes, shown as a gray pentagon (forward
primer) or purple hexagon (reverse primer). The DNA duplex at the
bottom of the Figure has incorporated multiple labeled 3'-thiol
nucleotides (As, Ts, Gs, Cs) following primer extension by DNA
polymerase.
[0030] FIG. 2B is a schematic depicting the selective cleavage of
DNA strands containing 3'-thiol nucleotides in the presence of
AgNO.sub.3. Cleavage occurs at the 3' end of each modified 3'-thiol
nucleotide and results in the generation of several labeled
cleavage products.
[0031] FIG. 2C is a schematic depicting the purification of 5'
extension products following AgNO.sub.3-induced cleavage of
forward- and reverse-primed DNA strands containing 3'-thiol
nucleotides. DNA cleavage products generated from the
forward-primed strand are isolated using a solid support (red
circle), which contains a molecule (blue cross) that binds to the
affinity tag on the primer (gray pentagon). DNA cleavage products
generated from the reverse-primed strand are isolated using a solid
support (red circle), which contains a molecule (blue sun) that
binds to the affinity tag on the reverse primer (purple hexagon).
Therefore, the 5'-most DNA fragments from both the forward and
reverse strands can be recovered separately and analyzed to
determine sequence.
[0032] FIG. 3A-FIG. 3E demonstrate the incorporation of
3'-deoxy-dithiomethyl thymidine (dTsTP) into a growing DNA strand
by a DNA polymerase.
[0033] FIG. 3A depicts the chemical structure of
3'-deoxy-dithiomethyl thymidine (dTsTP).
[0034] FIG. 3B is a representation showing a dual biotin-labeled
DNA template attached to a magnetic bead. The template is
hybridized to a primer, which is labeled with Cy5. Five adenine (A)
nucleotides are located downstream of the portion of the template
sequence that is complementary to the primer sequence.
[0035] FIG. 3C depicts the fluorescence profile generated by the
Cy5-labeled primer, shown in FIG. 3B. Fluorescence intensity is
indicated along the Y-axis, while the size of the labeled product
is indicated on the X-axis, where units 0, 1, 2, 3, 4 and 5,
indicate that the primer has been extended by 0, 1, 2, 3, 4, or 5
additional nucleotides, respectively. Fluorescence resulting from
the label on the primer is indicated by a blue peak. Orange peaks
represent the fluorescent profile of size standards.
[0036] FIG. 3D depicts the fluorescence profile of the DNA
extension products generated when DNA synthesis is conducted in the
presence of 3'-deoxy-dithiomethyl thymidine (dTTP) at a 5 .mu.M
concentration. Fluorescence intensity is indicated along the
Y-axis, while the size of the labeled product is indicated on the
X-axis, where units 0, 1, 2, 3, 4 and 5, indicate that the primer
has incorporated 0, 1, 2, 3, 4, or 5 additional nucleotides,
respectively. Fluorescence resulting from the label on the primer
is indicated by a blue peak. The products from this reaction
contain anywhere from 0 to 5 modified thymidine residues, with the
majority of products containing either 0 or 1 modified nucleotides.
Orange peaks represent the fluorescent profile of size
standards.
[0037] FIG. 3E depicts the fluorescence profile of the DNA
extension products generated when DNA synthesis is conducted in the
presence of 1.0 mM 3'-deoxy-dithiomethyl thymidine (dTsTP).
Fluorescence intensity is indicated along the Y-axis, while the
size of the labeled product is indicated on the X-axis, where units
0, 1, 2, 3, 4 and 5 indicate that the primer has incorporated 0, 1,
2, 3, 4, or 5 additional nucleotides, respectively. Fluorescence
resulting from the label on the primer is indicated by a blue peak.
Nearly all of the products from this reaction have incorporated 5
modified thymidine nucleotides. Orange peaks represent the
fluorescent profile of size standards.
[0038] FIG. 4A-FIG. 4E depict incorporation of
3'-deoxy-dithiomethyl thymidine
[0039] (dTsTP) nucleotides into primer extension products, followed
by subsequent cleavage of the nucleotides in the presence of
silver.
[0040] FIG. 4A depicts the fluorescence profile (blue) of uncleaved
primer extension products generated in the presence of 50 .mu.M
3'-deoxy-dithiomethyl thymidine (dTsTP) nucleoside
triphosphates.
[0041] FIG. 4B depicts the fluorescence profile of cleaved primer
extension products (blue peaks), which were generated in the
presence of 50 .mu.M 3'-deoxy-dithiomethyl thymidine (dTsTP)
nucleoside triphosphates, following treatment with AgNO.sub.3.
Orange peaks represent the fluorescent profile of size
standards.
[0042] FIG. 4C depicts the fluorescence profile (blue) of uncleaved
primer extension products generated in the presence of 500 .mu.M
3'-deoxy-dithiomethyl thymidine (dTTP) nucleoside
triphosphates.
[0043] FIG. 4D depicts the fluorescence profile of cleaved primer
extension products (blue peaks), which were generated in the
presence of 500 .mu.M 3'-deoxy-dithiomethyl thymidine (dTsTP)
nucleoside triphosphates, following treatment with AgNO.sub.3.
Orange peaks represent the fluorescent profile of size
standards.
[0044] FIG. 4E depicts the fluorescence profile generated by the
labeled-primer alone. Fluorescence resulting from the label on the
primer is indicated by a blue peak. Orange peaks represent the
fluorescent profile of size standards.
[0045] FIG. 5 depicts the chemical structure of a polynucleotide
that comprises a 3' phosphorothiolate linkage.
DETAILED DESCRIPTION OF THE INVENTION
[0046] The present invention is based, in part, on Applicants'
discovery that nucleic acid polymerases can incorporate
thiol-nucleoside triphosphates into a growing polynucleotide strand
to generate a polynucleotide that comprises one or more
phosphorothiolate linkages. Thus, a polynucleotide comprising a
phosphorothiolate linkage can be enzymatically synthesized using a
polymerase, rather than chemically synthesized. Therefore, the
present invention provides a method for producing a modified
polynucleotide sequence, wherein the modified polynucleotide
sequence comprises a (one or more) scissile internucleoside
linkage. As used herein, a "scissile internucleoside linkage" or
"scissile linkage" refers to an internucleoside linkage that can be
cleaved under conditions that will not substantially cleave
phosphodiester bonds. As described herein, nucleic acid polymerases
can be used to generate a polynucleotide sequence that comprises
one or more scissile internucleoside linkage(s). In a particular
embodiment, the scissile internucleoside linkage is a (one or more)
phosphorothiolate linkage.
