U.S. patent application number 12/370514 was filed with the patent office on 2009-08-13 for method for archiving and clonal expansion.
Invention is credited to Nurith Kurn.
Application Number | 20090203531 12/370514 |
Document ID | / |
Family ID | 40939211 |
Filed Date | 2009-08-13 |
United States Patent
Application |
20090203531 |
Kind Code |
A1 |
Kurn; Nurith |
August 13, 2009 |
Method for Archiving and Clonal Expansion
Abstract
The present method provides methods, libraries, and kits related
to the archiving and clonal expansion of sequences related to
target polynucleotide sequences. The method allow for the
attachment of polynucleotides with defined 3' and or 5' sequences
to solid surfaces. The polynucleotides attached to the solid
substrates can be stored or archived as libraries and can
subsequently be retrieved for analysis, for example by clonal
expansion. In some embodiments, nucleotides attached to solid
surfaces can be used for sequencing of nucleotide sequences related
to target RNA or target RNA. The methods are applicable to total
RNA and/or total DNA analysis.
Inventors: |
Kurn; Nurith; (Palo Alto,
CA) |
Correspondence
Address: |
WILSON SONSINI GOODRICH & ROSATI
650 PAGE MILL ROAD
PALO ALTO
CA
94304-1050
US
|
Family ID: |
40939211 |
Appl. No.: |
12/370514 |
Filed: |
February 12, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61028146 |
Feb 12, 2008 |
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61074991 |
Jun 23, 2008 |
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61085811 |
Aug 1, 2008 |
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Current U.S.
Class: |
506/6 ;
506/26 |
Current CPC
Class: |
C12Q 1/686 20130101;
C12Q 1/686 20130101; C12Q 2525/191 20130101; C12Q 2531/101
20130101; C12Q 2537/1373 20130101; C12Q 1/686 20130101; C12Q
2525/191 20130101; C12Q 2525/301 20130101 |
Class at
Publication: |
506/6 ;
506/26 |
International
Class: |
C40B 20/08 20060101
C40B020/08; C40B 50/06 20060101 C40B050/06 |
Claims
1-329. (canceled)
330. A method for amplifying a DNA or RNA target nucleic acid
sequence or its complement on a solid support to form a plurality
of amplified products comprising clonally amplifying said target
sequence or its complement by: (i). linear amplification; (ii).
amplification using a single primer; (iii). amplification from a
double-stranded nucleic acid having a 3' overhang; (iv).
amplification using a DNA-RNA chimeric primer; or (v). (i), (ii),
(iii), and (iv); wherein said target nucleic acid sequence is
coupled to said solid support.
331. The method of claim 330 wherein said amplification is
isothermal.
332. The method of claim 330 wherein said solid support is a bead
or an array.
333. The method of claim 330 wherein said amplification results in
at least 50, 100, 500, 1,000, 5,000 copies of the target nucleic
acid sequence.
334. The method of claim 330, wherein said solid surface comprises
a plurality of primers of substantially identical sequence.
335. The method of claim 330, wherein said target nucleic acid
sequence is from a linear template.
336. The method of claim 330, wherein said target nucleic acid
sequence is greater than 200 base pairs.
337. A method for clonally amplifying a target nucleic acid
sequence or its complement comprising: delivering the target
nucleic acid sequence or its complement into an emulsion; and
performing linear amplification of said target nucleic acid
sequence inside said emulsion.
338. The method of claim 337 wherein said amplification results in
a plurality of non-multimerized amplified products.
339. The method of claim 330 or 337 wherein the amplified products
have fewer than 2 errors for every 100,000 nucleotides.
340. A method comprising: (a) extending a first primer or a set of
first primers hybridized to a target nucleic acid with a DNA
polymerase comprising a DNA segment and a 5' RNA segment, wherein a
3' portion of the primer, sequence (P) is complementary to a target
nucleic acid and a portion of the 5' RNA segment, sequence (A), is
not complementary to the target nucleic acid, to form a first
primer extension product or set of first primer extension products
hybridized to the target nucleic acid; (b) separating or removing
the first primer extension product or the set of first primer
extension products from the target nucleic acid; (c) extending a
second primer with a DNA polymerase comprising a 3' DNA segment and
a 5' DNA segment, wherein a portion of the 3' DNA segment is
complementary to the first primer extension product or set of first
primer extension products and a 5' portion, sequence (B), of the of
the second primer is not complementary to the first primer
extension product or the set of first primer extension products, to
produce a double-stranded DNA product or a set of double-stranded
DNA products, each product comprising the first primer extension
product hybridized to a second primer extension product, whereby
the second primer extension product has a DNA sequence (A') that is
complementary and hybridized to the RNA sequence (A) of the first
primer extension product at its 3' end, thereby forming the double
stranded nucleic acid product or products with an RNA/DNA
heteroduplex at one end; (d) cleaving the RNA from the RNA/DNA
heteroduplex; (e) annealing a chimeric oligonucleotide comprising a
5' end wherein the 5' end comprises sequence (C), and the 5' end
further comprises RNA, and a 3' end wherein the 3' end comprises
the DNA sequence (A), to the double stranded nucleic acid product
or products; and (f) extending the double stranded nucleic acid
product or products with a DNA polymerase to produce a sequence
(C)--(C') RNA-DNA heteroduplex.
341. The method of claim 340 wherein the second primer further
comprises a ligand at a 5' end of the 5' DNA segment.
342. The method of claim 340 further comprising binding the ligand
to a solid surface, whereby the first and second primer extension
product or products are bound to the solid surface.
343. The method of claims 340 or 342 further comprising an
amplification with the steps of: (g) cleaving the RNA portion of
the chimeric oligonucleotide in the DNA-RNA heteroduplex, whereby
sequence (C') of step (f) is single stranded. (h) annealing a
chimeric amplification primer to the single stranded portion of the
second primer extension product complementary to sequence (C),
wherein the amplification primer has a DNA portion and a 5' RNA
portion; (i) extending the amplification primer with a DNA
polymerase having strand displacement activity to produce a third
primer extension product hybridized to the second primer extension
product; (j) cleaving the RNA from the third primer extension
product hybridized to the second primer extension product in the
RNA-DNA heteroduplex; and (k) repeating steps (h) to (j) to produce
multiple copies of third primer extension product comprising a 5'
end and a 3' end, wherein the 5' end comprises sequence (A) and the
3' end comprises sequence (B').
344. The method of claim 340 wherein the 3' portion of the first
primer that is complementary to the target nucleic acid comprises a
random nucleotide sequence.
345. The method of claim 340 using a set of first primers wherein
the members of the set of first primers each comprises a distinct
3' DNA annealing sequence, each specific for a target or region of
template nucleic acid.
346. The method of claim 340 wherein the 3' portion of the first
primer that is complementary to the target nucleic acid comprises a
degenerate or a random sequence for annealing to multiple target
sequences.
347. The method of claim 340 wherein the target nucleic acid is
RNA.
348. The method of claim 340 wherein the target nucleic acid is
DNA.
349. The method of claim 340 wherein, in step (b), the target RNA
is cleaved by heat, enzyme treatment, or chemical treatment or
enzymes.
350. The method of claim 347 wherein the 3' portion of the first
primer that is complementary to the target RNA comprises a sequence
that is complementary to polyadenosine (poly-A).
351. The method of claim 347 wherein the first primer comprises a
mixture of a random and a poly-A-directed first primer, wherein the
3' portion of the random first primer comprises a random sequence,
and wherein the 3' portion of the poly-A-directed first primer
comprises a sequence that is complementary to polyadenosine
(poly-A).
352. The method of claim 347 wherein the RNA target is contained
within a sample that also comprises DNA, and actinomycin is added
prior to step (a) to selectively inhibit the production of
extension product complementary to the DNA during step (a).
353. The method of claim 340 wherein the 3' segment of the second
primer complementary to a portion of the first primer extension
product comprises one or more sequences selected from the group
consisting of a random nucleotide sequence, a specific sequence
complementary to a specific sequence of the first primer extension
product, and a sequence common to multiple first primer extension
products.
354. The method of claim 343 wherein the amplification is a clonal
amplification.
355. The method of claim 343 wherein the solid surface is a bead or
an isolated area on a surface.
356. The method of claim 355 wherein the bead or isolated area is
the only bead or isolated area within isolated liquid volume such
that the amplified product is contained within such liquid
volume.
357. The method of claim 356 wherein the liquid volume is an
aqueous droplet within a non-aqueous fluid.
358. The method of claim 357 wherein the droplet is part of a
microemulsion.
359. The method of claim 356 wherein the liquid volume is a well in
a plate.
360. The method of claim 356 wherein the bead or isolated area
comprises covalently attached at their 5' ends thereto multiple
oligonucleotides comprising sequence (B), whereby upon the
amplification of step (k) multiple copies of the third primer
extension product comprising sequence (B') at their 3' end are
hybridized to the bead or isolated area.
361. A method of producing a bead or isolated area with multiple
copies of a nucleotide sequence covalently attached thereto by
performing the method of claim 360 further comprising extending the
oligonucleotide comprising sequence (B) to produce a multiple
polynucleotides covalently attached to the bead or isolated area
that are substantially complementary to the third primer extension
product, wherein the multiple polynucleotides comprise sequence
(A') near their 3' ends and sequence (B) near their 5' ends.
362. A sequencing method comprising performing the method of claim
361, further comprising the steps of removing the third primer
extension product to render the covalently attached polynucleotides
single-stranded, and extending a sequencing primer, a portion of
which is complementary to a portion of sequence (A') to produce
detectable oligonucleotide fragments characteristic of the sequence
of the polynucleotide bound to the bead or isolated area.
363. The method of claim 362 wherein the sequencing method
comprises cleavable labeled terminators.
364. The method of claim 362 wherein the sequencing method
comprises pyrophosphate detection.
365. The method of claim 362 wherein the sequencing method is an
isothermal sequencing method.
366. The method of claim 362 wherein the sequencing method
comprises cycle sequencing.
367. The method of claim 362 wherein the sequencing method
comprises sequencing by ligation.
368. A method for generating an amplified nucleic acid of
comprising: (a) providing a first nucleic acid or a set of first
nucleic acids comprising DNA corresponding to a region of a target
nucleic acid and further comprising a 5' end and a 3' end, wherein
said 5' end of the first nucleic or the set of first nucleic acids
comprises a sequence (A) and a sequence (C), wherein said sequence
(A) is 3' of sequence (C), and wherein sequence (C) comprises RNA,
and wherein said 3' end of the first nucleic acid comprises
sequence (B'); (b) hybridizing said first nucleic acid or set of
first nucleic acids to a solid support comprising an
oligonucleotide comprising a sequence (B) complementary to the
sequence (B') of the first nucleic acid; (c) extending the
oligonucleotide to produce a double stranded product comprising a
second nucleic acid hybridized to the first nucleic acid, wherein
the second nucleic acid comprises a 3' segment complementary to a
portion of the first nucleic acid and a 5' segment complementary to
a portion of the first nucleic acid, whereby a portion of the 3'
segment comprises a sequence (A') and a portion of the 3' segment
comprises a sequence (C'), and wherein the sequence (A') is 5' of
the sequence (C'), and whereby a portion of the 5' segment
comprises a sequence (B).
