U.S. patent application number 11/404695 was filed with the patent office on 2007-10-18 for methods for increasing accuracy of nucleic acid sequencing.
Invention is credited to Timothy D. Harris.
Application Number | 20070243535 11/404695 |
Document ID | / |
Family ID | 38997463 |
Filed Date | 2007-10-18 |
United States Patent
Application |
20070243535 |
Kind Code |
A1 |
Harris; Timothy D. |
October 18, 2007 |
Methods for increasing accuracy of nucleic acid sequencing
Abstract
The invention provides methods for improving the accuracy of a
sequencing-by-synthesis reaction by sequencing at least a portion
of a template and at least a portion of template complementary
sequence.
Inventors: |
Harris; Timothy D.; (Toms
River, NJ) |
Correspondence
Address: |
EDWARDS ANGELL PALMER & DODGE LLP
P.O. BOX 55874
BOSTON
MA
02205
US
|
Family ID: |
38997463 |
Appl. No.: |
11/404695 |
Filed: |
April 14, 2006 |
Current U.S.
Class: |
435/6.1 ;
435/287.2; 536/25.32 |
Current CPC
Class: |
C12Q 1/6869 20130101;
C12Q 1/6874 20130101; C12Q 1/6874 20130101; C12Q 2533/101 20130101;
C12Q 2525/155 20130101; C12Q 2565/501 20130101; C12Q 2525/155
20130101; C12Q 2565/537 20130101; C12Q 2535/119 20130101; C12Q
2535/119 20130101; C12Q 2533/101 20130101; C12Q 2535/119 20130101;
C12Q 2535/119 20130101; C12Q 1/6869 20130101; C12Q 1/6869 20130101;
C12Q 1/6874 20130101 |
Class at
Publication: |
435/006 ;
536/025.32; 435/287.2 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12M 1/34 20060101 C12M001/34; C07H 21/04 20060101
C07H021/04 |
Claims
1. A method of increasing accuracy of nucleic acid sequencing, the
method comprising the steps of: a) exposing a duplex comprising a
template and a primer to a polymerase and one or more nucleotide
comprising a detectable label under conditions sufficient for
template-dependent nucleotide addition to said primer, wherein said
template is individually optically resolvable; b) identifying
nucleotide incorporated into said primer; c) neutralizing said
detectable label or removing said detectable label from said
incorporated nucleotide; d) repeating steps a)-c), thereby
determining a nucleotide sequence incorporated into said primer of
said duplex; e) removing the template from the primer of step d);
f) adding a polynucleotide to a 3' terminus of the primer from step
e) to form a template; g) exposing the template of step f) to a
primer capable of hybridizing to said added polynucleotide to form
template/primer duplex, and repeating steps a) through d) to
sequence a portion of the template, wherein at least a portion of
the sequence obtained is complementary to the nucleotide sequence
of d), thereby increasing the accuracy of nucleic acid
sequencing.
2. The method of claim 1, wherein the sequence obtained in c) is
compared with a complement of the sequence obtained in f).
3. The method of claim 1, further comprising repeating steps d) and
f).
4. The method of claim 1, wherein said label is an
optically-detectable label.
5. The method of claim 4, wherein said optically-detectable label
is a fluorescent label.
6. The method of claim 5, wherein said fluorescent label is
selected from the group consisting of fluorescein, rhodamine,
cyanine, Cy5, Cy3, BODIPY, alexa, and derivatives thereof.
7. The method of claim 1, wherein said duplex is attached to a
surface.
8. The method of claim 1, wherein said primer is attached to a
surface.
Description
TECHNICAL FIELD OF THE INVENTION
[0001] The invention generally relates to methods for increasing
accuracy in nucleic acid synthesis reactions.
BACKGROUND OF THE INVENTION
[0002] In vitro nucleic acid synthesis is a foundation of many
fundamental research and diagnostic tools, such as nucleic acid
amplification and sequencing. In a template-dependent nucleic acid
synthesis reaction, the sequential addition of nucleotides is
catalyzed by a nucleic acid polymerase. Depending on the template
and the nature of the reaction, the nucleic acid polymerase may be
a DNA polymerase, an RNA polymerase, or a reverse
transcriptase.
[0003] The accuracy of template-dependent nucleic acid synthesis
depends in part on the ability of the polymerase to discriminate
between complementary and non-complementary nucleotides. Normally,
the conformation of the polymerase enzyme favors incorporation of
the complementary nucleotide. However, there is still an
identifiable rate of misincorporation that depends upon factors
such as local sequence and the base to be incorporated.
[0004] Synthetic or modified nucleotides and analogs, such as
labeled nucleotides, tend to be incorporated into a primer less
efficiently than naturally-occurring nucleotides. The reduced
efficiency with which the unconventional nucleotides are
incorporated by the polymerase can adversely affect the performance
of sequencing techniques that depend upon faithful incorporation of
such unconventional nucleotides.
[0005] Single molecule sequencing techniques allow the evaluation
of individual nucleic acid molecules in order to identify changes
and/or differences affecting genomic function. In single molecule
techniques, a nucleic acid fragment is attached to a solid support
such that at least a portion of the nucleic acid fragment is
individually optically-resolvable. Sequencing is conducted using
the fragments as templates. Sequencing events are detected and
correlated to the individual strands. See Braslavsky et al., Proc.
Natl. Acad. Sci., 100: 3960-64 (2003), incorporated by reference
herein. Because single molecule techniques do not rely on ensemble
averaging as do bulk techniques, errors due to misincorporation can
have a significant deleterious effect on the sequencing results.
The incorporation of a nucleotide that is incorrectly paired, under
standard Watson and Crick base-pairing, with a corresponding
template nucleotide during primer extension may result in
sequencing errors. Furthermore, where the template being sequenced
is present in only one or a few copies in the sample (a rare
template), misincorporations can have a great impact on the
sequence obtained because fewer sequences are obtained with which
to compare to each other or with a reference sequence.
[0006] There is, therefore, a need in the art for improved methods
for improving the accuracy of nucleic acid synthesis reactions,
especially in single molecule sequencing.