[0047] Accordingly, the present invention provides methods for
producing a modified polynucleotide sequence. As used herein, the
term "modified polynucleotide sequence" refers to a polynucleotide
sequence that comprises one or more phosphorothiolate linkages. The
method comprises annealing at least one primer to a template
polynucleotide sequence and extending the primer in the presence of
one or more nucleoside triphosphates, wherein at least one
nucleoside triphosphate is modified. As used herein, the phrase
"modified nucleoside triphosphate" or "nucleoside triphosphate that
is modified" refers to a thiol-nucleoside triphosphate that can be
incorporated into a nascent polynucleotide by a nucleic acid
polymerase, thereby producing a modified polynucleotide sequence
that comprises one or more phosphorothiolate linkages. The term
"phosphorothiolate linkage", as used herein, refers to a covalent
bond between a sulfur and a phosphorus atom. As used herein, the
phrase "polynucleotide sequence comprising a phosphorothiolate
linkage" refers to a polynucleotide sequence that comprises a
sulfur-phosphorus covalent bond. In particular, a phosphorothiolate
linkage results when a sulfur atom replaces one of the bridging
oxygen atoms in a phosphodiester bond.
[0048] In a particular embodiment, the polynucleotide sequence
comprises a 3' phosphorothiolate linkage and is represented by the
general structure [II]:
##STR00011##
[0049] A modified polynucleotide sequence comprising a 3'
phosphorothiolate linkage can be generated by incorporating one or
more 3' thiol-nucleoside triphosphates into a growing
polynucleotide strand. As used herein, a "3' thiol-nucleoside
triphosphate" refers to a molecule having the general structure
[I]:
##STR00012## [0050] wherein R.sub.1 is hydrogen, a substituted or
non-substituted: alkyl, akenyl, alkynyl or aryl group, or R.sub.2;
[0051] wherein R.sub.2 is --SH, or --SR.sub.3; and [0052] wherein
R.sub.3 is a substituted or non-substituted: alkyl, akenyl, alkynyl
or aryl group.
[0053] As used herein, "alkyl", "alkenyl" and "alkynyl" means a
group that is saturated or unsaturated, straight-chain, branched,
or cyclic, and is derived from a hydrocarbon radical derived by the
removal of one hydrogen atom from a single carbon atom of a parent
alkane, alkene, or alkyne, respectively. Typical alkyl (e.g.,
alkyl, cycloalkyl, heteroalkyl), alkenyl (e.g., alkenyl,
cycloalkenyl, heteroalkenyl) and alkynyl (e.g., alkynyl,
cycloalkynyl, heteroalkynyl) groups, consist of 1 to 12 saturated
and/or unsaturated carbons, including, but are not limited to,
methyl, ethyl, propyl, butyl, and the like.
[0054] As used herein, "aryl" means a monovalent aromatic
hydrocarbon radical of 6 to 20 carbon atoms derived by the removal
of one hydrogen atom from a single carbon atom of a parent aromatic
ring system. Typical aryl groups, which include, for example,
cycloaryl and heteroaryl groups, can be substituted or
non-substituted and include, but are not limited to, radicals
derived from benzene, substituted benzene, naphthalene, anthracene,
biphenyl, and the like.
[0055] In another embodiment, the polynucleotide sequence comprises
a 5' phosphorothiolate linkage and is represented by the general
structure [IV]:
##STR00013##
[0056] A modified polynucleotide sequence comprising a 5'
phosphorothiolate linkage can be generated by incorporating one or
more 5' thiol-nucleoside triphosphates into a growing
polynucleotide strand. As used herein, a "5' thiol-nucleoside
triphosphate" refers to a molecule having the general structure
[III]:
##STR00014##
[0057] A "nucleoside" comprises a nitrogenous base linked to a
sugar molecule. As used herein, the term includes natural
nucleosides in their 2'-deoxy and 2'-hydroxyl forms as described in
Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman, San
Francisco, 1992) and nucleoside analogs. For example, natural
nucleosides include adenosine, thymidine, guanosine, cytidine,
uridine, inosine, deoxyadenosine, deoxythymidine, deoxyguanosine,
and deoxycytidine. Nucleoside "analogs" refers to synthetic
nucleosides having modified base moieties and/or modified sugar
moieties, e.g., as described generally by Scheit, Nucleotide
Analogs (John Wiley, New York, 1980). Such analogs include
synthetic nucleosides designed to enhance binding properties,
reduce degeneracy, increase specificity, and the like. Nucleoside
analogs include 2-aminoadenosine, 2-thiothymidine,
pyrrolo-pyrimidine, 3-methyl adenosine, C5-propynylcytidine,
C5-propynyluridine, C5-bromouridine, C5-fluorouridine,
C5-iodouridine, C5-methylcytidine, 7-deazaadenosine,
7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine,
O(6)-methylguanine, 2-thiocytidine, etc. Nucleoside analogs may
comprise any of the universal bases mentioned herein.
[0058] As used herein, the term "base" refers to the heterocyclic
nitrogenous base of a nucleotide or nucleotide analog (e.g., a
purine, a pyrimidine, a 7-deazapurine). Suitable bases for use in
the methods of the invention include, but are not limited to,
adenine, cytosine, guanine, thymine, uracil, hypoxanthine and
7-deaza-guanine. These and other suitable bases will permit a
nucleotide bearing the base to be enzymatically-incorporated into a
polynucleotide chain or sequence. The base will also be capable of
forming a base pair involving hydrogen bonding with a base on
another nucleotide or nucleotide analog. The base pair can be
either a conventional (standard) Watson-Crick base pair or a
non-conventional (non-standard) non-Watson-Crick base pair, for
example, a Hoogstein base pair or bidentate base pair.
[0059] As used herein, "Watson-Crick base pair" refers to a pair of
hydrogen-bonded bases on opposite antiparallel strands of a nucleic
acid. The rules of base pairing, which were first elaborated by
Watson and Crick, are well known to those of skill in the art. For
example, these rules require that adenine (A) pairs with thymine
(T) or uracil (U), and guanine (G) pairs with cytosine (C), with
the complementary strands anti-parallel to one another. As used
herein, the term "Watson-Crick base pair" encompasses not only the
standard AT, AU or GC base pairs, but also base pairs formed
between non-standard or modified bases of nucleotide analogs
capable of hydrogen bonding to a standard base or to another
complementary non-standard base. One example of such non-standard
Watson-Crick base pairing is the base pairing which involves the
nucleotide analog inosine, wherein its hypoxanthine base forms two
hydrogen bonds with adenine, cytosine or uracil of other
nucleotides.