369. The method of claim 368 wherein the solid support comprises a
substantially planar array.
370. The method of claim 368 wherein the solid support comprises a
bead.
371. The method of claim 370 wherein the bead comprises a magnetic
bead.
372. The method of claim 370 wherein the bead comprises only one
copy of the first and second nucleic acid.
373. The method of claim 368, wherein the target nucleic acid is
RNA.
374. The method of claim 368, wherein the target nucleic acid is
DNA.
375. The method of claim 368, wherein a plurality of first and
second nucleic acids are provided corresponding to different
sequences in the target nucleic acid.
376. The method of claim 375 wherein the plurality of first and
second nucleic acids are bound to one or a plurality of beads or a
plurality of isolated areas on a surface.
377. The method of claim 376 wherein the plurality of first and
second nucleic acids are bound to a plurality of beads under
conditions such that generally, one or fewer first and second
nucleic acid is bound to one bead or one isolated area on a
surface.
378. The method of claim 368 further comprising amplification by
treating the beads or isolated areas with reagents to produce
multiple copies of an amplification product complementary to all or
a portion of the second nucleic acid.
379. The method of claim 378 wherein the plurality of beads or
isolated areas are contained within a plurality of isolated volumes
such that generally one or fewer beads or isolated area is
associated with each isolated volume, and whereby the production of
multiple copies of amplification product results in multiple copies
of substantially one amplification product in each volume.
380. The method of claim 379 wherein the amplification is carried
out with a reaction mixture comprising RNase H, an amplification
primer comprising a DNA portion and a 5' RNA portion, and a DNA
polymerase with strand displacement activity.
381. The method of claim 377 further comprising the step of storing
the beads or isolated areas comprising generally one or fewer
primer extension products per bead or isolated area.
382. The method of claim 381 further comprising amplification by
treating the beads or isolated areas with reagents to produce
multiple copies of an amplification product complementary to all or
a portion of the second nucleic acid after the step of storing the
beads or isolated areas, wherein the amplification is carried out
with a reaction mixture comprising RNase H, an amplification primer
comprising a DNA portion and a 5' RNA portion, and a DNA polymerase
with strand displacement activity.
Description
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional
Application Nos. 61/028,146, filed Feb. 12, 2008, 61/074,991, filed
Jun. 23, 2008, and 61/085,811, filed Aug. 1, 2008, which
applications are incorporated herein by reference in their
entirety. This application is also related to the co-pending patent
application [Attorney Docket No 25115-731.201] filed Feb. 12, 2009,
which is incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] The quality and quantity of nucleic acid sample is important
for many studies. High-throughput genomic analysis requires large
amounts of template for testing, yet typically the yield of nucleic
acids from individual patient samples is limited. Forensic and
paleoarcheology work also can be severely limited by nucleic acid
sample size. The limitation of starting material impacts the
ability to carry out large scale analysis of multiple parameters,
as is required for, for example, the genotyping of multiple loci in
the study of complex diseases. Moreover, it is well accepted that
molecular analysis determination of genomic instability in various
pathological condition such as cancer, is most precisely carried
out in well defined cell populations, such as that obtained by
laser capture micro-dissection or cell sorting. Nucleic acid
amplification technologies that provide global amplification of
very small polynucleotide samples, for example, from one or a very
few cells, may provide a solution to the limited starting materials
generally available for analysis.
[0003] Likewise, the ability to amplify ribonucleic acid (RNA) is
an important aspect of efforts to elucidate biological processes.
Total cellular mRNA represents gene expression activity at a
defined time. Gene expression is affected by cell cycle
progression, developmental regulation, response to internal and
external stimuli and the like. The profile of expressed genes for
any cell type in an organism reflects normal or disease states,
response to various stimuli, developmental stages, cell
differentiation, and the like. Non-coding RNAs have been shown to
be of great importance in regulation of various cellular functions
and in certain disease pathologies. Such RNAs are often present in
very low levels. Thus, amplification methods capable of amplifying
low abundance RNAs, are of great importance.
[0004] In addition to the need for amplifying RNA and DNA, there is
a need for being able to archive samples, and later retrieve the
samples for analysis.
[0005] Various methods for global amplification of DNA target
molecules (e.g., whole genome amplification) have been described,
including methods based on the polymerase chain reaction (PCR).
See, e.g., U.S. Pat. Nos. 5,731,171; 6,365,375; Daigo et al.,
(2001) Am. J. Pathol. 158 (5):1623-1631; Wang et al, (2001); Cancer
Res. 61:4169-4174; Zheng et al, (2001) Cancer Epidemiol.
10:697-700; Dietmaier et al (1999) Am. J. Pathol. 154 (1) 83-95;
Stoecklein et al (2002) Am. J. Pathol. 161 (1):43-51; U.S. Pat.
Nos. 6,124,120; 6,280,949; Dean et al (2002) PNAS 99 (8):5261-5266.
However, PCR-based global amplification methods, such as whole
genome amplification (WGA), may generate non-specific amplification
artifacts, give incomplete coverage of loci, or generate DNA of
insufficient length that cannot be used in many applications.
PCR-based methods also suffer from the propensity of the PCR
reaction to generate products that are preferentially amplified,
and thus resulting in biased representation of genomic sequences in
the products of the amplification reaction. Methods of global
amplification of DNA using composite primers have been described.
See e.g. U.S. patent application Ser. No. 10/824,829.
[0006] Additionally, a number of methods for the analysis of gene
expression have been developed in recent years. See, for example,
U.S. Pat. Nos. 6,251,639, 6,692,918, 6,686,156, 5,744,308;
6,143,495; 5,824,517; 5,829,547; 5,888,779; 5,545,522; 5,716,785;
5,409,818; EP 0971039A2; EP0878553A2; and U.S. published patent
applications nos. 2002/0115088, 2003/0186234, 2003/0087251, and
2004/0023271. These include quantification of specific mRNAs, and
the simultaneous quantification of a large number of mRNAs, as well
as the detection and quantification of patterns of expression of
known and unknown genes. RNA amplification is most commonly
performed using the reverse transcriptase-polymerase chain reaction
(RT-PCR) method and variations thereof. These methods are based on
replication of RNA by reverse transcriptase to form single stranded
DNA complementary to the RNA (cDNA), which is followed by
polymerase chain reaction (PCR) amplification to produce multiple
copies of double stranded DNA. However, the total amount of sample
RNA that is available is frequently limited by the amount of
biological sample from which it is derived. Biological samples are
often limited in amount and precious. Moreover, the amount of the
various RNA species is not equal; some species are more abundant
than others are, and these are more likely and easier, to analyze.
The ability to amplify RNA sequences enables the analysis of less
abundant, rare RNA species. The ability to analyze small samples,
by means of nucleic acid amplification, is also advantageous for
design parameters of large scale screening of effector molecule
libraries, for which reduction in sample volume is a major concern
both for the ability to perform very large scale screening or ultra
high throughput screening, and in view of the limiting amounts of
library components. Methods of amplification from RNA templates
have been described, for example in U.S. Pat. No. 6,946,251.
[0007] Sequencing of nucleic acids continues to be one of the most
important and useful ways to analyze DNA and RNA samples. Recent
developments have made possible highly parallel high throughput
sequencing. Many of these approaches use an in vitro cloning step
to generate many copies of each individual molecule. Emulsion PCR
is one method, isolating individual DNA molecules along with
primer-coated beads in aqueous bubbles within an oil phase. A
polymerase chain reaction (PCR) then coats each bead with clonal
copies of the isolated library molecule and these beads are
subsequently immobilized for later sequencing. See, e.g.
WO04069849A2, WO05010145A2. In other cases, surface methods of
clonal amplification have been developed, for example, by the use
of polonies (PCR colonies), or by bridge PCR where fragments are
amplified upon primers attached to a solid surface. These methods
produce many physically isolated locations which each contain many
copies of a single fragment. While these methods have provided
improvements in sequencing throughput, there is a continuing need
to improve the methods of obtaining samples appropriate for
sequencing, and of handling, storing, and amplifying such
samples.
[0008] Therefore, there is a need for improved methods of
obtaining, storing, amplifying, and analyzing DNA and RNA samples,
including methods which can globally amplify DNA or RNA
polynucleotide targets. The invention described herein fulfills
this need.
SUMMARY OF THE INVENTION
[0009] One aspect of the invention comprises amplifying a target
nucleic acid sequence (DNA or RNA) or the complement of a target
nucleic acid sequence on a solid support such as a bead, a magnetic
bead, a substantially planar array, an isolated surface, or a well
in a plate to form a plurality (e.g. 2; 3; 4; 5; 10; 25; 50; 100;
150; 500; 1,000; 5,000; 25,000 or more) of amplified products. In
some cases, the plurality of amplified products comprise clonally
amplified products such that a given bead, isolated surface, or
well contains a plurality of amplified products of substantially
identical sequence. The amplification may be performed by linear
amplification such as for example single primer isothermal
amplification (SPIA), amplification using a single primer such as
for example single primer PCR, rolling circle amplification, or
SPIA, amplification from a double stranded nucleic acid having a
single stranded 3' overhang, amplification using a DNA-RNA chimeric
amplification primer, or any combination thereof. In some
embodiments of the method, the amplification step results in at
least 1,000; 5,000; 10,000; 100,000; 1,000,000; or more copies of
the target nucleic acid or a portion thereof. In some embodiments
of the method, the amplification step is performed on a linear
template. In some embodiments of the method, the target nucleic
acid sequence and/or the amplified product is greater than 100;
200; 400; 500; 1,000; 2,000; 5,000 or more base pairs in
length.
[0010] One aspect of the invention comprises a method for clonally
amplifying a target nucleic acid sequence or its complement by
delivering a target nucleic acid sequence or a set of target
nucleic acid sequences into the aqueous phase of a microdroplet of
an emulsion and performing linear amplification of the target
nucleic acid or a portion thereof in the aqueous phase of the
emulsion such that on average each microdroplet amplifies one or
less than one of the target nucleic acid sequence or sequences, or
such that on average the step of amplification provides
microdroplets that comprise a plurality of amplified products that
are substantially identical in sequence. In some cases, the
amplification within an emulsion is performed in the presence of a
solid substrate such as a bead or isolated surface. In some cases,
the solid substrate may comprise capture moieties useful for
capturing target nucleic acid and/or amplified product. In some
cases, the amplification is performed such that on average, a given
bead or isolated surface captures one or less than one target
nucleic acid, or such that a given bead or isolated surface
captures a plurality of amplified products that are substantially
identical in sequence.
[0011] One aspect of the invention comprises a kit for performing
the methods of the present invention. The kit may be useful for
amplifying DNA or RNA for subsequent analysis such as expression
analysis including quantitative PCR or microarray analysis;
sequencing including thermocycle sequencing, dye terminator
sequencing, sequencing using the methods of Illumina/Solexa, SOLiD
(ABI), Roche/454 LifeSciences, Helicos, or Sequenom; alternatively
the kit may be useful for archiving DNA or RNA sequencing. In
another aspect, the kit may be useful for generating clonally
expanded target sequences. In yet another aspect, the kit may be
useful for generating one or a plurality of amplified products
having a defined 3' and 5' end. The kit comprises an RNA-DNA
chimeric first primer for creating a first primer extension
product, and an RNA-DNA chimeric oligonucleotide for annealing to
an end of a double stranded nucleic acid product having a single
stranded 3' overhang at one end. In some embodiments, the kit may
further comprise a polymerase having substantial
strand-displacement activity. In some embodiments, the kit may
further comprise a second primer. In some embodiments, the kit may
further comprise RNase H.