SUMMARY OF THE INVENTION
[0007] The invention improves the accuracy of nucleic acid
sequencing reactions. According to the invention, a template
nucleic acid is hybridized to a primer and template-dependent
sequencing-by-synthesis is conducted to extend the 3' end of the
primer. The template is then removed from the extended primer and
the primer is then "primed" and re-sequenced. Practice of the
invention allows resequencing of the same sequence and its
complement in situ, and results in increased accuracy of sequence
determination.
[0008] According to the invention, a polymerization reaction is
conducted on a nucleic acid duplex that comprises a primer
hybridized to a template nucleic acid. The reaction is conducted in
the presence of a polymerase, and at least one nucleotide
comprising a detectable label. If the nucleotide is complementary
to the next nucleotide in the template, it is added to the primer
by the polymerase. The added nucleotide is detected and the
reaction is then repeated at least once. Thus, the primer is
extended by one or more nucleotides corresponding to sequence that
is complementary to at least a portion of the template. The
template is removed from the duplex, leaving the extended
primer.
[0009] In one embodiment, one or more primer/template duplexes are
bound to a solid support such that a least some of the duplexes are
individually optically detectable. The duplexes are exposed to a
polymerase, and at least one detectably-labeled nucleotide under
conditions sufficient for template-dependent nucleotide addition to
the primer. Unincorporated labeled nucleotides are optionally
washed away. The incorporation of the labeled nucleotide is
detected, thereby identifying the added nucleotide and the
complementary template nucleotide. Base addition, washing, and
identification steps can be serially repeated in the presence of
detectably labeled nucleotide that corresponds to each of the other
nucleotide species. As a result, the primer is extended by the
added nucleotides. The added nucleotides correspond to sequence
that is complementary to at least a portion of the template.
[0010] After one or more primer extension steps, the template is
removed from the duplex. The template can be removed by any
suitable means, for example by raising the temperature of the
surface or the flow cell such that the duplex is melted, or by
changing the buffer conditions to destabilize the duplex, or
combination thereof. Methods for melting template/primer duplexes
are well known in the art and are described, for example, in
chapter 10 of Molecular Cloning, a Laboratory Manual, 3.sup.rd
Edition, J. Sambrook, and D. W. Russell, Cold Spring Harbor Press
(2001), the teachings of which are incorporated herein by
reference. The template can then be removed from the surface, for
example, by rinsing the surface with a suitable rinsing
solution.
[0011] After removing the template, the extended primer used in the
polymerization reaction remains on the surface. The 3' terminus of
the primer is then modified by addition of a short polynucleotide.
The polynucleotide is added to the primer by enzymatic catalysis. A
preferred enzyme is a ligase or a polymerase. Suitable ligases
include, for example, T4 DNA ligase and T4 RNA ligase (such ligases
are available commercially, from New England BioLabs (on the World
Wide Web at NEB.com) and others capable of adding nucleotides to
the 3' terminus of the primer. In a preferred embodiment, a
dephosphorylated polynucleotide is added to the primer. Methods for
using ligases and dephosphorylating oligonucleotides are well known
in the art.
[0012] If polymerization is used to add polynucleotides to the 3'
terminus of the primer, any suitable enzyme can be used. For
example, a polymerase, such as poly(A) polymerase, including yeast
poly(A) polymerase, commercially available from USB (on the World
Wide Web at USBweb.com), terminal deoxyribonucleotidyl transferase
(TdT), and the like are useful. The polymerases can be used
according to the manufacturer's instructions.
[0013] Having been modified as described above, the primer is then
used as a template for template-dependent sequencing-by-synthesis
as described generally above.
[0014] The polynucleotide added to the primer is chosen such that
it is complementary to a new primer (or at least a portion
thereof). In a preferred embodiment, the polynucleotide is a
homopolymer, such as oligo(dA), and the corresponding primer
includes an oligo(dT) sequence. The complementary sequences are of
a length suitable for hybridization. The added polynucleotide and
its complementary new primer can be about 10 to about 100
nucleotides in length, and preferably about 50 nucleotides in
length. The added polynucleotide and new primer can be of the same
length or of different lengths. It is routine in the art to adjust
primer length and/or oligonucleotide length to optimize
hybridization.
[0015] Once a polynucleotide is added to the 3' end of the primer
and a new primer sequence is hybridized to the polynucleotide (or
portion thereof), template-dependent sequencing-by-synthesis is
conducted on the primer in the opposite direction of the original
sequencing reaction (i.e., toward to surface to which the primer is
bound).
[0016] After conducting the sequencing reaction back toward to the
surface, the "new" extended primer can be melted off, leaving a
template having the complementary sequence as the original template
for optional resequencing in the 3' to 5' direction (i.e., toward
the surface).
[0017] Sequencing and/or resequencing at least a portion of the
complement of the original template increases the accuracy of the
sequence information obtained from a given template by providing
more than one set of sequence information to compare, for example,
to a reference sequence. In another embodiment, the sequence
initially obtained can be compared to the sequence obtained from
the new template.
[0018] Sequencing methods of the invention preferably comprise
template/primer duplex attached to a surface. Individual
nucleotides added to the surface comprise a detectable
label--preferably an optically-detectable label, such as a
fluorescent label. Each nucleotide species can comprise a different
label, or can comprise the same label. In a preferred embodiment,
each duplex is individually optically resolvable in order to
facilitate single molecule sequence discrimination. The choice of a
surface for attachment of duplex depends upon the detection method
employed. Preferred surfaces for methods of the invention include
epoxide surfaces and polyelectrolyte multilayer surfaces, such as
those described in Braslavsky, et al., supra. Surfaces preferably
are deposited on a substrate that is amenable to optical detection
of the surface chemistry, such as glass or silica.
[0019] Nucleotides useful in the invention include any nucleotide
or nucleotide analog, whether naturally-occurring or synthetic. For
example, preferred nucleotides include phosphate esters of
deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine,
adenosine, cytidine, guanosine, and uridine.