[0060] As used herein, the term "polynucleotide sequence" refers to
a nucleic acid molecule (e.g., DNA, RNA) that is produced by the
incorporation of two or more nucleoside triphosphates into a single
molecule via one or more covalent linkages (e.g., a phosphodiester
bond, a phosphorothiolate linkage). A "template polynucleotide
sequence" can be any nucleotide sequence for which it is desirable
to produce or to obtain sequence information using the methods
described herein. The template polynucleotide sequence may be a
polynucleotide sequence (e.g., oligonucleotide sequence) and may be
single-stranded or double-stranded. A template that is initially
provided in double-stranded form can be treated to separate the two
strands (e.g., the DNA will be denatured). The template
polynucleotide also may be naturally-occurring, isolated or
synthetic. Examples of suitable templates include, but are not
limited to, genomic DNA, mitochondrial DNA, complementary DNA
(cDNA), a PCR product and other amplified nucleotides. RNA may also
be used as a template. For example, RNA can be reverse transcribed
to yield cDNA, using methods known in the art such as RT-PCR. The
template polynucleotide sequence may be used in any convenient
form, according to techniques known in the art (e.g., isolated,
cloned, amplified), and may be prepared for the sequencing
reaction, as desired, according to techniques known in the art. In
a particular embodiment, the template polynucleotide sequence
comprises DNA. In a further embodiment, the template polynucleotide
sequence comprises a sense DNA strand and an antisense DNA strand,
wherein at least one primer is annealed to at least one strand
(e.g., sense strand, antisense strand) or to both sense and
antisense strands.
[0061] Template polynucleotides can be obtained from any of a
variety of sources. For example, DNA may be isolated from a sample,
which may be obtained or derived from a subject. The word "sample"
is used in a broad sense to denote any source of a template on
which sequence determination is to be performed. The source of a
sample may be of any viral, prokaryotic, archaebacterial, or
eukaryotic species. The sample may be blood or another bodily fluid
containing cells; sperm; and a biopsy (e.g., tissue) sample, among
others.
[0062] As used herein, the term "primer" refers to an
oligonucleotide, which is complementary to the template
polynucleotide sequence and is capable of acting as a point for the
initiation of synthesis of a primer extension product. In one
embodiment, the primer is complementary to the sense strand of a
polynucleotide sequence and acts as a point of initiation for
synthesis of a forward extension product. In another embodiment,
the primer is complementary to the antisense strand of a
polynucleotide sequence and acts as a point of initiation for
synthesis of a reverse extension product.
[0063] The primer may occur naturally, as in a purified restriction
digest, or be produced synthetically. The appropriate length of a
primer depends on the intended use of the primer, but typically
ranges from about 5 to about 200; from about 5 to about 100; from
about 5 to about 75; from about 5 to about 50; from about 10 to
about 35; from about 18 to about 22 nucleotides. A primer need not
reflect the exact sequence of the template but must be sufficiently
complementary to hybridize with a template for primer elongation to
occur, i.e., the primer is sufficiently complementary to the
template polynucleotide sequence such that the primer will anneal
to the template under conditions that permit primer extension. As
used herein, the phrase "conditions that permit primer extension"
refers to those conditions, e.g., salt concentration (metallic and
non-metallic salts), pH, temperature, and necessary cofactor
concentration, among others, under which a given polymerase enzyme
catalyzes the extension of an annealed primer. Conditions for the
primer extension activity of a wide range of polymerase enzymes are
known in the art. As one example, conditions permitting the
extension of a nucleic acid primer by Taq polymerase include the
following (for any given enzyme, there can and often will be more
than one set of such conditions): reactions are conducted in a
buffer containing 50 mM KCl, 10 mM Tris (pH 8.3), 4 mM MgCl.sub.2,
(200 .mu.M of one or more dNTPs and/or a chain terminator may be
included, depending upon the type of primer extension or sequencing
being performed); reactions are performed at 72.degree. C.
[0064] It will be clear to persons skilled in the art that the size
of the primer and the stability of hybridization will be dependent
to some degree on the ratio of A-T to C-G base pairings, since more
hydrogen bonding is available in a C-G pairing. Also, the skilled
person will consider the degree of homology between the extension
primer to other parts of the amplified sequence and choose the
degree of stringency accordingly. Guidance for such routine
experimentation can be found in the literature, for example,
Molecular Cloning: A Laboratory Manual by Sambrook, J., Fritsch E.
F. and Maniatis, T. (1989).
[0065] In the methods of the present invention, tags can be used to
facilitate the production and/or sequencing of polypeptide
sequences. As used herein, a "tag" or "label" are used
interchangeably to refer to any moiety that is capable of being
specifically detected (e.g., by binding to an isolating means
(e.g., a partner moiety)), either directly or indirectly, and
therefore, can be used to identify and/or isolate a polynucleotide
sequence that comprises the tag. Suitable tags for use in the
methods of the present invention can be present on a primer, a
modified polynucleotide sequence, a template, or on one or more
nucleoside triphosphates (e.g., non-modified or standard nucleoside
triphosphates, modified nucleoside triphosphates) and include,
among others, affinity tags (e.g., biotin, avidin, streptavidin),
haptens, ligands, peptides, nucleic acids, fluorophores,
chromophores, and epitope tags that are recognized by an antibody
(e.g., digoxigenin (DIG), hemagglutinin (HA), myc, FLAG) (Andrus,
A. "Chemical methods for 5' non-isotopic labelling of PCR probes
and primers" (1995) in PCR 2: A Practical Approach, Oxford
University Press, Oxford, pp. 39-54). Other suitable tags include,
but are not limited to, chromophores, fluorophores, haptens,
radionuclides (e.g., .sup.32P, .sup.33P, .sup.35S), fluorescence
quenchers, enzymes, enzyme substrates, affinity tags (e.g., biotin,
avidin, streptavidin, etc.), mass tags, electrophoretic tags and
epitope tags that are recognized by an antibody. In certain
embodiments, the label is present on the 5 carbon position of a
pyrimidine base or on the 3 carbon deaza position of a purine base.
In a particular embodiment, the primer comprises at least one tag
or label.
[0066] In a further embodiment, the primer comprises at least one
affinity tag. As defined herein, an "affinity tag" refers to a
moiety that can be attached to a nucleoside or nucleoside analog,
which can be specifically-bound by a partner moiety. The
interaction of the affinity tag and its partner permits the
isolation (i.e., specific capture and purification) of molecules
bearing the affinity tag. Suitable examples include, but are not
limited to, biotin or iminobiotin and avidin or streptavidin. A
sub-class of affinity tag is the "epitope tag," which refers to a
tag that is recognized and specifically bound by an antibody or an
antigen-binding fragment thereof. Examples of epitope tags include
the Myc tag, recognized by monoclonal anti-Myc antibodies; FLAG.TM.
tag, recognized by anti-FLAG.TM. antibodies; and digoxigenin,
recognized by anti-digoxigenin antibodies. In one embodiment, the
primer comprises a biotin tag. In another embodiment, the primer
comprises a digoxigenin tag.