[0012] One aspect of the invention comprises a method for creating
a double stranded nucleic acid comprising an RNA-DNA heteroduplex,
wherein the double stranded nucleic acid is suitable for creating
amplified products by single primer isothermal linear amplification
that have a defined 5' and 3' end. The method comprises (a)
annealing to a template nucleic acid and extending a first primer
or a set of first primers with a DNA polymerase comprising a DNA
segment and a 5' RNA segment, wherein a 3' portion of the primer is
complementary to a target nucleic acid sequence of interest, and a
portion of the 5' RNA segment, sequence (A), is not complementary
to the target nucleic acid. The annealing and the extension
produces a first primer extension product or set of products that
is hybridized to the target nucleic acid. The extension step may be
performed with an RNA-dependant DNA polymerase from an RNA template
or a DNA-dependent DNA polymerase from a DNA template. In some
cases, step (a) may be performed in the presence of RNA and DNA and
an inhibitor such as for example actinomycin to selectively inhibit
the formation of an extension product complementary to the DNA.
[0013] The method further comprises (b) separating or removing the
first primer extension product from the target nucleic acid. The
separating step may be performed by heat, enzyme, chemical
treatment, or a combination thereof.
[0014] The method further comprises step (c) annealing and
extending a second primer with a DNA-dependent DNA polymerase. The
second primer may comprise a 3' DNA segment and a 5' DNA segment,
wherein a portion of the 3' DNA segment is complementary to the
first primer extension product or set of first primer extension
products and a 5' portion, sequence (B), is not complementary to
the first primer extension product or the set of first primer
extension products, to produce a double-stranded DNA product or a
set of double-stranded DNA products, each product comprising the
first primer extension product hybridized to a second primer
extension product. The second primer extension product may comprise
a DNA sequence (A') that is complementary to and hybridized to the
RNA sequence (A) of the first primer extension product at its 3'
end, thereby forming a double stranded nucleic acid product or
products with an RNA-DNA heteroduplex at one end.
[0015] The method further comprises step (d) cleaving the RNA from
the RNA-DNA heteroduplex. The cleaving step may be performed using
an enzyme that is specific for the RNA portion of an RNA-DNA
heteroduplex such as for example RNase H. The cleaving step may
provide a double stranded nucleic acid product or products with a
single stranded 3' overhang at one end.
[0016] The method further comprises step (e) annealing a chimeric
oligonucleotide comprising a 5' end, wherein the 5' end comprises
sequence (C), and the 5' end further comprises RNA, and a 3' end
wherein the 3' end comprises the DNA sequence (A), to the single
stranded 3' overhang of the double stranded nucleic acid product or
products formed in step (d).
[0017] The method further comprises step (f), extending the double
stranded nucleic acid product or products with a DNA polymerase to
produce a double stranded nucleic acid product with a (C)-(C')
RNA-DNA heteroduplex at one end and a (B)-(B') double stranded DNA
sequence at the other end.
[0018] In some embodiments of the method, the second primer further
comprises a ligand at a 5' end of the 5' DNA segment.
[0019] In some embodiments of the method, the method further
comprises binding the ligand to a solid surface, whereby the first
and second primer extension product or products are bound to the
solid surface.
[0020] In some embodiments of the method, the method further
comprises step (g), cleaving the RNA portion of the chimeric
oligonucleotide in the DNA-RNA heteroduplex to generate a double
stranded nucleic acid or a set of double stranded nucleic acids
with a single stranded 3' overhang at one end of sequence (C'). The
method may further comprise step (h), annealing a DNA-RNA chimeric
amplification primer to the single stranded portion of the second
primer extension product. The chimeric amplification primer may
comprise a sequence (C) that is complementary to the 3' overhang
formed in step (g). The chimeric amplification primer may comprise
a DNA portion and a 5' RNA portion. The method may further
comprises step (i), extending the amplification primer with a DNA
polymerase having strand displacement activity to produce an
amplified product hybridized to the second primer extension
product. The method may further comprise step (j), cleaving the RNA
from the amplified product hybridized to the second primer
extension product in the RNA-DNA heteroduplex; and step (k),
repeating steps (h) to (j) to produce multiple copies of amplified
product comprising a 5' end and a 3' end, wherein the 5' end
comprises sequence (A) and the end comprises sequence (B').
[0021] In some embodiments of the method, the method comprises use
of a first primer that comprises a random annealing sequence such
as for example random hexamers or random decamers. Alternatively,
the method comprises use of a set of first primers, wherein each
member of the set of first primers comprises a distinct 3' DNA
annealing sequence, each specific for a target or a region of
template nucleic acid. Alternatively, the method comprises use of a
first primer that comprises a degenerate annealing sequence for
binding to multiple related target sequences. Alternatively, the
method comprises use of a first primer that comprises a poly-T
sequence, or any sequence that substantially hybridizes to the
poly-sequence of messenger RNA. Alternatively, the first primer or
set of first primers may comprise a combination thereof.
[0022] In some embodiments of the method, the method comprises use
of a second primer that comprises a random annealing sequence such
as for example random hexamers or random decamers. Alternatively,
the method comprises use of a set of second primers, wherein each
member of the set of first primers comprises a distinct 3' DNA
annealing sequence, each specific for a target or a region of
template nucleic acid. Alternatively, the method comprises use of a
second primer that comprises a degenerate annealing sequence for
binding to multiple related target sequences. Alternatively, the
second primer or set of second primers may comprise a combination
thereof.
[0023] In some embodiments of the method, the amplified products
are attached, bound, or covalently linked to a solid surface such
as a bead. In some cases, the attached, bound, or covalently linked
amplified products comprise a clonally amplified sequence on the
surface such as a bead or an isolated area on a surface. In some
cases, the bead or isolated area on a surface is the only bead or
isolated area on a surface within an isolated liquid volume such as
an aqueous droplet in a water/oil emulsion or a liquid volume in
the well of a plate, such that the amplified product is contained
with such a liquid volume. In some cases, the bead or isolated area
comprises covalently attached oligonucleotides comprising sequence
(B), at their 3' ends, whereby upon the amplification of step (k)
multiple copies of amplified product comprising sequence (B') at
their 5' end are hybridized to the bead or isolated area.
[0024] In some embodiments of the method, the sequence (B)
covalently attached to the bead or isolated area is extended to
produce multiple polynucleotides covalently attached to the bead or
isolated area that are substantially complementary to the amplified
product, wherein the multiple polynucleotides comprise sequence
(A') near their 5' ends and sequence (B) near their 3' ends.
[0025] In some embodiments of the method, the method further
comprises removing the amplified product from the bead or isolated
area to render the covalently attached polynucleotides single
stranded, and extending a sequencing primer, a portion of which is
complementary to a portion of sequence (C') to produce a detectable
signal or detectable oligonucleotide fragments characteristic of
the sequence of the polynucleotide bound to the bead or isolated
area, and thereby perform sequencing. In some cases, the sequencing
method comprises the use of cleavable labeled terminators, dye
terminators, pyrophosphate detection, an isothermal sequencing
method, cycle sequencing, or sequencing by ligation.
[0026] One aspect of the invention comprises a method for attaching
a target nucleic acid (DNA or RNA) to a solid surface such as a
bead or an isolated area. The target nucleic acid attached thereto
may be useful for example for clonally amplifying the sequence or a
portion thereof. The method comprises step (a), providing a first
nucleic acid or a set of first nucleic acids comprising DNA
corresponding to a region of a target nucleic acid and further
comprising a 5' end and a 3' end, wherein said 5' end of the first
nucleic acid or the set of first nucleic acids comprises a sequence
(A) and a sequence (C), wherein said sequence (A) is 3' of sequence
(C), and wherein sequence (C) comprises RNA, and wherein said 3'
end of the first nucleic acid comprises sequence (B'). The first
nucleic acid may be from a variety of sources including but not
limited to the methods provided herein, single primer polymerase
chain reaction products, products, and endonuclease treated rolling
circle amplification products. The method further comprises step
(b), hybridizing the first nucleic acid or set of first nucleic
acids to a solid support comprising an oligonucleotide comprising
sequence (B) complementary to sequence (B') of the first nucleic
acid. The solid support may be a bead, a substantially planar
array, or a magnetic bead. In some cases, the bead or an isolated
area of the solid support may comprise only one copy of the first
nucleic acid. The method further comprises step (c), extending the
oligonucleotide to produce a double stranded product comprising a
second nucleic acid hybridized to the first nucleic acid, wherein
the second nucleic acid comprises a 3' segment complementary to a
portion of the first nucleic acid and a 5' segment complementary to
a portion of the first nucleic acid, whereby a portion of the 3'
segment comprises a sequence (A') and a portion of the 3' segment
comprises a sequence (C'), and wherein the sequence (A') is 5' of
the sequence (C'), and whereby a portion of the 5' segment
comprises sequence (B). In some cases, the bead or an isolated area
of the solid support may thus comprise only one copy of the first
and second nucleic acid.
[0027] In some embodiments, a plurality of first and second nucleic
acids are provided corresponding to different sequences in the
target nucleic acid. In some cases, the plurality of first and
second nucleic acids are bound to one or a plurality of beads or
isolated areas on a surface. In some cases, the plurality of first
and second nucleic acids are bound to a plurality of beads or
isolated areas on a surface such that generally, or on average, one
or fewer than one first and second nucleic acids are bound to one
bead or isolated area.
[0028] In some embodiments, the method further comprises amplifying
the target nucleic acid by treating the first and second nucleic
acids bound to the beads or isolated areas with reagents to produce
multiple copies of amplification product in an clonal fashion (i.e.
multiple copies of substantially one amplification product on each
bead or isolated area) that are complementary to all or a portion
of the second nucleic acid product. In some cases, the reagents
comprise SPIA amplification reagents including but not limited to a
DNA-RNA chimeric amplification primer, a DNA-polymerase with
substantial strand-displacement activity, and RNase H. In some
cases, the bead or isolated area are stored prior to the step of
amplification.
[0029] One aspect of the invention comprises a method for attaching
a polynucleotide sequence that is representative of a sequence
within a nucleic acid target molecule to a solid surface
comprising: (a) extending a first primer comprising a DNA segment
and a 5' RNA segment, wherein a 3' portion of the primer, sequence
(P), is complementary to a target nucleic acid and a 5' portion of
the of the primer, sequence (A), is not complementary to the target
nucleic acid, to form a first primer extension product hybridized
to the target nucleic acid; (b) separating or removing the first
primer extension product from the target nucleic acid; (c)
extending a second primer to produce a double stranded product
comprising a second primer extension product hybridized to the
first primer extension product, wherein the second primer comprises
a 3' segment complementary to a portion of the first primer
extension product and a ligand, whereby a portion of the 3' end of
the second primer extension product comprises a sequence (A') that
is complementary to the sequence (A) of the of the first primer;
and (d) binding the ligand to a receptor bound to a solid surface
whereby the second primer extension product is attached to the
solid surface.