[0020] Polymerases useful in the invention include any nucleic acid
polymerase capable of catalyzing a template-dependent addition of a
nucleotide or nucleotide analog to a primer. Depending on the
characteristics of the target nucleic acid, a DNA polymerase, an
RNA polymerase, a reverse transcriptase, or a mutant or altered
form of any of the foregoing can be used. According to one aspect
of the invention, a thermophilic polymerase is used, such as
ThermoSequenase.RTM., 9.degree.N.TM., Therminator.TM., Taq, Tne,
Tma, Pfu, Tfl, Tth, Tli, Stoffel fragment, Vent.TM. and Deep
Vent.TM. DNA polymerase.
BRIEF DESCRIPTION OF THE DRAWING
[0021] The FIGURE shows a schematic representation of one
embodiment of the present invention.
DETAILED DESCRIPTION
[0022] The invention provides methods and compositions for
improving the accuracy of nucleic acid sequencing-by-synthesis
reactions by re-sequencing a least a portion of a template nucleic
acid. While applicable to bulk sequencing methods, the invention is
particularly useful in connection with single molecule sequencing
methods. According to the invention, the methods comprise the steps
of exposing a duplex comprising a template and a primer to a
polymerase and one or more nucleotide comprising a detectable label
under conditions sufficient for template-dependent nucleotide
addition to the primer. In one embodiment, the template is
individually optically resolvable. Any unincorporated labeled
nucleotide is optionally washed way. Any nucleotide incorporated
into the primer is identified by detecting the label associated
with the incorporated nucleotide. The steps of exposing duplex to
polymerase and another nucleotide comprising a detectable label and
polymerizing, optional washing, and identification are repeated,
thereby determining a nucleotide sequence. As a result of the
exposing and polymerizing steps, the primer is extended by the
addition of one or more nucleotides that are complementary to the
corresponding positions of the template. The template is then
removed from the duplex, leaving the extended primer. A
polynucleotide is added to the 3' terminus of the primer or
extended primer, thereby forming a modified primer. The modified
primer is used as the template in subsequent sequencing
reactions.
[0023] The FIGURE is a schematic representation of one embodiment
of the invention. In this embodiment, a primer, 2, is attached to a
solid support, 3. A template, 1, is hybridized to the primer,
forming a template/primer duplex. In step A, the template
primer/duplex is exposed to a polymerase and at least one
nucleotide comprising a detectable label, under conditions
sufficient for template-dependent nucleotide addition to said
primer. If the nucleotide is complementary to the template
nucleotide immediately downstream of the primer, a nucleotide is
added to the primer. After identifying nucleotide incorporated into
the primer, the process is repeated, thereby adding a second
nucleotide to the primer in a template dependent manner, and so on.
As shown in the FIGURE, as a result of repeating the process,
template complementary sequence, 4, is added to the primer. After
the process has been repeated the desired number of times, the
template is removed as shown in step B, leaving the extended
primer. In step C, a polynucleotide, 6, is added to the extended
primer at the 3' terminus (e.g., downstream of the previously added
template complementary sequence) forming a new template for
sequencing in the opposite direction. In step D, a primer capable
of hybridizing to the polynucleotide is added, forming a
template/primer duplex, 7. The process of adding nucleotide and
polymerase, detecting incorporated nucleotide and repeating the
desired number of times is then repeated using the modified
extended primer as a template, as shown in step E, thereby
sequencing the template complementary sequence 4, 8. As a result,
the primer, 6, is extended by the addition of sequence 8 that
corresponds to at least a portion of the original template, 1. The
extended primer (6, 8) can be removed, F, and the template can be
resequenced, as described above.
[0024] In a preferred embodiment of the invention, direct amine
attachment is used to attach primer or template to an epoxide
surface. The primer or the template can comprise an
optically-detectable label in order to determine the location of
duplex on the surface. At least a portion of the duplex is
optically resolvable from other duplexes on the surface. The
surface is preferably passivated with a reagent that occupies
portions of the surface that might, absent passivation, fluoresce.
Optimal passivation reagents include amines, phosphate, water,
sulfates, detergents, and other reagents that reduce native or
accumulating surface fluorescence. Sequencing is then accomplished
by presenting one or more labeled nucleotide in the presence of a
polymerase under conditions that promote complementary base
incorporation in the primer. In a preferred embodiment, one base at
a time (per cycle) is added and all bases have the same label.
There is a wash step after each incorporation cycle, and the label
is either neutralized without removal or removed from incorporated
nucleotides. After the completion of a predetermined number of
cycles of base addition, the linear sequence data for each
individual duplex is compiled. Numerous algorithms are available
for sequence compilation and alignment as discussed below.
[0025] In general, epoxide-coated glass surfaces are used for
direct amine attachment of templates, primers, or both. Amine
attachment to the termini of template and primer molecules is
accomplished using terminal transferase. Primer molecules can be
custom-synthesized to hybridize to templates for duplex
formation.
[0026] A full-cycle is conducted as many times as necessary to
complete sequencing of a desired length of template, or
resequencing of the desired length of the template complementary
sequence. Once the desired number of cycles is complete, the result
is a stack of images represented in a computer database. For each
spot on the surface that contained an initial individual duplex,
there will be a series of light and dark image coordinates,
corresponding to whether a base was incorporated in any given
cycle. For example, if the template sequence was TACGTACG and
nucleotides were presented in the order CAGU(T), then the duplex
would be "dark" (i.e., no detectable signal) for the first cycle
(presentation of C), but would show signal in the second cycle
(presentation of A, which is complementary to the first T in the
template sequence). The same duplex would produce signal upon
presentation of the G, as that nucleotide is complementary to the
next available base in the template, C. Upon the next cycle
(presentation of U), the duplex would be dark, as the next base in
the template is G. Upon presentation of numerous cycles, the
sequence of the template would be built up through the image stack.
The sequencing data are then fed into an aligner as described below
for resequencing, or are compiled for de novo sequencing as the
linear order of nucleotides incorporated into the primer.
[0027] The imaging system used in practice of the invention can be
any system that provides sufficient illumination of the sequencing
surface at a magnification such that single fluorescent molecules
can be resolved.