[0067] In another embodiment, the primer comprises a tag (e.g., a
label) that is a fluorophore. Suitable fluorophores can be provided
as fluorescent dyes, including, but not limited to Alexa Fluor dyes
(Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor
546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor
660 and Alexa Fluor 680), AMCA, AMCA-S, BODIPY dyes (BODIPY FL,
BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530/550, BODIPY 558/568,
BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650,
BODIPY 650/665), CAL dyes, Carboxyrhodamine 6G, carboxy-X-rhodamine
(ROX), Cascade Blue, Cascade Yellow, Cyanine dyes (Cy3, Cy5, Cy3.5,
Cy5.5), Dansyl, Dapoxyl, Dialkylaminocoumarin,
4',5'-Dichloro-2',7'-dimethoxy-fluorescein, DM-NERF, Eosin,
Erythrosin, Fluorescein, Carboxy-fluorescein (FAM),
Hydroxycoumarin, IRDyes (IRD40, IRD 700, IRD 800), JOE, Lissamine
rhodamine B, Marina Blue, Methoxycoumarin, Naphthofluorescein,
Oregon Green 488, Oregon Green 500, Oregon Green 514, Oyster dyes,
Pacific Blue, PyMPO, Pyrene, Rhodamine 6G, Rhodamine Green,
Rhodamine Red, Rhodol Green,
2',4',5',7'-Tetra-bromosulfone-fluorescein, Tetramethyl-rhodamine
(TMR), Carboxytetramethylrhodamine (TAMRA), Texas Red, and Texas
Red-X. In other embodiments, the label is a mass tag or an
electrophoretic tag.
[0068] In addition to the various detectable moieties mentioned
above, the present invention also comprehends the use of tags or
labels, such as spectrally resolvable quantum dots, metal
nanoparticles or nanoclusters, etc., which may either be directly
attached to an oligonucleotide primer or may be embedded in or
associated with a polymeric matrix which is then attached to the
primer. As mentioned above, detectable moieties need not themselves
be directly detectable. For example, they may act on a substrate
which is detected, or they may require modification to become
detectable.
[0069] As described herein, the primers and nucleotide
triphosphates can comprise one or more tags or labels (e.g., a
first tag, second tag, third tag, fourth tag, fifth tag, sixth tag,
seventh tag, eighth tag). The various tags used in the methods can
be the same or different (distinct). In a particular embodiment,
when more than one tag is used in the method, the tags (e.g.,
first, second) are different (distinct), such that the primers or
nucleoside triphosphates can be separated from one another using
the distinct tag or tags.
[0070] As will be appreciated by one of ordinary skill in the art,
references to templates, primers, etc., generally mean populations
or pools of nucleic acid molecules that are substantially identical
within a relevant region rather than single molecules. Thus, for
example, a "template" generally means a plurality of substantially
identical template molecules.
[0071] According to the methods of the invention, primer extension
is carried out in the presence of one or more nucleoside
triphosphates. In one embodiment, the nucleoside triphosphates are
a mixture of standard deoxynucleo side triphosphates (also referred
to herein as dNTPs (e.g., dATP, dCTP, dGTP, dTTP, dUTP) and pppNs
(e.g., pppA, pppC, pppG, pppT)) and modified thiol-deoxynucleoside
triphosphates (e.g., 3' and 5' thiol-nucleoside triphosphates (also
referred to herein as sdNTPs (e.g., sdATP, sdCTP, sdGTP, sdTTP,
sdUTP) and dNsTPs (e.g., dAsTP, dCsTP, dGsTP, dTsTP, dUsTP))). In a
particular embodiment, the mixture of nucleoside triphosphates
comprises four standard deoxynucleotide triphosphates and one or
more thiol-nucleoside triphosphates. Suitable thiol-nucleoside
triphosphates include, but are not limited to, those which comprise
a base that is either adenine, cytosine, thymine, guanine or
uracil. In particular embodiments of the methods of the invention,
each standard dNTP is present at a higher concentration than its
corresponding modified dNTP (e.g, dCTP is present in the mixture at
a higher concentration than the modified thiol-dCTP in the same
mixture) so that the modified dNTP will be incorporated less
frequently than the standard dNTP, as will be understood by the
person of skill in the art.
[0072] The modified thiol-nucleoside triphosphates in the mixture
of nucleoside triphosphates can be unlabeled or can comprise one or
more labels, as described herein.
[0073] In a particular embodiment, the mixture of nucleotides
comprises four labeled thiol-nucleoside triphosphates (e.g., sdCTP,
sdTTP, sdATP, and sdGTP). In a particular embodiment, each of the
four thiol-nucleoside triphosphates comprises a distinct label,
which is not present on any other nucleotide in the mixture. Thus,
in certain embodiments, the label is present on the 5 carbon
position of a pyrimidine base or on the 7 carbon deaza position of
a purine base. A person of skill in the art will recognize that a
polynucleotide sequence can be determined, according to the methods
of the present invention, by performing a single reaction that
utilizes a mixture of four conventional nucleoside triphosphates
and four modified 3' thiol-nucleoside triphosphates, wherein each
of the four modified 3' thiol-nucleoside triphosphates comprises a
distinct label that is not present on any other nucleotide in the
mixture.
[0074] Extension of a primer (e.g., DNA synthesis) can be
accomplished using a nucleic acid polymerase which is capable of
enzymatically-incorporating both standard (dNTPs) and modified
thiol deoxynucleotides (sdNTPs) into a growing nucleic acid strand.
As used herein, the phrase "nucleic acid polymerase enzyme" refers
to an enzyme (e.g., naturally-occurring, recombinant, synthetic)
that catalyzes the template-dependent polymerization of nucleoside
triphosphates to form primer extension products that are
complementary to one of the nucleic acid strands of the template
nucleic acid sequence. Numerous nucleic acid polymerases are known
in the art and are commercially available. Nucleic acid polymerases
that are thermostable, i.e., they retain function after being
subjected to temperatures sufficient to denature annealed strands
of complementary nucleic acids, are particularly useful for the
methods of the present invention.
[0075] Suitable polymerases for the methods of the present
invention include any polymerase known in the art to be useful for
recognizing and incorporating standard deoxynucleotides. Examples
of such polymerases are disclosed in Table 1 of U.S. Pat. No.
6,858,393, the contents of which are incorporated herein by
reference. Many polymerases are known by those of skill in the art
to possess a proof-reading, or exonucleolytic activity, which can
result in digestion of 3' ends that are available for primer
extension. In order to avoid this potential problem, it may be
desirable to use polymerase enzyme which lack this activity (e.g.,
exonuclease-deficient polymerases, referred to herein as
exo-polymerases). Such polymerases are well known to those of skill
in the art and include, for example, Klenow fragment of E. Coli DNA
polymerase I, Sequenase, exo-Thermus aquaticus (Taq) DNA polymerase
and exo-Bacillus stearothermophilus (Bst) DNA polymerase. In a
particular embodiment, incorporation of modified thiol
deoxynucleotides (sdNTPs) into DNA is accomplished using a DNA
amplification reaction, such as PCR. Therefore, especially suitable
polymerases for the methods of the present invention include those
that are stable and function at high temperatures (i.e.,
thermostable polymerases useful in PCR thermal cycling). Examples
of such polymerases include, but are not limited to, Thermus
aquaticus (Taq) DNA polymerase, TaqFS DNA polymerase,
thermosequenase, Therminator DNA polymerase, Tth DNA polymerase,
Pfu DNA polymerase and Vent (exo-)DNA polymerase. In another
embodiment, incorporation of modified thiol-nucleoside
triphosphates into RNA is accomplished using an RNA polymerase.