[0030] One aspect of the invention comprises a method comprising:
(a) extending a first primer comprising a DNA segment and a 5' RNA
segment, wherein a 3' portion of the primer is complementary to a
target RNA and a 5' portion, sequence (A), of the of the primer is
not complementary to the target RNA; to form a first primer
extension product hybridized to the target RNA, forming an RNA/DNA
hybrid; (b) cleaving the target RNA from the RNA/DNA hybrid; and
(c) extending a second primer, comprising a ligand and a 3' segment
complementary to a portion of the first primer extension product,
to produce a double stranded product with a DNA/RNA heteroduplex at
one end; wherein the double stranded product comprises a second
primer extension product hybridized to the first primer extension
product, and whereby a portion of the 3' end of the second primer
extension product comprises a sequence (A') that is complementary
to the sequence (A) of the of the first primer.
[0031] In some embodiments of the method, the 3' portion of the
primer that is complementary to the target RNA comprises a random
nucleotide sequence. In some embodiments of the method the 3'
portion of the primer that is complementary to the target RNA
comprises a sequence that is complementary to polyadenosine
(poly-A). In some embodiments of the method the 3' portion of the
primer that is complementary to the target RNA comprises a specific
sequence that is complementary to a multiplicity of targets. In
some embodiments of the method, in step (b), the target RNA is
cleaved by heat, enzyme treatment, or chemical treatment.
[0032] In some embodiments of the method the RNA target is in a
sample that also comprises DNA, and wherein actinomycin is added
prior to step (a) to selectively inhibit the production of
extension product complementary to the DNA during step (a).
[0033] In some embodiments of the method the 3' segment of the
second primer complementary to a portion of the first primer
extension product comprises a random nucleotide sequence, a
specific sequence complementary to a specific sequence of the first
primer extension product, or a sequence common to multiple first
primer extension products. In some embodiments of the method the
second primer further comprises a nucleotide sequence (B) that is
not complementary to the first primer extension product sequence.
In some embodiments of the method the method further comprises step
(d) of binding the ligand to a solid surface, whereby the second
primer extension product becomes bound to the solid surface. In
some embodiments of the method the binding of the ligand results in
the double stranded product being bound to the solid surface. In
some embodiments of the method the binding of the ligand results in
the single stranded second primer extension product being bound to
the solid surface.
[0034] In some embodiments of the method the method further
comprises treating the solid surface with reagents to produce
multiple copies of an amplification product that are substantially
complementary the second primer extension product. In some
embodiments of the method the solid surface comprises a
substantially planar array. In some embodiments of the method the
solid surface comprises a bead. In some embodiments of the method
the bead comprises a magnetic bead. In some embodiments of the
method the bead comprises only one copy of second primer extension
product. In some embodiments of the method the second primer
extension product is single-stranded. In some embodiments of the
method the second primer extension product is double-stranded.
[0035] In some embodiments of the method a plurality of second
primer extension products are produced corresponding to different
sequences in the target RNA. In some embodiments of the method the
plurality of second primer extension products is bound to one or a
plurality of beads or a plurality of isolated areas on a surface.
In some embodiments of the method the plurality of second primer
extension products is bound to a plurality of beads under
conditions such that generally, one or fewer second primer
extension products is bound to one bead or one isolated area on a
surface.
[0036] In some embodiments of the method the method further
comprises treating the beads or isolated areas with reagents to
produce multiple copies of an amplification product complementary
to all or a portion of the second primer extension products. In
some embodiments of the method the plurality of beads or isolated
areas are contained within a plurality of isolated volumes such
that generally one or fewer beads or isolated area is associated
with each isolated volume, and whereby the production of multiple
copies of amplification product results in multiple copies of
substantially one amplification product in each volume. In some
embodiments of the method the amplification is carried out with a
reaction mixture comprising RNase H, an amplification primer
comprising a DNA portion and a 5' RNA portion, and a DNA polymerase
with strand displacement activity.
[0037] In some embodiments of the method the method further
comprises the step of storing the beads or isolated areas
comprising generally one or fewer primer extension products per
bead or isolated area.
[0038] In some embodiments of the method the method further
comprises clonally amplifying the primer extension product bound to
the beads or isolated areas after storing them.
[0039] One aspect of the invention comprises a method for
amplifying a nucleic acid representative of a target RNA comprising
carrying out steps (a) through (d) above and further comprises the
steps of: (e) cleaving the RNA in the heteroduplex from the first
primer extension product such that a portion of the second primer
extension product that is complementary to sequence (A) is single
stranded; (f) annealing an amplification primer to the single
stranded portion of the second primer extension product
complementary to sequence (A), wherein the amplification primer has
a DNA portion and a 5' RNA portion; (g) extending the amplification
primer with a DNA polymerase having strand displacement activity to
produce an amplified product hybridized to the second primer
extension product; (h) cleaving the RNA from the amplified product
hybridized to the second primer extension product; and repeating
steps (f) to (h) to produce multiple copies of amplified
product.
[0040] In some embodiments of the method the second primer further
comprises a sequence (B that is not complementary to the first
primer extension product sequence, whereby the amplification
product comprises a sequence at its 3' end which is complementary
to (B). In some embodiments of the method the target RNA comprises
messenger RNA.
[0041] One aspect of the invention comprises a method comprising:
(a) denaturing a double-stranded target DNA; (b) annealing to the
target DNA and extending with a DNA polymerase comprising strand
displacement activity, a first primer comprising a DNA segment and
a 5' RNA segment, wherein a 3' portion of the primer comprises a
random sequence, and a 5' portion of the of the primer comprises
sequence (A), which is not complementary to the target DNA; to form
a plurality of first primer extension products, each with sequence
(A) at its 5' end; and (c) extending a second primer comprising a
ligand and a 3' DNA region that comprises a random sequence,
wherein the primer is optionally a tailed primer comprising a
nucleic acid sequence (B) that is 5' of the random sequence, to
form a plurality of double-stranded products each comprising a
first primer extension product and a second primer extension
product whereby the second primer extension product comprises a
ligand;
[0042] In some embodiments of the method step (b) comprises a first
incubation at a temperature below about 30.degree. C., and a second
incubation at a temperature above about 40.degree. C. In some
embodiments of the method a DNA polymerase which is active at
temperatures above about 45.degree. C. is used to extend the first
primer.
[0043] In some embodiments of the method the method further
comprises step (d) of binding the ligand to a solid surface,
whereby the plurality of second primer extension products become
bound to the solid surface.
[0044] In some embodiments of the method the binding of the ligand
results in the double stranded product being bound to the solid
surface. In some embodiments of the method the binding of the
ligand results in the single stranded second primer extension
product being bound to the solid surface.
[0045] In some embodiments of the method the method further
comprises treating the solid surface with reagents to produce
multiple copies of amplification products that are substantially
complementary to the plurality of second primer extension products.
In some embodiments of the method the solid surface comprises a
substantially planar array. In some embodiments of the method the
solid surface comprises a plurality of beads or a plurality of
isolated areas on a surface. In some embodiments of the method the
plurality of second primer extension products is bound to a
plurality of beads or isolated areas under conditions such that
generally, one or fewer copies of a single second primer extension
product is bound to one bead or one isolated area.
[0046] In some embodiments of the method the method further
comprises treating the beads or isolated areas with reagents to
produce multiple copies of an amplification product substantially
complementary to the second primer extension products. In some
embodiments of the method the plurality of beads or isolated areas
are contained within a plurality of isolated volumes such that
generally one or fewer beads or isolated area is associated with
each isolated volume, and whereby the production of multiple copies
of amplification product results in multiple copies of
substantially one amplification product in each volume. In some
embodiments of the method the amplification is carried out with a
reaction mixture comprising RNase H, an amplification primer with a
DNA portion and a 5' RNA portion, and a DNA polymerase with strand
displacement activity.
[0047] In some embodiments of the method the method further
comprises the step of storing the beads or isolated areas
comprising generally one or fewer primer extension products.
[0048] In some embodiments of the method the method further
comprises clonally amplifying the primer extension product bound to
the beads or isolated areas after storing them.
[0049] One aspect of the invention comprises a method for
amplifying a nucleic acid representative of a target DNA comprising
carrying out steps (a) through (d) and further comprising the steps
of: (e) cleaving the RNA from the first primer extension products
such that a portion of the second primer extension product that is
complementary to sequence (A) is single stranded; (f) annealing an
amplification primer to the single stranded portion of the second
primer extension products complementary to sequence (A), wherein
the amplification primer has a DNA portion and a 5' RNA portion;
(g) extending the amplification primer with a DNA polymerase having
strand displacement activity to produce an amplified product
hybridized to the second primer extension product; (h) cleaving the
RNA from the amplified product hybridized to the second primer
extension product; and repeating steps (f) to (h) to produce
multiple copies of amplified product
[0050] In some embodiments of the method the second primer
comprises a tailed primer comprising a nucleic acid sequence (B)
that is 5' of the random sequence, whereby the amplified product
comprises a portion complementary to sequence (B) at or near its 3'
end. In some embodiments of the method the target DNA is genomic
DNA. In some embodiments of the method the target DNA comprises
multiple genomes.
[0051] One aspect of the invention comprises a method for archiving
and/or clonal expansion of a nucleotide sequence comprising the
steps of: (a) obtaining a plurality of partially double-stranded
DNA products comprising a first polynucleotide and a second
polynucleotide, wherein the second polynucleotide comprises a
sequence (A') at its 3' end and a ligand, wherein the sequence (A')
portion of the second polynucleotide is single-stranded, wherein
optionally the second polynucleotide comprises a sequence (B) at or
near its 5' end; (b) attaching the partially double stranded DNA
products to a plurality of beads or a plurality of isolated areas
on a surface by binding the ligands to the bead or isolated area;
(c) annealing an amplification primer to the single stranded
portion of the second polynucleotide complementary to sequence
(A'), wherein the amplification primer has a DNA portion and a 5'
RNA portion; (d) extending the amplification primer with an enzyme
having strand displacement activity to produce a plurality of
amplified products hybridized to the second polynucleotide
products; (e) cleaving the RNA from the amplified product
hybridized to the second polynucleotide products using RNase H; and
repeating steps (c) to (e) to produce multiple copies of amplified
products.
[0052] In some embodiments of the method the ligands of the DNA
products are attached to beads or isolated areas, and on average,
one DNA product is attached to one or fewer beads or isolated
areas.
[0053] In some embodiments of the method the method further
comprises storing the plurality of beads or isolated areas for
later analysis.
[0054] In some embodiments of the method the beads or isolated
areas are stored after step (b), then later amplified with steps
(c) through (f). In some embodiments of the method the
amplification is a clonal amplification carried out in multiple
isolated volumes wherein on average, one isolated volume has one or
fewer beads or isolated areas. In some embodiments of the method
the multiple isolated volumes are droplets in a non-aqueous
phase.