General Considerations
[0028] A. Nucleic Acid Templates
[0029] Nucleic acid templates include deoxyribonucleic acid (DNA)
and/or ribonucleic acid (RNA). Nucleic acid template molecules can
be isolated from a biological sample containing a variety of other
components, such as proteins, lipids and non-template nucleic
acids. Nucleic acid template molecules can be obtained from any
cellular material, obtained from an animal, plant, bacterium,
fungus, or any other cellular organism. Biological samples for use
in the invention also include viral particles or samples prepared
from viral material. Nucleic acid template molecules may be
obtained directly from an organism or from a biological sample
obtained from an organism, e.g., from blood, urine, cerebrospinal
fluid, seminal fluid, saliva, sputum, stool and tissue. Any tissue
or body fluid specimen may be used as a source for nucleic acid for
use in the invention. Nucleic acid template molecules may also be
isolated from cultured cells, such as a primary cell culture or a
cell line. The cells or tissues from which template nucleic acids
are obtained can be infected with a virus or other intracellular
pathogen. A sample can also be total RNA extracted from a
biological specimen, a cDNA library, viral, or genomic DNA.
[0030] Nucleic acid obtained from biological samples typically is
fragmented to produce suitable fragments for analysis. In one
embodiment, nucleic acid from a biological sample is fragmented by
sonication. Nucleic acid template molecules can be obtained as
described in U.S. Patent Application 2002/0190663 A1, published
Oct. 9, 2003, the teachings of which are incorporated herein in
their entirety. Generally, nucleic acid can be extracted from a
biological sample by a variety of techniques such as those
described by Maniatis, et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor, N.Y., pp. 280-281 (1982). Generally,
individual nucleic acid template molecules can be from about 5
bases to about 20 kb. Nucleic acid molecules may be
single-stranded, double-stranded, or double-stranded with
single-stranded regions (for example, stem- and
loop-structures).
[0031] A biological sample as described herein may be homogenized
or fractionated in the presence of a detergent or surfactant. The
concentration of the detergent in the buffer may be about 0.05% to
about 10.0%. The concentration of the detergent can be up to an
amount where the detergent remains soluble in the solution. In a
preferred embodiment, the concentration of the detergent is between
0.1% to about 2%. The detergent, particularly a mild one that is
nondenaturing, can act to solubilize the sample. Detergents may be
ionic or nonionic. Examples of nonionic detergents include triton,
such as the Triton.RTM. X series (Triton.RTM. X-100
t-Oct-C.sub.6H.sub.4--(OCH.sub.2--CH.sub.2).sub.xOH, x=9-10,
Triton.RTM. X-100R, Triton.RTM. X-114 x=7-8), octyl glucoside,
polyoxyethylene(9)dodecyl ether, digitonin, IGEPAL.RTM. CA630
octylphenyl polyethylene glycol, n-octyl-beta-D-glucopyranoside
(betaOG), n-dodecyl-beta, Tween.RTM. 20 polyethylene glycol
sorbitan monolaurate, Tween.RTM. 80 polyethylene glycol sorbitan
monooleate, polidocanol, n-dodecyl beta-D-maltoside (DDM), NP-40
nonylphenyl polyethylene glycol, C12E8 (octaethylene glycol
n-dodecyl monoether), hexaethyleneglycol mono-n-tetradecyl ether
(C14EO6), octyl-beta-thioglucopyranoside (octyl thioglucoside,
OTG), Emulgen, and polyoxyethylene 10 lauryl ether (C12E10).
Examples of ionic detergents (anionic or cationic) include
deoxycholate, sodium dodecyl sulfate (SDS), N-lauroylsarcosine, and
cetyltrimethylammoniumbromide (CTAB). A zwitterionic reagent may
also be used in the purification schemes of the present invention,
such as Chaps, zwitterion 3-14, and
3-[(3-cholamidopropyl)dimethylammonio)-1-propanesulf-onate. It is
contemplated also that urea may be added with or without another
detergent or surfactant.
[0032] Lysis or homogenization solutions may further contain other
agents, such as reducing agents. Examples of such reducing agents
include dithiothreitol (DTT), .beta.-mercaptoethanol, DTE, GSH,
cysteine, cysteamine, tricarboxyethyl phosphine (TCEP), or salts of
sulfurous acid.
[0033] B. Nucleotides
[0034] Nucleotides useful in the invention include any nucleotide
or nucleotide analog, whether naturally-occurring or synthetic. For
example, preferred nucleotides include phosphate esters of
deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine,
adenosine, cytidine, guanosine, and uridine. Other nucleotides
useful in the invention comprise an adenine, cytosine, guanine,
thymine base, a xanthine or hypoxanthine; 5-bromouracil,
2-aminopurine, deoxyinosine, or methylated cytosine, such as
5-methylcytosine, and N4-methoxydeoxycytosine. Also included are
bases of polynucleotide mimetics, such as methylated nucleic acids,
e.g., 2'-O-methRNA, peptide nucleic acids, modified peptide nucleic
acids, locked nucleic acids and any other structural moiety that
can act substantially like a nucleotide or base, for example, by
exhibiting base-complementarity with one or more bases that occur
in DNA or RNA and/or being capable of base-complementary
incorporation, and includes chain-terminating analogs. A nucleotide
corresponds to a specific nucleotide species if they share
base-complementarity with respect to at least one base.