Examples of RNA polymerases include, but are not limited to, E.
coli RNA polymerase, T7 RNA polymerase and T3 RNA polymerases.
[0076] The present invention also encompasses a method for
determining all or a portion of a polynucleotide sequence
comprising: annealing a plurality of primers to a plurality of
template polynucleotide sequences; and extending the plurality of
the primers in the presence of one or more nucleoside triphosphates
wherein at least one of the nucleoside triphosphates is modified,
thereby producing a plurality of extension products that comprise a
modified nucleotide sequence having one or more phosphorothiolate
linkages. The phosphorothiolate linkages in the extension products
are cleaved under conditions in which a plurality of fragments are
produced; and the fragments of the of the extension products that
comprise a primer are then identified (e.g., using tags, labels,
solid supports and other means described herein), and the
nucleotide at the 3' end of the fragments is subsequently
identified, such that the polynucleotide sequence can be
determined.
[0077] In one embodiment, the at least one modified nucleotide
comprises a general structure [I]:
##STR00015##
such that the modified nucleotide sequences that are produced
comprise a general structure [II]:
##STR00016##
wherein the general structure [II] comprises at least one 3'
phosphorothiolate linkage.
[0078] In another embodiment, the at least one modified nucleotide
comprises a general structure [III]:
##STR00017##
such that the modified nucleotide sequences that are produced
comprise a general structure [IV]:
##STR00018##
wherein the general structure [IV] comprises at least one 5'
phosphorothiolate linkage.
[0079] One of skill in the art will recognize that, in order to
determine the sequence of a polynucleotide using the methods of the
present invention, a ladder of fragments in which each fragment
comprises a primer can be produced by cleavage of a plurality of
extension products. One of skill in the art will appreciate that
four separate extension reactions can be performed in which a
different modified dNTP (e.g., thiol-nucleoside triphosphate) is
used in each of the four reactions. Upon cleaving the extension
products to produce fragments of various sizes and resolving the
fragments (e.g., on a gel), four distinct ladders are produced,
wherein each fragment in an individual ladder has at its 3' end the
same modified nucleotide that was used for the extension reaction.
By determining the size of each fragment that has a known
nucleotide at its 3' end and comparing the size of the fragments in
the four individual ladders, the sequence of the extension product
can be determined. Once the sequence of the extension product is
known, the sequence of the template polynucleotide, which is the
reverse complement of the sequence of the extension product, can be
determined.
[0080] Alternatively, one of skill in the art will recognize that a
single extension reaction comprising 4 modified dNTPs (e.g.,
thiol-nucleoside triphosphates), wherein each reaction comprises
four distinct labels corresponding to the four bases (e.g., a
distinct label on more than one modified nucleoside triphosphate,
more than one dNTP, more than one primer, more than one etc., and
combinations thereof), wherein each distinct label can be used to
generate a single sequence ladder representing the different bases.
Thus, the ladder comprises fragments that represent the full-length
extension product and various 3' truncations thereof. Preferably,
all possible 3' truncations of the extension product are produced,
such that the complete sequence of the polynucleotide can be
determined. By resolving the ladder of fragments (e.g., on a gel),
identifying the nucleotide at the 3' end of each fragment (e.g.,
using the distinct label or tag on the nucleotide, such as a
fluorophore) and reading the sequence ladder (e.g., on a gel),
beginning with the nucleotide at the 3' end of the smallest
fragment and ending with the nucleotide at the 3' end of the
largest fragment, the sequence of the polynucleotide can be
determined. Once the sequence of the extension product is known,
the sequence of the template polynucleotide, which is the reverse
complement of the sequence of the extension product, can be
determined.
[0081] As used herein, the phrase "determining a polynucleotide
sequence", "sequencing", and like terms, in reference to
polynucleotides, includes determination of partial as well as full
sequence information of the polynucleotide. That is, the term
includes sequence comparisons, fingerprinting, and like levels of
information about a target polynucleotide, as well as the express
identification and ordering of each nucleoside of the target
polynucleotide within a region of interest. In certain embodiments
of the invention "determining a polynucleotide sequence" comprises
identifying a single nucleotide, while in other embodiments more
than one nucleotide is identified. In certain embodiments of the
invention, sequence information that is insufficient by itself to
identify any nucleotide in a single cycle is gathered.
Identification of nucleosides, nucleotides, and/or bases are
considered equivalent herein. It is noted that performing sequence
determination on a polynucleotide typically yields equivalent
information regarding the sequence of a perfectly complementary
(100% complementary) polynucleotide, and thus, is equivalent to
sequence determination performed directly on a perfectly
complementary polynucleotide. The methods described herein allow
partial determination of a sequence, e.g., the identification of
individual nucleotides spaced apart from one another in a template.
In certain embodiments of the invention, in order to gather more
complete information, a plurality of reactions is performed.
[0082] In one embodiment of the invention, the identity of one or
more nucleotides is determined using the methods described herein,
for the purpose of detecting a polymorphism. The term
"polymorphism" is given its ordinary meaning in the art and refers
to a difference in a nucleotide sequence (e.g., genomic sequence)
among individuals (e.g., of the same species). In a particular
embodiment, the polymorphism is a "single nucleotide polymorphism"
(SNP), which refers to a polymorphism at a single position. In
other embodiments of the invention, the methods for determining a
polynucleotide sequence are employed to determine the identity of
multiple nucleotides (e.g., more than one) in a template
polynucleotide sequence.
[0083] In particular embodiments, a plurality of extension products
that comprises a modified nucleotide sequence having one or more
phosphorothiolate linkages are produced using polymerase chain
reaction (PCR). Methods for performing PCR are well known in the
art and are described, for example, in U.S. Pat. Nos. 4,683,195,
4,683,202, and 4,965,188, and in Dieffenbach, C. and Dveksler, G S,
PCR Primer: A Laboratory Manual, 2.sup.nd ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, 2003.
[0084] The methods for determining a polynucleotide sequence
comprise cleaving phosphorothiolate linkages in the extension
products under conditions in which a plurality of fragments of the
modified nucleotide sequence attached to a plurality of primers are
produced. One of skill in the art will recognize that such
conditions can also result in the separation of the cleaved
fragments from the template polynucleotide sequence to which they
are annealed, thereby producing a single-stranded polynucleotide. A
phosphorothiolate linkage (3' or 5') in a polynucleotide sequence
can be efficiently cleaved according to methods known in the art
(Vyle, J. S., et al., Biochemistry 31: 3012-3018 (1992);
Sontheimer, E. J., et al., Methods 18: 29-37 (1999); Mag, M., et
al., Nucleic Acids Res., 19(7):1437-1441 (1991)). For example,
cleavage of a phosphorothiolate linkage can be accomplished
chemically, by exposing (e.g., contacting) the polynucleotide
sequence to certain metal agents. The metal can be, for example,
silver (Ag), mercury (Hg), copper (Cu), manganese (Mn), zinc (Zn)
or cadmium (Cd), among others. Water-soluble salts that provide
Ag.sup.+, Hg.sup.++, Cu.sup.++, Mn.sup.++, Zn.sup.+ or Cd.sup.+
anions (salts that provide ions of other oxidation states can also
be used) are particularly useful. Iodide (I.sub.2) can also be
used. Silver-containing salts such as silver nitrate (AgNO.sub.3),
or other salts that provide Ag.sup.+ ions, are particularly useful
in the methods of the present invention.