[0055] One aspect of the invention comprises a method comprising:
(a) extending a first primer comprising a DNA segment and a 5' RNA
segment, wherein a 3' portion of the primer is complementary to a
target RNA and a 5' portion, sequence (A), of the of the primer is
not complementary to the target RNA; to form a first primer
extension product hybridized to the target RNA, forming an RNA/DNA
hybrid; (b) cleaving the target RNA from the RNA/DNA hybrid; (c)
extending a second primer, comprising a ligand and a 3' segment
complementary to a portion of the first primer extension product,
to produce a double stranded product with a DNA/RNA heteroduplex at
one end; wherein the double stranded product comprises a second
primer extension product hybridized to the first primer extension
product, and wherein a portion of the 3' end of the second primer
extension product comprises a sequence (A') that is complementary
to the sequence (A) of the of the first primer; (d) cleaving the
RNA in the heteroduplex from the first primer extension product
such that a portion of the second primer extension product that is
complementary to sequence (A) is single stranded; (e) annealing to
the second primer extension product an oligonucleotide comprising a
3'-DNA segment that is complementary to sequence (A') and a 5' RNA
segment comprising sequence (C); (f) optionally extending the
oligonucleotide to form an oligonucleotide extension product
hybridized to the second primer extension product; (g) extending
the second primer extension product to create a heteroduplex such
that the second primer comprises a DNA sequence (C') that is
complementary to sequence (C); and (h) cleaving the RNA from the
heteroduplex created in step (g) to produce a single-stranded
portion of the second primer extension product corresponding to
sequence (C').
[0056] In some embodiments of the method the 3' portion of the
first primer that is complementary to the target RNA comprises a
random nucleotide sequence. In some embodiments of the method the
3' portion of the first primer that is complementary to the target
RNA comprises a sequence that is complementary to polyadenosine
(poly-A). In some embodiments of the method the 3' portion of the
primer that is complementary to the target RNA comprises a specific
sequence that is complementary to a multiplicity of targets.
[0057] In some embodiments of the method the RNA target is
contained within a sample that also comprises DNA, and actinomycin
is added prior to step (a) to selectively inhibit the production of
extension product complementary to the DNA during step (a).
[0058] In some embodiments of the method, in step (b), the target
RNA is cleaved by chemical heat, or enzyme treatment.
[0059] In some embodiments of the method the 3' segment of the
second primer complementary to a portion of the first primer
extension product comprises a random nucleotide sequence, a
specific sequence complementary to a specific sequence of the first
primer extension product, or a sequence common to multiple first
primer extension products.
[0060] In some embodiments of the method the method further
comprises: (i) binding the ligand on the second primer extension
product to a solid surface. In some embodiments of the method, step
(i) of binding the ligand to the solid surface is performed before
step (h). In some embodiments of the method, step (i) of binding
the ligand to the solid surface is performed after step (h).
[0061] One aspect of the invention comprises a method of amplifying
a sequence representative of an sequence within an RNA target
molecule comprising carrying out steps (a) through (i), and further
comprising the steps of: (j) annealing an amplification primer,
wherein the amplification primer has a DNA portion and a 5' RNA
portion, to the single stranded portion of the second primer
extension product complementary to sequence (C'); (k) extending the
amplification primer with an enzyme having strand displacement
activity to produce an amplified product; (l) cleaving the RNA from
the amplified product; and (m) repeating steps (j) to (l) to
produce multiple copies of amplified product wherein the 5' portion
of the amplified product has a sequence complementary to sequence
(A').
[0062] In some embodiments of the method, the second primer further
comprises a segment (B) that is not complementary to the first
primer extension product sequence, whereby the amplified product
comprises a sequence (B') at or near its 3' end that is
substantially complementary to sequence (B), and a sequence (A)
near its 5' end that is complementary to sequence (A'). In some
embodiments of the method, the amplification is a clonal
amplification. In some embodiments of the method, the solid surface
is a bead or an isolated area on a surface. In some embodiments of
the method, the bead or isolated area is the only bead or isolated
area associated with a isolated liquid volume such that the
amplified product is contained within such liquid volume. In some
embodiments of the method, the liquid volume is an aqueous droplet
within a non-aqueous fluid. In some embodiments of the method, the
solid surface is a bead and the droplet is part of a microemulsion.
In some embodiments of the method, the liquid volume is a well in a
plate. In some embodiments of the method, the solid surface is a
substantially planar substrate.
[0063] In some embodiments of the method, the bead or isolated area
comprises covalently attached thereto multiple oligonucleotides
comprising the sequence (B) at their 3' ends, whereby upon the
amplification of step (m) multiple copies of amplified product
comprising sequence (B') at their 5' end are hybridized to the bead
or isolated area.
[0064] One aspect of the invention comprises a method of producing
a bead or isolated area with multiple copies of a nucleotide
sequence covalently attached thereto by attaching the amplified
product as described above, and further comprising extending the
(B) sequences to produce a multiple polynucleotides covalently
attached to the bead or isolated area that are substantially
complementary to the amplified product and that comprise sequence
(A') near their 5' ends.
[0065] One aspect of the invention comprises a sequencing method
comprising extending the (B) sequences as described above, further
comprising the steps of removing the amplified product to render
the covalently attached polynucleotides single-stranded, and
extending a primer to sequence (A') to produce detectable
oligonucleotide fragments characteristic of the sequence of the
polynucleotide bound to the bead or isolated area. In some
embodiments of the method, the sequencing method comprises
cleavable labeled terminators. In some embodiments of the method,
the sequencing method comprises pyrophosphate detection. In some
embodiments of the method, the sequencing method is an isothermal
sequencing method. In some embodiments of the method the sequencing
method comprises cycle sequencing.
[0066] One aspect of the invention comprises a method of performing
bridge PCR comprising creating amplified product with defined 3'
and 5' ends as described herein, and further comprising the steps
of exposing the amplified product to a solid substrate comprising
oligonucleotide sequences attached thereto complementary to the A
and B' sequences on the amplified product in the presence of
components necessary for polymerase chain reaction, and thermal
cycling the system to perform bridge PCR amplification.
[0067] One aspect of the invention comprises a method of performing
rolling circle amplification comprising creating amplified product
with defined 3' and 5' ends as described herein, and further
comprising the steps of: (n) hybridizing the amplified product to a
nucleic acid sequence comprising regions complementary to A and B'
sequences in close proximity; (o) optionally extending the gap with
a DNA polymerase enzyme; (p) ligating to form a circular nucleic
acid comprising the amplified product, and performing rolling
circle amplification by extending a primer that is complementary to
a sequence in the circular nucleic acid. In some embodiments of the
method, the primer is complementary to sequence (A), sequence (B'),
or a sequence that was between sequences (A) and (B') in the
amplified product. In some embodiments of the method, the primer is
an oligonucleotide attached to a solid surface.
[0068] One aspect of the invention comprises a method of PCR
amplification comprising creating amplified product with defined 3'
and 5' ends as described herein further comprising the steps of
amplifying the amplified product using primers complementary to
sequences (A) and (B), or using primers complementary to sequences
(A') and (B').
[0069] One aspect of the invention comprises a method of strand
displacement amplification (SDA) creating amplified product with
defined 3' and 5' ends as described herein wherein sequences (A)
and (B') in the amplified product are designed to be cleaved by a
restriction enzyme, and performing strand displacement
amplification on the amplified product.
[0070] One aspect of the invention comprises a method comprising:
(a) denaturing a double-stranded target DNA; (b) annealing to the
target DNA and extending with and enzyme comprising strand
displacement activity, a first primer comprising a DNA segment and
a 5' RNA segment, wherein a 3' portion of the primer comprises a
random sequence, and a 5' portion of the of the primer comprises
sequence (A), which is not complementary to the target DNA; to form
a plurality of first primer extension products, each with sequence
(A) at its 5' end; (c) extending a second primer comprising a
ligand and a 3' DNA region that comprises a random sequence,
wherein the primer is optionally a tailed primer comprising a
nucleic acid sequence (B) that is 5' of the random sequence, to
form a plurality of double-stranded products each comprising a
first primer extension product and a second primer extension
product whereby the second primer extension product comprises a
ligand; (d) cleaving the RNA from the first primer extension
products such that a portion of the second primer extension
products that are complementary to sequence (A) are single
stranded; (e) annealing to the second primer extension product an
oligonucleotide comprising a 3'-DNA segment that is complementary
to sequence (A') and a 5'-RNA segment comprising sequence (C); (f)
optionally extending the oligonucleotide to form a plurality of
oligonucleotide extension products hybridized to the second primer
extension products; (g) extending the second primer extensions
product to create a heteroduplex such that the second primer
extension products comprise a DNA sequence (C') that is
complementary to sequence (C); and (h) cleaving the RNA from the
heteroduplex created in step (g).
[0071] In some embodiments of the method the method further
comprises: (i) binding the ligand on the second primer extension
products to a solid surface. In some embodiments of the method,
step (i) of binding the ligand to the solid surface is performed
before step (h). In some embodiments of the method, step (i) of
binding the ligand to the solid surface is performed after step
(h).
[0072] One aspect of the invention comprises a method of amplifying
a sequence representative of an sequence within n DNA target
molecule carrying out steps (a) through (h) above, and further
comprising the steps of: (j) annealing an amplification primer,
wherein the amplification primer has a DNA portion and a 5' RNA
portion, to the single stranded portion of the second primer
extension products complementary to sequence (C'); (k) extending
the amplification primer with an enzyme having strand displacement
activity to produce a amplified products; (l) cleaving the RNA from
the amplified products; and (m) repeating steps (j) to (l) to
produce multiple copies of amplified products wherein the 5'
portion of the amplified product has a sequence complementary to
sequence (A').
[0073] In some embodiments of the method, the second primer
comprises the segment (B) that is not complementary to the first
primer extension product sequence, whereby the amplified products
comprise a sequence (B') at or near their 3' ends that is
substantially complementary to sequence (B), and a sequence (A)
near their 5' ends that is complementary to sequence (A'). In some
embodiments of the method, the amplification is a clonal
amplification. In some embodiments of the method, the solid surface
is a bead or isolated area on a surface. In some embodiments of the
method, the bead or isolated area is the only bead or isolated area
within isolated liquid volume such that the amplified product is
contained within such liquid volume. In some embodiments of the
method, the liquid volume is an aqueous droplet within a
non-aqueous fluid. In some embodiments of the method, the solid
surface is a bead and the droplet is part of a microemulsion. In
some embodiments of the method, the liquid volume is a well in a
plate. In some embodiments of the method, the solid surface is a
substantially planar substrate.
[0074] In some embodiments of the method, the bead or isolated area
comprises covalently attached multiple oligonucleotides comprising
the sequence (B) at their 3' ends, whereby upon the amplification
of step (m) multiple copies of amplified products comprising
sequence (B') at their 5' end are hybridized to the bead or
isolated area.
[0075] One aspect of the invention comprises a method of producing
a bead or isolated area with multiple copies of a nucleotide
sequence covalently attached thereto by comprising hybridizing
amplified product as described herein further comprising extending
the (B) sequences to produce a multiple polynucleotides covalently
attached to the bead or isolated area that are substantially
complementary to the amplified product and that comprise sequence
(A') near their 5' ends.