[0035] Nucleotides for nucleic acid sequencing according to the
invention preferably comprise a detectable label that is directly
or indirectly detectable. Preferred labels include
optically-detectable labels, such as fluorescent labels. Examples
of fluorescent labels include, but are not limited to,
4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid; acridine
and derivatives: acridine, acridine isothiocyanate;
5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);
4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate;
N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY;
Brilliant Yellow; coumarin and derivatives; coumarin,
7-amino-4-methylcoumarin (AMC, Coumarin 120),
7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanine dyes;
cyanosine; 4',6-diaminidino-2-phenylindole (DAPI);
5'5''-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);
7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin;
diethylenetriamine pentaacetate;
4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid;
4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid;
5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS,
dansylchloride); 4-dimethylaminophenylazophenyl-4'-isothiocyanate
(DABITC); eosin and derivatives; eosin, eosin isothiocyanate,
erythrosin and derivatives; erythrosin B, erythrosin,
isothiocyanate; ethidium; fluorescein and derivatives;
5-carboxyfluorescein (FAM),
5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),
2',7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein, fluorescein,
fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144;
IR1446; Malachite Green isothiocyanate; 4-methylumbelliferoneortho
cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;
B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives:
pyrene, pyrene butyrate, succinimidyl 1-pyrene; butyrate quantum
dots; Reactive Red 4 (Cibacron.TM. Brilliant Red 3B-A) rhodamine
and derivatives: 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine
(R6G), lissamine rhodamine B sulfonyl chloride rhodamine (Rhod),
rhodamine B, rhodamine 123, rhodamine X isothiocyanate,
sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative
of sulforhodamine 101 (Texas Red);
N,N,N',N'tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl
rhodamine; tetramethyl rhodamine isothiocyanate (TRITC);
riboflavin; rosolic acid; terbium chelate derivatives; Cy3; Cy5;
Cy5.5; Cy7; IRD 700; IRD 800; La Jolta Blue; phthalo cyanine; and
naphthalo cyanine. Preferred fluorescent labels are cyanine-3 and
cyanine-5. Labels other than fluorescent labels are contemplated by
the invention, including other optically-detectable labels.
[0036] C. Nucleic Acid Polymerases
[0037] Nucleic acid polymerases generally useful in the invention
include DNA polymerases, RNA polymerases, reverse transcriptases,
and mutant or altered forms of any of the foregoing. DNA
polymerases and their properties are described in detail in, among
other places, DNA Replication 2nd edition, Komberg and Baker, W. H.
Freeman, New York, N.Y. (1991). Known conventional DNA polymerases
useful in the invention include, but are not limited to, Pyrococcus
furiosus (Pfu) DNA polymerase (Lundberg et al., 1991, Gene, 108: 1,
Stratagene), Pyrococcus woesei (Pwo) DNA polymerase (Hinnisdaels et
al., 1996, Biotechniques, 20:186-8, Boehringer Mannheim), Thermus
thermophilus (Tth) DNA polymerase (Myers and Gelfand 1991,
Biochemistry 30:7661), Bacillus stearothermophilus DNA polymerase
(Stenesh and McGowan, 1977, Biochim Biophys Acta 475:32),
Thermococcus litoralis (Tli) DNA polymerase (also referred to as
Vent.TM. DNA polymerase, Cariello et al., 1991, Polynucleotides
Res, 19: 4193, New England Biolabs), 9.degree.Nm.TM. DNA polymerase
(New England Biolabs), Stoffel fragment, ThermoSequenase.RTM.
(Amersham Pharmacia Biotech UK), Therminator.TM. (New England
Biolabs), Thermotoga maritima (Tma) DNA polymerase (Diaz and
Sabino, 1998 Braz J Med. Res, 31:1239), Thermus aquaticus (Taq) DNA
polymerase (Chien et al., 1976, J. Bacteoriol, 127: 1550), DNA
polymerase, Pyrococcus kodakaraensis KOD DNA polymerase (Takagi et
al., 1997, Appl. Environ. Microbiol. 63:4504), JDF-3 DNA polymerase
(from thermococcus sp. JDF-3, Patent application WO 0132887),
Pyrococcus GB-D (PGB-D) DNA polymerase (also referred as Deep
Vent.TM. DNA polymerase, Juncosa-Ginesta et al., 1994,
Biotechniques, 16:820, New England Biolabs), UlTma DNA polymerase
(from thermophile Thermotoga maritima; Diaz and Sabino, 1998 Braz
J. Med. Res, 31:1239; PE Applied Biosystems), Tgo DNA polymerase
(from thermococcus gorgonarius, Roche Molecular Biochemicals), E.
coli DNA polymerase I (Lecomte and Doubleday, 1983, Polynucleotides
Res. 11:7505), T7 DNA polymerase (Nordstrom et al., 1981, J Biol.
Chem. 256:3112), and archaeal DP1I/DP2 DNA polymerase II (Cann et
al., 1998, Proc Natl Acad. Sci. USA 95:14250.fwdarw.5).
[0038] While mesophilic polymerases are contemplated by the
invention, preferred polymerases are thermophilic. Thermophilic DNA
polymerases include, but are not limited to, ThermoSequenase.RTM.,
9.degree.Nm.TM., Therminator.TM., Taq, Tne, Tma, Pfu, Tfl, Tth,
Tli, Stoffel fragment, Vent.TM. and Deep Vent.TM. DNA polymerase,
KOD DNA polymerase, Tgo, JDF-3, and mutants, variants and
derivatives thereof.
[0039] Reverse transcriptases useful in the invention include, but
are not limited to, reverse transcriptases from HIV, HTLV-1,
HTLV-II, FeLV, FIV, SIV, AMV, MMTV, MoMuLV and other retroviruses
(see Levin, Cell 88:5-8 (1997); Verma, Biochim Biophys Acta.
473:1-38 (1977); Wu et al., CRC Crit Rev Biochem.
3:289-347(1975)).
[0040] D. Surfaces
[0041] In a preferred embodiment, nucleic acid template molecules
are attached to a substrate (also referred to herein as a surface)
and subjected to analysis by sequencing as taught herein. Nucleic
acid template molecules are attached to the surface such that the
template/primer duplexes are individually optically resolvable.
Substrates for use in the invention can be two- or
three-dimensional and can comprise a planar surface (e.g., a glass
slide) or can be shaped. A substrate can include glass (e.g.,
controlled pore glass (CPG)), quartz, plastic (such as polystyrene
(low cross-linked and high cross-linked polystyrene),
polycarbonate, polypropylene and poly(methymethacrylate)), acrylic
copolymer, polyamide, silicon, metal (e.g.,
alkanethiolate-derivatized gold), cellulose, nylon, latex, dextran,
gel matrix (e.g., silica gel), polyacrolein, or composites.