[0085] Suitable conditions for cleaving a phosphorothiolate linkage
present in a polynucleotide sequence include, but are not limited
to, incubating the polynucleotide sequence with a metal agent, such
as Ag.sup.+ ions, at a pH in the range of from about 4.0 to about
10.0, from about 5.0 to about 9.0 or from about 6.0 to about 8.0,
and at a temperature in the range of from about 15.degree. C. to
about 50.degree. C., from about 20.degree. C. to about 45.degree.
C., from about 25.degree. C. to about 40.degree. C., from about
22.degree. C. to about 37.degree. C., or from about 24.degree. C.
to about 32.degree. C. Particular suitable conditions include, for
example, incubation in the presence of 50 mM AgNO.sub.3 at about 22
to about 37.degree. C. for at least about 10 minutes at a pH of
about 7.0. An example of conditions for a cleavage reaction are
described in Example 2. Such conditions can optionally comprise an
additional step in which the cleaved fragments are separated from
the template polynucleotide sequence to which they are annealed
(e.g., incubation at a temperature from about 90.degree. C. to
about 100.degree. C. for about 30 seconds to about 60 seconds)
prior to or at the same time the phosphorothiolate linkages are
cleaved.
[0086] In a further embodiment of the invention, the method for
determining a polynucleotide sequence additionally comprises
isolating a cleaved fragment (e.g., using an isolating means)
subsequent to the cleavage reaction. In a particular embodiment,
the cleaved fragment that is isolated comprises the primer that was
extended in the extension reaction. As used herein, the term
"isolated fragment" refers to a preparation of fragments that is
purified from, or otherwise substantially free of, other components
from the extension and/or cleavage reactions, including, but not
limited to, cleavage fragments that are not attached to a primer,
buffers, unincorporated nucleotides, nucleic acid templates and
enzymes. Such fragments can be isolated using an isolating means,
for example, a support (e.g., magnetic beads, agarose or sepharose
beads, among others) that comprises a moiety which recognizes and
binds to a tag (e.g., a tag on a primer). Examples of pairs of
partner moieties that are suitable for the present invention
include, but are not limited to, biotin and streptavidin/avidin, or
an epitope (e.g., digoxigenin (DIG)) and an antibody that
recognizes and binds the epitope (e.g., an anti-DIG antibody).
[0087] "Support", as used herein, refers to a matrix on or in which
nucleic acid molecules, microparticles, and the like may be
immobilized, e.g., to which they may be covalently or noncovalently
attached or, in or on which they may be partially or completely
embedded so that they are largely or entirely prevented from
diffusing freely or moving with respect to one another. The term
"microparticle" is used herein to refer to particles having a
smallest cross-sectional dimension of 50 microns or less,
preferably 10 microns or less. Microparticles may be made of a
variety of inorganic or organic materials including, but not
limited to, glass (e.g., controlled pore glass), silica, zirconia,
cross-linked polystyrene, polyacrylate, poly-methylmethacrylate,
titanium dioxide, latex, polystyrene, etc. See, e.g., U.S. Pat. No.
6,406,848, for various suitable materials and other considerations.
Magnetically responsive microparticles can be used.
[0088] The magnetic responsiveness of certain preferred
microparticles permits facile collection and concentration of the
microparticle-attached templates after amplification, and
facilitates additional steps (e.g., washes, reagent removal, etc.).
In certain embodiments of the invention a population of
microparticles having different shapes (e.g., some spherical and
others nonspherical) is employed. In general, any pair of molecules
that exhibit affinity for one another such that they form a binding
pair may be used to attach microparticles or templates to a
substrate. The first member of the binding pair is attached
covalently or noncovalently to the substrate, and the second member
of the binding pair is attached covalently or noncovalently to the
microparticles or templates.
[0089] In other embodiments of the invention, the templates are
amplified by polymerase chain reaction (PCR) in a semi-solid
support, such as a gel having suitable amplification primers
immobilized therein. Templates, additional amplification primers,
and reagents needed for the PCR reaction are present within the
semi-solid support. One or both of a pair of amplification primers
is attached to the semi-solid support via a suitable linking
moiety, e.g., an acrydite group. Attachment may occur during
polymerization. Additional reagents (e.g., templates, second
amplification primer, polymerase, nucleotides, cofactors, etc.) may
be present prior to formation of the semi-solid support (e.g., in a
liquid prior to gel formation), or one or more of the reagents may
be diffused into the semi-solid support after its formation. The
pore size of the semi-solid support is selected to allow such
diffusion. As is well known in the art, in the case of a
polyacrylamide gel, pore size is determined mainly by the
concentration of acrylamide monomer and to a lesser extent by the
crosslinking agent. Similar considerations apply in the case of
other semi-solid support materials. Appropriate cross-linkers and
concentrations to achieve a desired pore size can be selected.
[0090] In certain embodiments of the invention an additive such as
a cationic lipid, polyamine, polycation, etc., is included in the
solution prior to polymerization, which forms in-gel micelles or
aggregates surrounding the microparticles. Methods disclosed in
U.S. Pat. Nos. 5,705,628, 5,898,071, and 6,534,262 and U.S. Patent
Application Publication No. 2002/0106686, each of which are
incorporated herein by reference, may also be used. For example,
various "crowding reagents" can be used to crowd DNA near beads for
clonal PCR. SPRI.RTM. magnetic bead technology and/or conditions
can also be employed. See, e.g., U.S. Pat. No. 5,665,572,
demonstrating effective PCR amplification in the presence of 10%
polyethylene glycol (PEG). In certain embodiments of the inventive
methods amplification (e.g., PCR), ligation, or both, are performed
in the presence of a reagent such as betaine, polyethylene glycol,
PVP-40, or the like. These reagents may be added to a solution,
present in an emulsion, and/or diffused into a semi-solid
support.
[0091] Numerous other supports are known in the art, some of which
are described in U.S. Pat. No. 6,828,100, the contents of which are
herein incorporated by reference. In general, any of a wide variety
of methods known in the art can be used to modify nucleic acids
such as oligonucleotide primers, probes, templates, etc., to
facilitate the attachment of such nucleic acids to microparticles
or to other supports or substrates.