[0076] One aspect of the invention comprises a sequencing method
comprising hybridizing amplified product to a solid surface as
described herein, further comprising the steps of removing the
amplified product to render the covalently attached polynucleotides
single-stranded, and extending a primer to sequence (A') to produce
detectable oligonucleotide fragments characteristic of the sequence
of the polynucleotide bound to the bead or isolated area. In some
embodiments of the method, the sequencing method comprises
cleavable labeled terminators. In some embodiments of the method
the sequencing method comprises pyrophosphate detection. In some
embodiments of the method, the sequencing method is an isothermal
sequencing method. In some embodiments of the method, the
sequencing method comprises cycle sequencing.
[0077] One aspect of the invention comprises a method of performing
bridge PCR comprising creating amplified product with defined 3'
and 5' ends as described herein further comprising the steps of
exposing the amplified products to a solid substrate comprising
oligonucleotide sequences attached thereto complementary to the A
and B' sequences on the amplified products in the presence of
components necessary for polymerase chain reaction, and thermal
cycling the system to perform bridge PCR amplification.
[0078] One aspect of the invention comprises a method of performing
rolling circle amplification comprising creating amplified product
with defined 3' and 5' ends as described above further comprising
the steps of: (n) hybridizing the amplified products to a target
nucleic acid comprising regions complementary to A and B' sequences
in close proximity; (o) optionally extending the gap with a
polymerase enzyme; (p) ligating to form a circular nucleic acid
comprising the amplified product, and performing rolling circle
amplification by extending a primer that is complementary to a
sequence in the circular nucleic acid. In some embodiments of the
method, the primer is complementary to sequence (A), sequence (B'),
or a sequence that was between sequences (A) and (B') in the
amplified product. In some embodiments of the method, the primer is
an oligonucleotide attached to a solid surface.
[0079] One aspect of the invention comprises a method of PCR
amplification comprising creating amplified product with defined 3'
and 5' ends as described above further comprising the steps of
amplifying the amplified product using primers complementary to
sequences (A) and (B), or using primers complementary to sequences
(A') and (B').
[0080] One aspect of the invention comprises a method of strand
displacement amplification (SDA) comprising creating amplified
product with defined 3' and 5' ends as described above wherein
sequences (A) and (B') in the amplified product are designed to be
cleaved by a restriction enzyme, and performing strand displacement
amplification on the amplified product.
[0081] One aspect of the invention provides an alternative method
to produce DNA with defined 3' and 5' sequences from RNA.
[0082] One aspect of the invention comprises a method comprising:
(a) extending a first primer comprising a 3' portion complementary
to a target RNA and a 5' portion, sequence (D), not complementary
to the target RNA, to form a first primer extension product
hybridized to the target RNA, forming an RNA/DNA hybrid; (b)
cleaving the target RNA from the RNA/DNA hybrid; (c) extending a
second primer comprising a DNA segment and a 5' RNA segment,
wherein a 3' portion of the primer is complementary to the first
primer extension product and a 5' portion, sequence (E), of the of
the second primer is not complementary to the first primer
extension product, to produce a double-stranded DNA product
comprising the first primer extension product hybridized to a
second primer extension product, whereby the second primer
extension product has a sequence (D') that is complementary to
sequence (D) at its 3' end; (d) denaturing the double-stranded DNA
product; (e) annealing to the second primer extension product and
extending a third primer comprising, from its 5' end, a ligand,
optionally a sequence (F), and a sequence (D), wherein sequence (D)
is complementary to sequence (D') on the second primer extension
product to produce a double-stranded DNA product comprising the
second primer extension product hybridized to a third primer
extension product, whereby the third primer extension product
comprises a sequence (E') at its 3' end complementary to sequence
(E).
[0083] In some embodiments of the method the method further
comprises binding the ligand to a solid surface, whereby the third
primer extension product is bound to the solid surface.
[0084] One aspect of the invention comprises an amplification
method comprising carrying out steps (a) through (e) further
comprising the steps of: (f) cleaving the RNA portion of the second
primer extension product in the DNA-RNA heteroduplex, whereby
sequence (E') of the third primer extension product is single
stranded. (g) annealing an oligonucleotide comprising a 3' DNA
segment (E) that is complementary to sequence (E') and a 5' RNA
segment comprising sequence (G); (h) extending the third primer
extension product to produce a sequence (G') at its 3' end
complementary to sequence (G); cleaving the RNA from the
heteroduplex created in step (h) to produce a single-stranded
portion of the third primer extension product corresponding to
sequence (G'). In some embodiments of the method the method further
comprises binding the ligand to a solid surface, whereby the third
primer extension product comprising sequence (G') is bound to the
solid surface. In some embodiments of the method, the 3' portion of
the first primer that is complementary to the target RNA comprises
a random nucleotide sequence. In some embodiments of the method,
the 3' portion of the first primer that is complementary to the
target RNA comprises a sequence that is complementary to
polyadenosine (poly-A). In some embodiments of the method, the RNA
target is contained within a sample that also comprises DNA, and
actinomycin is added prior to step (a) to selectively inhibit the
production of extension product complementary to the DNA during
step (a).
[0085] In some embodiments of the method, in step (b), the target
RNA is cleaved by heat, enzyme treatment, or chemical treatment or
enzymes.
[0086] In some embodiments of the method, the 3' segment of the
second primer complementary to a portion of the first primer
extension product comprises a random nucleotide sequence, a
specific sequence complementary to a specific sequence of the first
primer extension product, or a sequence common to multiple first
primer extension products.
[0087] One aspect of the invention comprises a method of amplifying
a sequence representative of an sequence within an RNA target
molecule carrying out steps (a) through (i) and further comprising
the steps of: (j) annealing an amplification primer, wherein the
amplification primer has a DNA portion and a 5' RNA portion, to the
single stranded portion of the third primer extension product
complementary to sequence (G'); (k) extending the amplification
primer with an enzyme having strand displacement activity to
produce an amplified product; (l) cleaving the RNA from the
amplified product; and (m) repeating steps (j) to (l) to produce
multiple copies of amplified product wherein the 5' portion of the
amplified product has a sequence (E) complementary to sequence (E')
and the 3' end of the amplified product has sequence (D')
complementary to sequence (D) and optionally sequence (F')
complementary to sequence (F).
[0088] In some embodiments of the method, the amplification is a
clonal amplification. In some embodiments of the method, the solid
surface is a bead or an isolated area on a surface. In some
embodiments of the method, the bead or isolated area is the only
bead or isolated area within isolated liquid volume such that the
amplified product is contained within such liquid volume. In some
embodiments of the method, the liquid volume is an aqueous droplet
within a non-aqueous fluid. In some embodiments of the method, the
droplet is part of a microemulsion. In some embodiments of the
method, the liquid volume is a well in a plate. In some embodiments
of the method, the solid surface is a substantially planar
substrate.
[0089] In some embodiments of the method, the bead or isolated area
comprises covalently attached thereto multiple oligonucleotides
comprising the sequence (D), and/or sequence (F) at their 3' ends,
whereby upon the amplification of step (m) multiple copies of
amplified product comprising sequence (D') (and/or F') at their 5'
end are hybridized to the bead or isolated area.
[0090] One aspect of the invention comprises a method of producing
a bead or isolated area with multiple copies of a nucleotide
sequence covalently attached thereto by hybridizing amplified
product as described above, further comprising extending the
oligonucleotide at the (D), and/or (F) sequences to produce a
multiple polynucleotides covalently attached to the bead or
isolated area that are substantially complementary to the amplified
product comprising sequence (E') near their 5' ends.
[0091] One aspect of the invention comprises a sequencing method
comprising producing a bead with multiple copies of a nucleotide
sequence covalently attached thereto, further comprising the steps
of removing the amplified product to render the covalently attached
polynucleotides single-stranded, and extending a primer to sequence
(E') to produce detectable oligonucleotide fragments characteristic
of the sequence of the polynucleotide bound to the bead or isolated
area. In some embodiments of the method, the sequencing method
comprises cleavable labeled terminators. In some embodiments of the
method, the sequencing method comprises pyrophosphate detection. In
some embodiments of the method, the sequencing method is an
isothermal sequencing method. In some embodiments of the method,
the sequencing method comprises cycle sequencing.
[0092] One aspect of the invention comprises alternative methods to
produce DNA with defined 3' and 5' sequences from a DNA target
[0093] One aspect of the invention comprises a method comprising:
(a) denaturing a double-stranded target DNA; (b) annealing to the
target DNA and extending a first primer comprising a 3' portion
comprising a random sequence and a 5' portion, sequence (D), which
is not complementary to the target DNA, to form a plurality of
first primer extension products, each comprising sequence (D) at
its 3' end; (c) extending a second primer comprising a DNA segment
and a 5' RNA segment, wherein a 3' portion comprises a random
sequence, and a 5' portion, sequence (E), of the of the second
primer is not complementary to the first primer extension products,
to produce a plurality of double-stranded DNA products comprising a
first primer extension product hybridized to a second primer
extension product, whereby the second primer extension products
have a sequence (D') that is complementary to sequence (D) at their
3' ends; (d) denaturing the double-stranded DNA products; e)
annealing to the second primer extension products and extending a
third primer comprising, from its 5' end, a ligand, optionally a
sequence (F), and a sequence (D), wherein sequence (D) is
complementary to sequence (D') on the second primer extension
products to produce double-stranded DNA products comprising second
primer extension products hybridized to third primer extension
products, whereby the third primer extension products comprise a
sequence (E') at its 3' end complementary to sequence (E) in a
DNA-RNA heteroduplex.
[0094] In some embodiments of the method the method further
comprises binding the ligand to a solid surface, whereby the third
primer extension products are bound to the solid surface.
[0095] In some embodiments of the method the method further
comprises the steps of: (f) cleaving the RNA portion of the second
primer extension products in the DNA-RNA heteroduplex, whereby
sequence (E') of the third primer extension products is single
stranded. (g) annealing an oligonucleotide comprising a 3' DNA
segment (E) that is complementary to sequence (E') and a 5' RNA
segment comprising sequence (G); (h) extending the third primer
extension products to produce a sequence (G') at their 3' ends
complementary to sequence (G); (i) cleaving the RNA from the
heteroduplex created in step (h) to produce single-stranded
portions of the third primer extension products corresponding to
sequence (G').
[0096] In some embodiments of the method the method further
comprises binding the ligand to a solid surface, whereby the third
primer extension products comprising sequence (G') are bound to the
solid surface.
[0097] One aspect of the invention comprises a method of amplifying
a sequence representative of a sequence within a DNA target
molecule comprising following the steps (a) through (i), further
comprising the steps of: (j) annealing an amplification primer to
the single stranded portion of the third primer extension products
complementary to sequence (G'); wherein the amplification primer
has a DNA portion and a 5' RNA portion, (k) extending the
amplification primer with an enzyme having strand displacement
activity to produce a amplified products; (l) cleaving the RNA from
the amplified products; and (m) repeating steps (j) to (l) to
produce multiple copies of amplified products wherein the 5'
portion of the amplified products have a sequence (E) complementary
to sequence (E') and the 3' end of the amplified products have
sequence (D') complementary to sequence (D) and optionally sequence
(F') complementary to sequence (F).
[0098] In some embodiments of the method, the amplification is a
clonal amplification. In some embodiments of the method, the solid
surface is a plurality of beads or a plurality of isolated areas on
a surface.