[0042] Suitable three-dimensional substrates include, for example,
spheres, microparticles, beads, membranes, slides, plates,
micromachined chips, tubes (e.g., capillary tubes), microwells,
microfluidic devices, channels, filters, or any other structure
suitable for anchoring a nucleic acid. Substrates can include
planar arrays or matrices capable of having regions that include
populations of template nucleic acids or primers. Examples include
nucleoside-derivatized CPG and polystyrene slides; derivatized
magnetic slides; polystyrene grafted with polyethylene glycol, and
the like.
[0043] In one embodiment, a substrate is coated to allow optimum
optical processing and nucleic acid attachment. Substrates for use
in the invention can also be treated to reduce background.
Exemplary coatings include epoxides, and derivatized epoxides
(e.g., with a binding molecule, such as streptavidin). The surface
can also be treated to improve the positioning of attached nucleic
acids (e.g., nucleic acid template molecules, primers, or template
molecule/primer duplexes) for analysis. As such, a surface
according to the invention can be treated with one or more charge
layers (e.g., a negative charge) to repel a charged molecule (e.g.,
a negatively charged labeled nucleotide). For example, a substrate
according to the invention can be treated with polyallylamine
followed by polyacrylic acid to form a polyelectrolyte multilayer.
The carboxyl groups of the polyacrylic acid layer are negatively
charged and thus repel negatively charged labeled nucleotides,
improving the positioning of the label for detection. Coatings or
films applied to the substrate should be able to withstand
subsequent treatment steps (e.g., photoexposure, boiling, baking,
soaking in warm detergent-containing liquids, and the like) without
substantial degradation or disassociation from the substrate.
[0044] Examples of substrate coatings include, vapor phase coatings
of 3-aminopropyltrimethoxysilane, as applied to glass slide
products, for example, from Molecular Dynamics, Sunnyvale, Calif.
In addition, generally, hydrophobic substrate coatings and films
aid in the uniform distribution of hydrophilic molecules on the
substrate surfaces. Importantly, in those embodiments of the
invention that employ substrate coatings or films, the coatings or
films that are substantially non-interfering with primer extension
and detection steps are preferred. Additionally, it is preferable
that any coatings or films applied to the substrates either
increase template molecule binding to the substrate or, at least,
do not substantially impair template binding.
[0045] Various methods can be used to anchor or immobilize the
primer to the surface of the substrate. The immobilization can be
achieved through direct or indirect bonding to the surface. The
bonding can be by covalent linkage. See, Joos et al., Analytical
Biochemistry 247:96-101, 1997; Oroskar et al., Clin. Chem.
42:1547-1555, 1996; and Khandjian, Mol. Bio. Rep. 11:107-115, 1986.
A preferred attachment is direct amine bonding of a terminal
nucleotide of the template or the primer to an epoxide integrated
on the surface. The bonding also can be through non-covalent
linkage. For example, biotin-streptavidin (Taylor et al., J Phys.
D. Appl. Phys. 24:1443, 1991) and digoxigenin with anti-digoxigenin
(Smith et al., Science 253:1122, 1992) are common tools for
anchoring nucleic acids to surfaces and parallels. Alternatively,
the attachment can be achieved by anchoring a hydrophobic chain
into a lipid monolayer or bilayer. Other methods for known in the
art for attaching nucleic acid molecules to substrates also can be
used.
[0046] E. Detection
[0047] Any detection method may be used that is suitable for the
type of label employed. Thus, exemplary detection methods include
radioactive detection, optical absorbance detection, e.g.,
UV-visible absorbance detection, optical emission detection, e.g.,
fluorescence or chemiluminescence. For example, extended primers
can be detected on a substrate by scanning all or portions of each
substrate simultaneously or serially, depending on the scanning
method used. For fluorescence labeling, selected regions on a
substrate may be serially scanned one-by-one or row-by-row using a
fluorescence microscope apparatus, such as described in Fodor (U.S.
Pat. No. 5,445,934) and Mathies et al. (U.S. Pat. No. 5,091,652).
Devices capable of sensing fluorescence from a single molecule
include scanning tunneling microscope (siM) and the atomic force
microscope (AFM). Hybridization patterns may also be scanned using
a CCD camera (e.g., Model TE/CCD512SF, Princeton Instruments,
Trenton, N.J.) with suitable optics (Ploem, in Fluorescent and
Luminescent Probes for Biological Activity Mason, T.G. Ed.,
Academic Press, Landon, pp. 1-11 (1993), such as described in
Yershov et al., Proc. Natl. Aca. Sci. 93:4913 (1996), or may be
imaged by TV monitoring. For radioactive signals, a phosphorimager
device can be used (Johnston et al., Electrophoresis, 13:566, 1990;
Drmanac et al., Electrophoresis, 13:566, 1992; 1993). Other
commercial suppliers of imaging instruments include General
Scanning Inc., (Watertown, Mass. on the World Wide Web at
genscan.com), Genix Technologies (Waterloo, Ontario, Canada; on the
World Wide Web at confocal.com), and Applied Precision Inc. Such
detection methods are particularly useful to achieve simultaneous
scanning of multiple attached template nucleic acids.
[0048] A number of approaches can be used to detect incorporation
of fluorescently-labeled nucleotides into a single nucleic acid
molecule. Optical setups include near-field scanning microscopy,
far-field confocal microscopy, wide-field epi-illumination, light
scattering, dark field microscopy, photoconversion, single and/or
multiphoton excitation, spectral wavelength discrimination,
fluorophore identification, evanescent wave illumination, and total
internal reflection fluorescence (TIRF) microscopy. In general,
certain methods involve detection of laser-activated fluorescence
using a microscope equipped with a camera. Suitable photon
detection systems include, but are not limited to, photodiodes and
intensified CCD cameras. For example, an intensified charge couple
device (ICCD) camera can be used. The use of an ICCD camera to
image individual fluorescent dye molecules in a fluid near a
surface provides numerous advantages. For example, with an ICCD
optical setup, it is possible to acquire a sequence of images
(movies) of fluorophores.