[0092] As will be understood by a person of skill in the art,
isolated extension products can be identified, either directly or
indirectly, using one of many standard and well-known detection
methods and/or techniques. Such methods and/or techniques include,
but are not limited to, fluorescence detection, spectrophotometric
detection, chemical detection and/or electrophoretic detection. In
one embodiment, detection of isolated extension products is
accomplished by resolving the primer extension products by means
of, for example, high-resolution denaturing polyacrylamide/urea gel
electrophoresis, capillary separation, or other resolving means;
followed by detecting the fragments, for example, using a scanning
spectrophotometer or fluorometer. In a particular embodiment,
fluorescently-labeled primer extension products are resolved by gel
electrophoresis, according to procedures that are well known in the
art, and are subsequently detected in the gel using a standard
fluorometer.
[0093] Electrophoretic separation of the isolated cleavage
fragments produces a "ladder" of extension fragments, each fragment
starting with the primer and ending with one of the four modified
thiol-nucleotides at its 3' end. The sequence of the complement
(i.e., the primer extension product), from which the sequence of
the template can be deduced, is read directly from the order of
fragments on the gel.
[0094] Techniques for detecting nucleic acid fragments on a gel are
well known in the art. Furthermore, one of skill in the art will
recognize that the particular method of detection will depend on
the specific label comprised by the resolved fragments. For
example, if the fragments are labeled with a fluorophore, then
standard fluorescence-based techniques can be utilized to detect
the fragments in a gel.
[0095] The skilled artisan will recognize that a polynucleotide
sequence can be determined, according to the methods of the present
invention, by performing a single reaction that utilizes a mixture
of four conventional nucleoside triphosphates and four modified 3'
thiol-nucleoside triphosphates, wherein each of the four modified
3' thiol-nucleoside triphosphates comprises a distinct label that
is not present on any other nucleotide in the mixture (e.g., four
color sequencing).
[0096] Alternatively, one of skill in the art will also recognize
that a polynucleotide sequence can be determined, according to the
methods of the present invention, by performing four separate
reactions to determine the nucleotide sequence of a template when
5' thiol-nucleoside triphosphates are utilized to generate modified
polynucleotide sequences comprising one or more 5'
phosphorothiolate linkages. In a particular embodiment, each of the
four reactions comprises four conventional nucleoside triphosphates
and only one of four modified 5' thiol-nucleoside triphosphates,
for example, sdCTP, sdATP, sdGTP, or sdTTP. When four reactions are
performed using the same template polynucleotide sequence, each
with one of the four modified 5' thiol-nucleoside triphosphates,
for example, sdCTP, sdATP, sdGTP or sdTTP, the products of the
reactions can be cleaved and detected, according to the methods
described herein, and analyzed to determine the sequence of the
template polynucleotide (see, for example, Sanger, F., et al. Proc.
Natl. Acad. Sci. USA 74: 5463-5467 (1977) and Maxam, A. M. and
Gilbert, W. Proc. Natl. Acad. Sci. USA 74: 560-564 (1977), the
contents of each are incorporated by reference herein).
[0097] In other embodiments of the invention, the methods described
herein can be performed using a template polynucleotide sequence
comprising a sense and antisense nucleotide strand and two primers,
a forward primer and a reverse primer, such that sequence
information for both the sense and antisense strands of the
template polynucleotide sequence can be determined. In one
embodiment, each primer comprises at least one distinct tag that is
not present on the other primer. After primer extension is
performed to produce extension products that comprise modified
polynucleotide sequences having one or more phosphorothiolate
linkages, followed by cleavage of the phosphorothiolate linkages in
the modified polynucleotide sequences--both performed according to
methods described herein--two populations of cleaved extension
fragments can be isolated, also according to methods described
herein. The first population consists of cleavage fragments, each
of which comprises the forward primer, while the second population
consists of cleavage fragments, each of which comprises the reverse
primer. These populations can be separated from one another and the
sequences of the forward and reverse extension products can be
determined, as described herein, such that the sequences of both
the antisense and sense strands of the template polynucleotide
sequence can be determined.
[0098] Accordingly, the present invention also provides a method
for separating one or more forward extension products from one or
more reverse extension products comprising annealing a plurality of
first primers and a plurality of second primers to a plurality of
template polynucleotide sequences comprising a sense nucleotide
strand and an antisense nucleotide strand, wherein the first primer
anneals to the sense strand and the second primer anneals to the
antisense strand and wherein at least one primer comprises a tag.
The first and second primers are extended in the presence of one or
more nucleoside triphosphates, wherein at least one of the
nucleoside triphosphates is modified, thereby producing a plurality
of extension products that comprise a modified nucleotide sequence
having one or more phosphorothiolate linkages. The
phosphorothiolate linkages in the modified extension products are
cleaved under conditions in which a plurality of fragments are
produced; and the fragments attached to the first primers are
separated from the fragments attached to the second primers.
[0099] In a particular embodiment, the first primer and the second
primer each comprise a tag, wherein the tag on the first primer is
distinct from the tag on the second primer. Accordingly, the
fragments of the reverse extension product that comprise the first
primer can be separated from the fragments of the forward extension
product that comprise the second primer using the distinct tags on
the first and second primers. The fragments of the forward and
reverse extension products can be identified either simultaneously
or in succession.
[0100] In another embodiment, two primers, a forward and reverse
primer, are used to amplify the template polynucleotide sequence by
polymerase chain reaction (PCR), according to procedures that are
well known in the art. Forward and reverse primer extension
products with at least one phosphorothiolate linkage, whose
sequences correspond to the sense and antisense strands of the
template sequence, respectively, are generated. These products can
then be cleaved, isolated and detected, according to methods
described herein, in order to deduce the sequence of the template
polynucleotide.
[0101] The invention also encompasses a kit, which can comprise one
or more modified thiol-nucleoside triphosphates (sdNTPs),
conventional nucleoside triphosphates (dNTPs) and/or a nucleic acid
polymerase (e.g., Klenow fragment of E. Coli DNA polymerase I,
Sequenase, exo-Thermus aquaticus (Taq) DNA polymerase and
exo-Bacillus stearothermophilus (Bst) DNA polymerase). The modified
sdNTPs can be either labeled or unlabeled. In a particular
embodiment, the sdNTPs comprise a fluorescent label (e.g., a
fluorophore). In certain embodiments, the detectable label is
present on the 5 carbon position of a pyrimidine base or on the 7
carbon deaza position of a purine base. In another embodiment, the
standard and modified nucleotides comprise a base, such as, but not
limited to, adenine, guanine, cytosine, thymine, uracil,
hypoxanthine or 7-deaza-guanine. Such kits can be used, for
example, to produce and/or determine the sequence of a modified
polynucleotide that comprises a (e.g., one or more)
phosphorothiolate linkage.
[0102] The modified thiol nucleotides can be either 3' thiol
nucleotides or 5' thiol nucleotides. In a particular embodiment the
3' thiol nucleotides comprise either a 3' thiol group (--SH) or a
3' dithiomethyl group (--SSCH.sub.3), for example,
3'-deoxy-dithiomethyl thymidine (see FIG. 3B). In a another
embodiment, the modified 5' thiol nucleotides are 5'
phosphorothiolate dNTPs.