[0099] In some embodiments of the method, each bead or isolated
area in the plurality of beads or isolated areas is the only bead
or isolated area within isolated liquid volume such that the
amplified product corresponding to the sequence on that bead or
isolated area is associated with such liquid volume. In some
embodiments of the method, the liquid volume is an aqueous droplet
within a non-aqueous fluid. In some embodiments of the method, the
droplet is part of a microemulsion. In some embodiments of the
method, the liquid volume is a well in a plate. In some embodiments
of the method, the solid surface is a substantially planar
substrate.
[0100] In some embodiments of the method, the bead or isolated area
comprises covalently attached thereto multiple oligonucleotides
comprising the sequence (D), and/or sequence (F) at their 3' ends,
whereby upon the amplification of step (m) multiple copies of
amplified product comprising sequence (D') (and/or sequence (F') at
their 5' end are hybridized to the bead or isolated area.
[0101] One aspect of the invention comprises a method of producing
a bead or isolated area with multiple copies of a nucleotide
sequence covalently attached thereto further comprising extending
the (D), and/or (F) sequences to produce a multiple polynucleotides
covalently attached to the bead or isolated area that are
substantially complementary to the amplified product comprising
sequence (E') near their 5' ends.
[0102] One aspect of the invention comprises a sequencing method
comprising creating amplified product with defined 3' and 5' ends
as described above, further comprising the steps of removing the
amplified product to render the covalently attached polynucleotides
single-stranded, and extending a primer to sequence (E') to produce
detectable oligonucleotide fragments characteristic of the sequence
of the polynucleotide bound to the bead or isolated area. In some
embodiments of the method, the sequencing method comprises
cleavable labeled terminators. In some embodiments of the method,
the sequencing method comprises pyrophosphate detection. In some
embodiments of the method, the sequencing method is an isothermal
sequencing method. In some embodiments of the method, the
sequencing method comprises cycle sequencing.
[0103] In one aspect the invention comprises a library of nucleic
acid sequences.
[0104] One aspect of the invention comprises a library comprising a
plurality of double-stranded oligonucleotides each of the
oligonucleotides comprising: (a) a first strand comprising DNA
which has, proceeding from its 5' end (i) a ligand, (ii) a specific
sequence (B, D, or DF), (iii) a sequence corresponding to or
complementary to a sequence within a nucleic acid target, (iv) a
specific sequence (A' or E'); and a specific sequence (C' or G');
and (b) a second strand having from its 5' end, (i) a specific RNA
sequence (C or G) complementary to specific sequence C' or G', (ii)
a specific sequence (A or E) complementary to sequence (A' or E'),
(iii) a sequence complementary to or corresponding to a sequence
within a target nucleic acid. In some embodiments, the ligands are
bound to a solid surface. In some embodiments, the solid surface
comprises a plurality of beads. In some embodiments, each of the
plurality of beads comprises a single molecule of double-stranded
oligonucleotide. In some embodiments, the solid surface comprises a
plurality of isolated areas on a surface.
[0105] One aspect of the invention relates to kits comprising
reagents that can be used, for example, for carrying out the
methods of the invention.
[0106] One aspect of the invention comprises kit comprising: (a) a
first primer comprising a 3'-DNA portion and a 5'-RNA portion,
wherein the 5' RNA portion further comprises sequence (A); (b) a
second primer comprising a 5'-ligand; (c) an RNA dependent DNA
polymerase; (d) a DNA dependent DNA polymerase with strand
displacement activity; (e) RNase H; and (f) an amplification
chimeric primer comprising a 3'-DNA portion and a 5'-RNA portion
wherein the sequence of the amplification primer is the substantial
the same sequence as the (A) sequence. In some embodiments, the
3'-DNA portion of the first primer comprises a random sequence. In
some embodiments, the second primer is a DNA primer that comprises
a random sequence at its 3' end. In some embodiments, the 3'-DNA
portion of the first primer comprises a random sequence, and the
second primer is a DNA primer that comprises a random sequence at
its 3' end.
[0107] One aspect of the invention comprises a kit comprising: (a)
a first primer comprising a 3'-DNA portion and a 5'-RNA portion,
wherein the 5' RNA portion further comprises sequence (A); (b) a
second primer comprising a 5'-ligand; (c) an RNA dependent DNA
polymerase; (d) a DNA dependent DNA polymerase with strand
displacement activity; (e) RNase H; (f) a chimeric oligonucleotide
comprising a 3'-DNA portion substantially comprising sequence (A)
and a 5'-RNA sequence (C); and (g) a chimeric amplification primer
comprising a 3'-DNA portion and a 5'-RNA portion, wherein the
chimeric amplification primer comprises a sequence which is
substantially the same as sequence (C). In some embodiments, the
3'-DNA portion of the first primer comprises a random sequence. In
some embodiments, the second primer is a DNA primer that comprises
a random sequence at its 3' end. In some embodiments, the 3'-DNA
portion of the first primer comprises a random sequence, and the
second primer is a DNA primer that comprises a random sequence at
its 3' end. In some embodiments, the second primer further
comprises a sequence (B) at or near the 5'-end. In some
embodiments, the kit further comprises solid support with
immobilized receptor to the ligand on it surface. In some
embodiments, the kit further comprises solid surface with an
oligonucleotide attached to the surface by the 5'-end and
comprising a sequence substantially the same as sequence (B).
[0108] In some embodiments, the kit further comprises solid surface
with an oligonucleotide attached to the surface by the 5'-end
hybridizable to sequence (A).
[0109] In some embodiments, the kit further comprises an inhibitor
of the DNA dependent DNA polymerase. In some embodiments, the
inhibitor of the DNA dependent DNA polymerase is Actinomycin.
[0110] One aspect of the invention comprises a kit comprising: (a)
a first primer that is a tailed DNA primer comprising a 5'-tail
sequence (D); (b) a second primer that is a chimeric primer
comprising a 3'-DNA portion and a 5'-RNA portion wherein the 5'-end
comprises a tail sequence (E); (c) a third primer which is a tailed
primer comprising a 3'-sequence that comprises a sequence
substantially the same as sequence (D), optionally a 5'-tail
sequence (F), and 5'-ligand; (d) an RNA dependent DNA polymerase;
e) a DNA dependent DNA polymerase with strand displacement
activity; (f) RNase H; and (g) a chimeric amplification primer
comprising a 3'-DNA portion and a 5'-RNA portion wherein the
chimeric amplification primer comprises a sequence which is
substantially the same a sequence (E).
[0111] In some embodiments, the 3'-DNA portion of the first primer
comprises a random sequence. In some embodiments, the second primer
is a DNA primer that comprises a random sequence at its 3' end. In
some embodiments, the 3'-DNA portion of the first primer comprises
a random sequence, and the second primer is a DNA primer that
comprises a random sequence at its 3' end.
[0112] One aspect of the invention comprises a kit comprising: (a)
a first primer that is a tailed DNA primer comprising a 5'-tail
sequence (D); (b) a second primer that is a chimeric primer
comprising a 3'-DNA portion and a 5'-RNA portion wherein the 5'-end
comprises a tail sequence (E); (c) a third primer which is a tailed
primer comprising a 3'-sequence that comprises a sequence
substantially the same as sequence (D), optionally a 5'-tail
sequence (F), and 5'-ligand; (d) an RNA dependent DNA polymerase;
e) a DNA dependent DNA polymerase with strand displacement
activity; (f) RNase H; (g) a chimeric oligonucleotide comprising a
3'-DNA sequence (E) and a 5'-RNA sequence G; and (h) a chimeric
amplification primer comprising a 3'-DNA portion and a 5'-RNA
portion wherein the chimeric amplification primer comprises a
sequence which is substantially the same as sequence (G). In some
embodiments, the 3'-DNA portion of the first primer comprises a
random sequence. In some embodiments, the second primer is a DNA
primer that comprises a random sequence at its 3' end. In some
embodiments, the 3'-DNA portion of the first primer comprises a
random sequence, and the second primer is a DNA primer that
comprises a random sequence at its 3' end. In some embodiments, the
kit further comprises an inhibitor of the DNA dependent DNA
polymerase, In some embodiments, the inhibitor of the DNA dependent
DNA polymerase is Actinomycin.
[0113] One aspect of the invention comprises a method for attaching
a polynucleotide sequence that is representative of a sequence
within a nucleic acid target molecule to a solid surface
comprising: (a) extending a first primer comprising a DNA segment
and a 5' RNA segment, wherein a 3' portion of the primer, sequence
(P), is complementary to a target nucleic acid and a 5' portion of
the of the primer, sequence (A), is not complementary to the target
nucleic acid, to form a first primer extension product hybridized
to the target nucleic acid; (b) separating or removing the first
primer extension product from the target nucleic acid; (c)
extending a second primer to produce a double-stranded product
comprising a second primer extension product hybridized to the
first primer extension product, wherein the second primer comprises
a 3' segment complementary to a portion of the first primer
extension product and 5' segment non-complementary sequence (B) to
the first primer extension product, whereby a portion of the 3' end
of the second primer extension product comprises a sequence (A')
that is complementary to the sequence (A) of the of the first
primer and a portion of the 5' end of the second primer extension
product comprises non-complementary sequence (B); (d) adding an
exonuclease to the double-stranded DNA/RNA hybrid, whereby single
stranded 3' nucleotides are removed from the first primer extension
product; (e) extending the first primer extension product to
produce a sequence (B'), complementary to sequence (B) on the
second primer extension product; (f) denaturing the first and
second primer extension products; (g) binding the sequence (B') of
the first primer extension product to a third primer comprising
sequence (B) bound to a solid surface, whereby the first primer
extension product is attached to the solid surface; and (h)
extending the sequence (B) of the third primer to produce a
double-stranded product comprising a third primer extension product
hybridized to the first primer extension product, wherein the 5'
end of the third primer comprises a sequence (B) complementary to
the sequence (B') of the first primer extension product, whereby a
portion of the 3' end of the third primer extension product
comprises a sequence (A') that is complementary to the sequence (A)
of the of the first primer.
[0114] One aspect of the invention comprises a method comprising:
(a) extending a first primer comprising a DNA segment and a 5' RNA
segment, wherein a 3' portion of the primer is complementary to a
target RNA and a 5' portion, sequence (A), of the of the primer is
not complementary to the target RNA; to form a first primer
extension product hybridized to the target RNA, forming an RNA/DNA
hybrid; (b) removing the target RNA from the RNA/DNA hybrid; (c)
extending a second primer, comprising a 3' segment complementary to
a portion of the first primer extension product and a 5' segment
non-complementary to the first primer extension product comprising
sequence (B), to produce a double-stranded DNA product with a
DNA/RNA heteroduplex at one end, wherein the double-stranded
product comprises a second primer extension product hybridized to
the first primer extension product and wherein a portion of the 3'
end of the second primer extension product comprises a sequence
(A') that is complementary to the sequence (A) of the of the first
primer; (d) adding an exonuclease to the double-stranded DNA
product, whereby single stranded 3' nucleotides are removed from
the 3' region of the first primer extension product that is not
hybridized to the second primer extension product; (e) extending
the first primer extension product to produce a sequence (B'),
complementary to sequence (B) on the second primer extension
product; (f) denaturing the double-stranded DNA product; (g)
attaching the single-stranded first primer extension product to a
solid support by annealing sequence (B') to the solid support
comprising an oligonucleotide attached thereto, comprising a
sequence (B); and (h) extending sequence (B) on the solid support
to produce a third primer extension product, hybridized to the
first extension product, wherein the third primer extension product
comprises a 3' sequence (A'), whereby a DNA/RNA heteroduplex at one
end is generated.