[0049] Some embodiments of the present invention use TIRF
microscopy for two-dimensional imaging. TIRF microscopy uses
totally internally reflected excitation light and is well known in
the art. See, e g., the World Wide Web at
nikon-instruments.jp/eng/page/products/tirf.aspx. In certain
embodiments, detection is carried out using evanescent wave
illumination and total internal reflection fluorescence microscopy.
An evanescent light field can be set up at the surface, for
example, to image fluorescently-labeled nucleic acid molecules.
When a laser beam is totally reflected at the interface between a
liquid and a solid substrate (e.g., a glass), the excitation light
beam penetrates only a short distance into the liquid. The optical
field does not end abruptly at the reflective interface, but its
intensity falls off exponentially with distance. This surface
electromagnetic field, called the "evanescent wave", can
selectively excite fluorescent molecules in the liquid near the
interface. The thin evanescent optical field at the interface
provides low background and facilitates the detection of single
molecules with high signal-to-noise ratio at visible
wavelengths.
[0050] The evanescent field also can image fluorescently-labeled
nucleotides upon their incorporation into the attached
template/primer complex in the presence of a polymerase. Total
internal reflectance fluorescence microscopy is then used to
visualize the attached template/primer duplex and/or the
incorporated nucleotides with single molecule resolution.
[0051] F. Analysis
[0052] Alignment and/or compilation of sequence results obtained
from the image stacks produced as generally described above
utilizes look-up tables that take into account possible sequences
changes (due, e.g., to errors, mutations, etc.). Essentially,
sequencing results obtained as described herein are compared to a
look-up type table that contains all possible reference sequences
plus 1 or 2 base errors.
[0053] In resequencing, a preferred embodiment for sequence
alignment compares sequences obtained to a database of reference
sequences of the same length, or within 1 or 2 bases of the same
length, from the initially obtained sequence or the target sequence
contained in a look-up table format. In a preferred embodiment, the
look-up table contains exact matches with respect to the reference
sequence and sequences of the prescribed length or lengths that
have one or two errors (e.g., 9-mers with all possible 1-base or
2-base errors). The obtained sequences are then matched to the
sequences on the look-up table and given a score that reflects the
uniqueness of the match to sequence(s) in the table. The obtained
sequences are then aligned to the reference sequence based upon the
position at which the obtained sequence best matches a portion of
the reference sequence. More detail on the alignment process is
provided below in the Example.
EXAMPLE
[0054] The 7249 nucleotide genome of the bacteriophage M13mp 18 was
sequenced using single molecule methods of the invention. Purified,
single-stranded viral M13mp18 genomic DNA was obtained from New
England Biolabs. Approximately 25 ug of M13 DNA was digested to an
average fragment size of 40 bp with 0.1 U Dnase I (New England
Biolabs) for 10 minutes at 37.degree. C. Digested DNA fragment
sizes were estimated by running an aliquot of the digestion mixture
on a precast denaturing (TBE-Urea) 10% polyacrylamide gel (Novagen)
and staining with SYBR Gold (Invitrogen/Molecular Probes). The
DNase I-digested genomic DNA was filtered through a YM10
ultrafiltration spin column (Millipore) to remove small digestion
products less than about 30 nt. Approximately 20 pmol of the
filtered DNase I digest was then polyadenylated with terminal
transferase according to known methods (Roychoudhury, R and Wu, R.
1980, Terminal transferase-catalyzed addition of nucleotides to the
3' termini of DNA. Methods Enzymol. 65(1):43-62.). The average dA
tail length was 50.+-.5 nucleotides. Terminal transferase was then
used to label the fragments with Cy3-dUTP. Fragments were then
terminated with dideoxyTTP (also added using terminal transferase).
The resulting fragments were again filtered with a YM10
ultrafiltration spin column to remove free nucleotides and stored
in ddH.sub.2O at -20.degree. C.
[0055] Epoxide-coated glass slides were prepared for oligo
attachment. Epoxide-functionalized 40 mm diameter #1.5 glass cover
slips (slides) were obtained from Erie Scientific (Salem, N.H.).
The slides were preconditioned by soaking in 3.times.SSC for 15
minutes at 37.degree. C. Next, a 500 pM aliquot of 5' aminated
polydT(50) primer (polythymidine of 50 nucleotides in length with a
5' terminal amine) is incubated with each slide for 30 minutes at
room temperature in a volume of 80 ml. The resulting slides have
primer attached by direct amine linkage to the epoxide. The slides
are then treated with phosphate (1 M) for 4 hours at room
temperature in order to passivate the surface. Slides re then
stored in polymerase rinse buffer (20 mM Tris, 100 mM NaCl, 0.001%
Triton X-100, pH 8.0) until they are used for sequencing.
[0056] For sequencing, the slides are placed in a modified FCS2
flow cell (Bioptechs, Butler, Pa.) using a 50 um thick gasket The
flow cell is placed on a movable stage that is part of a
high-efficiency fluorescence imaging system built around a Nikon
TE-2000 inverted microscope equipped with a total internal
reflection (TIR) objective. The slide is then rinsed with HEPES
buffer with 100 mM NaCl and equilibrated to a temperature of
50.degree. C. An aliquot of poly(dT50) template is placed in the
flow cell and incubated on the slide for 15 minutes. After
incubation, the flow cell is rinsed with 1.times.SSC/HEPES/0.1% SDS
followed by HEPES/NaCl. A passive vacuum apparatus is used to pull
fluid across the flow cell. The resulting slide contains M13
template/primer duplex. The temperature of the flow cell is then
reduced to 37.degree. C. for sequencing and the objective is
brought into contact with the flow cell.
[0057] For sequencing, cytosine triphosphate, guanidine
triphosphate, adenine triphosphate, and uracil triphosphate, each
having a cyanine-5 label (at the 7-deaza position for ATP and GTP
and at the C5 position for CTP and UTP (PerkinElmer)) are stored
separately in buffer containing 20 mM Tris-HCl, pH 8.8, 10 mM
MgSO.sub.4, 10 mM (NH.sub.4).sub.2SO.sub.4, 10 mM HCl, and 0.1%
Triton X-100, and 100U Klenow exo.sup.- polymerase (NEN).