[0103] Other components that are suitable for the kits of the
invention include, but are not limited to, an extension buffer
(e.g., buffers, salts, magnesium (Mg)), a cleavage buffer,
pyrophosphate, one or more supports for isolating extension
products, one or more reagents for sample clean-up (e.g., CleanSeq,
AmPure) and manufacturer's instructions.
[0104] A suitable cleavage buffer comprises a source of one or more
metal ions, for example silver, mercury or copper. In a particular
embodiment, the cleavage buffer comprises a source of silver ions,
such as silver nitrate (AgNO.sub.3) or silver acetate. In a further
embodiment, the source of silver ions is provided at a
concentration in the range of 1-100 mM. Additionally, the cleavage
buffer can further comprise a source of magnesium ions (Mg.sup.++).
In a particular embodiment, the source of magnesium ions is
magnesium acetate.
Example 1
Incorporation of 3' Thiol-Nucleoside Triphosphates into a Growing
Strand of DNA by DNA Polymerase
Materials and Methods
[0105] DNA synthesis was performed on a single-stranded DNA
template (5'-TTT TTT CTA AGG TAG CGA CTG TCC TAT ACA GAC TGA CAA
AAA AAG AGA ATG AGG AAC CCG GGG CAG-3') (SEQ ID NO:1), which was
labeled at its 5' end with a dual biotin tag and was attached to a
magnetic bead (FIG. 3B). Synthesis was primed using a primer
(5'-CTG CCC CGG GTT CCT CAT TCT CT-3') (SEQ ID NO:2), which was
complementary to a portion of the DNA template. The primer was
labeled with Cy5 at its 5' end. The DNA template contained 5
adenine nucleotides immediately downstream of the primer sequence.
Primer extension reactions were performed using 12.5 U exo.sup.- E.
coli Polymerase I, Klenow fragment (Epicentre) with 500 .mu.m or 50
.mu.m of 3'-deoxy-dithiomethyl thymidine (dTsTP) at 37.degree. C.
for 4.0 min.
Results
[0106] Up to five 3'-deoxy-dithiomethyl thymidine nucleotides were
successfully incorporated into the DNA amplification product
following completion of the reaction. When present at a low
concentration (5.0 .mu.m), DNA products with 0, 1, 2, 3, 4 or 5
modified thymidine residues were recovered (FIG. 3D). The majority
of reaction products, however, contained either 0 or 1 modified
nucleotide. When present at a higher concentration (1.0 mM), the
vast majority of reaction products contained 5 modified thymidine
residues (FIG. 3E). Similar results were observed when reactions
were conducted using other polymerases, including Sequenase,
exo-Taq polymerase and exo-Bst polymerase. These data indicate that
modified 3'-deoxy-dithiomethyl thymidine nucleotides can be readily
incorporated into a growing DNA strand during synthesis reactions
involving DNA polymerase.
Example 2
Chemical Cleavage of DNA Containing 3' Thiol Modified Nucleotides
in the Presence of Silver Ions
[0107] Prior to cleavage, modified nucleotides containing five 3'
thiol modified thymidine nucleotides on one strand (see Example 1)
were washed with 25 mM Magnesium acetate. Cleavage was induced by
incubating the products with 10 .mu.l silver nitrate (AgNO.sub.3)
at a concentration of either 50 .mu.m (FIG. 4B) or 500 .mu.m (FIG.
4D) for 15 min at room temperature. Unbound cleavage fragments were
removed by washing in dH.sub.2O, and bound reaction products
containing the primer were analyzed using a standard gel shift
assay. Both concentrations of AgNO.sub.3 that were tested resulted
in cleavage of the DNA product containing 3' thiol modified
thymidine nucleotides (FIG. 4B and FIG. 4D).
Example 3
Prophetic Example of DNA Synthesis by Primer Extension with 3'
Thiol-Nucleoside Triphosphates (sdNTPs) and Recovery of
Chemically-Cleaved Extension Products
[0108] Primer extension on a DNA template is conducted in the
presence of both unmodified nucleoside triphosphates and modified
3'-thiol-nucleoside triphosphates (FIG. 1A). When the modified
nucleoside triphosphates are used at a low concentration, they
incorporate randomly into the growing DNA strand next to natural
dNTPs at a low frequency (FIG. 1B). Incorporation of these modified
nucleotides into the DNA introduces one or more
3'-phosphorothiolate linkages (FIG. 5) into the DNA strand. The
sulfur-phosphorus bond of a phosphorothiolate linkage are
specifically and rapidly cleaved by exposure to silver ions
(Ag.sup.+). As a consequence, the addition of silver nitrate
(AgNO.sub.3) to the reaction ensures that each strand containing a
phosphorothiolate linkage is cleaved into 2 or more fragments,
depending on the number of 3'-thiol nucleotides in the strand (FIG.
1C). If the primers used in the reaction are labeled with an
affinity tag, such as biotin, the 5'-most fragments, which contain
the primer sequence with the affinity tag, are readily isolated
using streptavidin magnetic beads (FIG. 1D). Once captured, the
remaining, untagged fragments are washed away. The purified
fragments can be resolved by gel electrophoresis, resulting in a
fragmentation ladder. If each of the four 3' thiol nucleotide
triphosphates are fluorescently-labeled with a distinct
fluorophore, the products are analyzed on a standard fluorescence
based sequencing instrument to read the sequence of the strand.
Furthermore, if polymerase chain reaction (PCR) is conducted using
two primers (F FIG. 2A-FIG. 2B), wherein each primer contains a
different tag, the 5'-most extension products from both strands are
recovered separately and the sequence of both strands could be
analyzed (FIG. 2C).
[0109] The relevant teachings of all publications cited herein that
have not explicitly been incorporated by reference, are
incorporated herein by reference in their entirety. While this
invention has been particularly shown and described with references
to particular embodiments thereof, it will be understood by those
skilled in the art that various changes in form and details may be
made therein without departing from the scope of the invention
encompassed by the appended claim
Sequence CWU 1
1
8166DNAArtificial Sequencetemplate sequence 1ttttttctaa ggtagcgact
gtcctataca gactgacaaa aaaagagaat gaggaacccg 60gggcag
66223DNAArtificial Sequenceoligonucleotide primer 2ctgccccggg
ttcctcattc tct 23325DNAArtificial Sequencehypothetical primer
extension product 3nnnnnannnn gnnncnnnnn ntnnn 25421DNAArtificial
Sequencehypothetical primer extension product 4nnnnnnnnnn
nnnnnnnnnn c 21510DNAArtificial Sequencehypothetical primer
extension product 5nnnnnnnnna 10611DNAArtificial
Sequencehypothetical primer extension product 6nnnnnnnnnn c
11725DNAArtificial Sequencehypothetical primer extension product
7nnnnnannnn gnnncnnnnn ntnnn 25810DNAArtificial
Sequencehypothetical primer extension product 8nnnnnnnnna 10
* * * * *