Sequencing proceeds as follows.
[0058] First, initial imaging is used to determine the positions of
duplex on the epoxide surface. The Cy3 label attached to the M13
templates is imaged by excitation using a laser tuned to 532 nm
radiation (Verdi V-2 Laser, Coherent, Inc., Santa Clara, Calif.) in
order to establish duplex position. For each slide only single
fluorescent molecules imaged in this step are counted. Imaging of
incorporated nucleotides as described below is accomplished by
excitation of a cyanine-5 dye using a 635 nm radiation laser
(Coherent). 5 uM Cy5CTP is placed into the flow cell and exposed to
the slide for 2 minutes. After incubation, the slide is rinsed in
1.times.SSC/15 mM HEPES/0.1% SDS/pH 7.0 ("SSC/HEPES/SDS") (15 times
in 60 ul volumes each, followed by 150 mM HEPES/150 mM NaCl/pH 7.0
("HEPES/NaCl") (10 times at 60 ul volumes). An oxygen scavenger
containing 30% acetonitrile and scavenger buffer (134 ul
HEPES/NaCl, 24 ul 100 mM Trolox in MES, pH6.1, 10 ul DABCO in MES,
pH6.1, 8 ul 2M glucose, 20 ul NaI (50 mM stock in water), and 4 ul
glucose oxidase) is next added. The slide is then imaged (500
frames) for 0.2 seconds using an Inova301K laser (Coherent) at 647
nm, followed by green imaging with a Verdi V-2 laser (Coherent) at
532 nm for 2 seconds to confirm duplex position. The positions
having detectable fluorescence are recorded. After imaging, the
flow cell is rinsed 5 times each with SSC/HEPES/SDS (60 ul) and
HEPES/NaCl (60 ul). Next, the cyanine-5 label is cleaved off
incorporated CTP by introduction into the flow cell of 50 mM TCEP
for 5 minutes, after which the flow cell is rinsed 5 times each
with SSC/HEPES/SDS (60 ul) and HEPES/NaCl (60 ul). The remaining
nucleotide is capped with 50 mM iodoacetamide for 5 minutes
followed by rinsing 5 times each with SSC/HEPES/SDS (60 ul) and
HEPES/NaCl (60 ul). The scavenger is applied again in the manner
described above, and the slide is again imaged to determine the
effectiveness of the cleave/cap steps and to identify
non-incorporated fluorescent objects.
[0059] The procedure described above is then conducted 100 nM
Cy5dATP, followed by 100 nM Cy5dGTP, and finally 500 nM Cy5dUTP.
The procedure (expose to nucleotide, polymerase, rinse, scavenger,
image, rinse, cleave, rinse, cap, rinse, scavenger, final image) is
repeated exactly as described for ATP, GTP, and UTP except that
Cy5dUTP is incubated for 5 minutes instead of 2 minutes. Uridine is
used instead of Thymidine due to the fact that the Cy5 label is
incorporated at the position normally occupied by the methyl group
in Thymidine triphosphate, thus turning the dTTP into dUTP. In all
64 cycles (C, A, G, U) are conducted as described in this and the
preceding paragraph.
[0060] Once the desired number of cycles are completed, the image
stack data (i.e., the single molecule sequences obtained from the
various surface-bound duplex) are aligned to the M13 reference
sequence. The image data obtained can be compressed to collapse
homopolymeric regions. Thus, the sequence "TCAAAGC" is represented
as "TCAGC" in the data tags used for alignment. Similarly,
homopolymeric regions in the reference sequence are collapsed for
alignment.
[0061] The alignment algorithm matches sequences obtained as
described above with the actual M13 linear sequence. Placement of
obtained sequence on M13 is based upon the best match between the
obtained sequence and a portion of M13 of the same length, taking
into consideration 0, 1, or 2 possible errors. All obtained 9-mers
with 0 errors (meaning that they exactly match a 9-mer in the M13
reference sequence) are first aligned with M13. Then 10-, 11-, and
12-mers with 0 or 1 error are aligned. Finally, all 13-mers or
greater with 0, 1, or 2 errors are aligned.
[0062] The template fragments are removed by increasing the
temperature of the flow cell above the melting temperature of the
duplex, thereby releasing the template fragments from the duplexes.
The free templates are removed from the flow cell by washing the
flow cell, for example the flow cell can be rinsed 5 times each
with SSC/HEPES/SDS (60 ul) and HEPES/NaCl (60 ul).
[0063] The primers are then modified by adding a polynucleotide
sequence to the 3' terminus of the primer. The
oligonucleotide-modified primers are then used as the template in
subsequent polymerization reactions. Free primer capable of
hybridizing to the added oligonucleotide is added to the flow cell
and incubated under conditions sufficient to allow hybridization
between the added oligonucleotide portion of the template and the
free primer. After incubation, the flow cell is rinsed with
1.times.SSC/HEPES/0.1% SDS followed by HEPES/NaCl. The resulting
slide contains template/primer duplexes where the template
comprises the original primer having M13 template complementary
sequences added thereto and modified with an oligonucleotide. The
temperature of the flow cell is then reduced to 37.degree. C. for
sequencing and the objective is brought into contact with the flow
cell. The procedure (expose to nucleotide, polymerase, rinse,
scavenger, image, rinse, cleave, rinse, cap, rinse, scavenger,
final image) is repeated as described above.
[0064] Once the desired number of cycles is completed, the image
stack data (i.e., the single molecule sequences obtained from the
various surface-bound duplex) are aligned to the M13 reference
sequence and/or are aligned to the sequence initially obtained as
described above. The image data obtained can be compressed to
collapse homopolymeric regions as described above.
[0065] The invention may be embodied in other specific forms
without departing from the spirit or essential characteristics
thereof. The foregoing embodiments are therefore to be considered
in all respects illustrative rather than limiting on the invention
described herein. Scope of the invention is thus indicated by the
appended claims rather than by the foregoing description, and all
changes which come within the meaning and range of equivalency of
the claims are therefore intended to be embraced therein.
* * * * *