[0115] One aspect of the invention comprises a method for
amplifying a nucleic acid representative of a target RNA comprising
carrying out steps (a) through (h) above and further comprising the
steps of: (i) cleaving the RNA region from the first polynucleotide
product hybridized to the third primer extension product using
RNase H; (j) annealing an amplification primer to sequence (A') on
the single-stranded portion of the third primer extension product,
wherein the amplification primer has a DNA portion and a 5' RNA
portion; (k) extending the amplification primer with an enzyme
having strand displacement activity to produce an amplified product
hybridized to the third primer extension product on the solid
support; (l) repeating steps (i) to (k) to produce multiple copies
of an amplified product wherein the amplified product comprises
sequence (B') at its 3' end; and (m) capturing the amplified
product on the solid support wherein the solid support comprises
sequence (B).
[0116] In some embodiments, the 3' portion of the primer that is
complementary to the target RNA comprises a random nucleotide
sequence. In some embodiments, the 3' portion of the primer that is
complementary to the target RNA comprises a sequence that is
complementary to polyadenosine (poly-A). In some embodiments, the
3' portion of the primer that is complementary to the target RNA
comprises a specific sequence that is complementary to a
multiplicity of targets. In some embodiments, the target RNA is
cleaved by heat, enzyme treatment, or chemical treatment in step
(b).
[0117] In some embodiments, the RNA target is in a sample that also
comprises DNA, and wherein actinomycin is added prior to step (a)
to selectively inhibit the production of extension product
complementary to the DNA during step (a). In some embodiments, the
3' portion of the second primer complementary to a portion of the
first primer extension product comprises a random nucleotide
sequence, a specific sequence complementary to a specific sequence
of the first primer extension product, or a sequence common to
multiple first primer extension products.
[0118] In some embodiments, the solid support comprises a bead. In
other embodiments, the solid support comprises an isolated area. In
some embodiments, the solid support comprises a plurality of beads
or a plurality of isolated areas on a surface. In some embodiments,
the solid support comprises a substantially planar array. In some
embodiments, the bead comprises a magnetic bead. In some
embodiments, the bead comprises only one copy of the first primer
extension product.
[0119] In some embodiments, the first primer extension product is
single-stranded. In some embodiments, a plurality of first primer
extension products are produced corresponding to different
sequences in the target RNA. In some embodiments, the plurality of
first primer extension products is bound to a solid support
comprising either one or a plurality of beads or a plurality of
isolated areas on a surface. In some embodiments, the plurality of
first primer extension products is bound to a plurality of beads
under conditions such that generally, one or fewer first primer
extension products is bound to one bead or one isolated area on a
surface. In some embodiments, the plurality of beads or isolated
areas are contained within a plurality of isolated volumes such
that generally one or fewer beads or isolated area is associated
with each isolated volume, and whereby the production of multiple
copies of amplification product results in multiple copies of
substantially one amplification product in each volume.
[0120] In some embodiments, the method further comprises the step
of storing the beads or isolated areas comprising generally one or
fewer primer extension products per bead or isolated area. In some
embodiments, the method further comprises clonally amplifying the
primer extension product bound to the beads or isolated areas after
storing them. In other embodiments, the target RNA comprises
messenger RNA.
[0121] One aspect of the invention comprises a method comprising:
(a) denaturing a double-stranded target DNA; (b) annealing to the
target DNA and extending with a DNA polymerase comprising strand
displacement activity, a first primer comprising a DNA segment and
a 5' RNA segment, wherein a 3' portion of the primer comprises a
random sequence, and a 5' portion of the primer comprises sequence
(A), which is not complementary to the target DNA; to form a
plurality of first primer extension product hybridized to the
target DNA and comprising sequence (A) at its 5' end; (c)
separating the first primer extension product from the target DNA;
(d) annealing to the first primer extension product and extending a
second primer comprising a 3' complementary DNA region that
comprises a random sequence, wherein the second primer is a tailed
primer comprising a 5' sequence (B), to form a double-stranded
product comprising a first primer extension product and a second
primer extension product, whereby a double-stranded product with a
DNA/RNA heteroduplex at one end is generated; (e) adding an
exonuclease to the double-stranded DNA product, whereby single
stranded 3' nucleotides are removed from the 3' region of the first
primer extension product that is not hybridized to the second
primer extension product; (f) extending the first primer extension
product to produce a sequence (B'), complementary to sequence (B)
on the second primer extension product; (g) denaturing the
double-stranded DNA product; (h) attaching the single-stranded
first primer extension product to a solid support by annealing
sequence (B') to the solid support comprising an oligonucleotide
attached thereto, comprising a sequence (B), whereby a plurality of
first primer extension products become bound to the solid surface;
and (i) extending sequence (B) on the solid support to produce a
third primer extension product, hybridized to the first primer
extension product, comprising a 3' sequence (A'), whereby a DNA/RNA
heteroduplex at one end is generated.
[0122] One aspect of the invention comprises a method for
amplifying a nucleic acid representative of a target RNA comprising
carrying out steps (a) through (i) above and further comprising the
steps of: (j) cleaving the RNA from the first polynucleotide
product hybridized to the amplified product using RNase H; (k)
annealing an amplification primer to the single-stranded portion of
the amplified product complementary to sequence (A'), wherein the
amplification primer has a DNA portion and a 5' RNA portion; (l)
extending the amplification primer with an enzyme having strand
displacement activity to produce an amplified product hybridized to
the third primer extension product on the bead or isolated area;
(m) repeating steps (j) to (l) to produce multiple copies of an
amplified product wherein the amplified product comprises sequence
(B') at its 3' end; and (n) capturing the amplified product on the
solid support comprising sequence (B).
[0123] In some embodiments, step (b) above comprises a first
incubation at a temperature below about 30.degree. C., and a second
incubation at a temperature above about 40.degree. C. In some
embodiments, a DNA polymerase which is active at temperatures above
about 45.degree. C. is used to extend the first primer.
[0124] In some embodiments, the solid support comprises a bead. In
some embodiments, the solid support comprises an isolated area. In
some embodiments, the solid support comprises a plurality of beads
or a plurality of isolated areas on a surface. In some embodiments,
the solid support comprises a substantially planar array. In some
embodiments, the bead comprises a magnetic bead.
[0125] In some embodiments, the plurality of first primer extension
products is bound to a plurality of beads or isolated areas under
conditions such that generally, one or fewer copies of a single
first primer extension product is bound to one bead or one isolated
area. In some embodiments, the plurality of beads or isolated areas
are contained within a plurality of isolated volumes such that
generally one or fewer beads or isolated area is associated with
each isolated volume, and whereby the production of multiple copies
of amplification product results in multiple copies of
substantially one amplification product in each volume.
[0126] In some embodiments, the method further comprises the step
of storing the beads or isolated areas comprising generally one or
fewer primer extension products. In some embodiments, the method
further comprises clonally amplifying the primer extension product
bound to the beads or isolated areas after storing them. In some
embodiments, the target DNA is genomic DNA. In some embodiments,
the target DNA comprises multiple genomes.
[0127] One aspect of the invention for preparing DNA with defined
3' and 5' sequences comprises a method comprising: (a) extending a
first primer comprising a DNA segment and a 5' RNA segment, wherein
a 3' portion of the primer is complementary to a target RNA and a
5' portion, sequence (A), of the of the primer is not complementary
to the target RNA; to form a first primer extension product
hybridized to the target RNA, forming an RNA/DNA hybrid; (b)
removing the target RNA from the RNA/DNA hybrid; (c) extending a
second primer, comprising a 3' segment complementary to a portion
of the first primer extension product and a 5' segment
non-complementary to the first primer extension product comprising
sequence (B), to produce a double-stranded product with a DNA/RNA
heteroduplex at one end; wherein the double-stranded product
comprises a second primer extension product hybridized to the first
primer extension product, and whereby a portion of the 3' end of
the second primer extension product comprises a sequence (A') that
is complementary to the sequence (A) of the of the first primer;
(d) cleaving the RNA in the heteroduplex from the first primer
extension product such that a portion of the second primer
extension product that is complementary to sequence (A) is
single-stranded; (e) annealing to the second primer extension
product an oligonucleotide comprising a 3'-DNA sequence (A) that is
complementary to sequence (A') and a 5'-RNA segment comprising
sequence (C) that is non-complementary to the second primer
extension product; (f) extending the oligonucleotide at the 3'
segment to form an oligonucleotide extension product hybridized to
the second primer extension product; (g) denaturing the
double-stranded DNA product; (h) attaching the single-stranded
first primer extension product to a solid support by annealing
sequence (B') to the solid support comprising a sequence (B); and
(i) extending sequence (B) on the solid support to produce a third
primer extension product, comprising a 3' sequence (A') and (C'),
whereby a DNA/RNA heteroduplex at one end is generated.
[0128] One aspect of the invention comprises a method for
amplifying a nucleic acid representative of a target RNA comprising
carrying out steps (a) through (i) above and further comprising the
steps of: (j) cleaving the RNA from the heteroduplex polynucleotide
product hybridized to the amplified product using RNase H to
produce a single-stranded portion of the third primer extension
product corresponding to sequence (C'); (k) annealing an
amplification primer to the single-stranded portion of the third
primer extension product complementary to sequence (C'), wherein
the amplification primer has a DNA portion and a 5' RNA portion;
(l) extending the amplification primer with an enzyme having strand
displacement activity to produce an amplified product hybridized to
the third primer extension product on the solid support; (m)
repeating steps (j) to (l) to produce multiple copies of the
amplified product comprising sequences (A) and (B'); and (n)
capturing the amplified product on the solid support wherein the
solid support comprises sequence (B).
[0129] In some embodiments, the method further comprises extending
sequence (B), whereby multiple copies are bound to solid support
through sequence (B) having sequence (A') at 3' end. In some
embodiments, the method further comprises sequencing by synthesis
using a sequence (A) complementary to (A') as the priming sequence.
In some embodiments, the 3' portion of the first primer that is
complementary to the target RNA comprises a random nucleotide
sequence. In some embodiments, the 3' portion of the first primer
that is complementary to the target RNA comprises a sequence that
is complementary to polyadenosine (poly-A). In some embodiments,
the 3' portion of the primer that is complementary to the target
RNA comprises a specific sequence that is complementary to a
multiplicity of targets. In some embodiments, the RNA target is
contained within a sample that also comprises DNA, and actinomycin
is added prior to step (a) to selectively inhibit the production of
extension product complementary to the DNA during step (a). In some
embodiments, the target RNA is cleaved by chemical heat, or enzyme
treatment in step (b)
[0130] In some embodiments, the 3' segment of the second primer
complementary to a portion of the first primer extension product
comprises a random nucleotide sequence, a specific sequence
complementary to a specific sequence of the first primer extension
product, or a sequence common to multiple first primer extension
products. In some embodiments, the amplification is a clonal
amplification.
[0131] In some embodiments, the solid support is a bead. In some
embodiments, the solid support is an isolated area on a surface. In
some embodiments, the bead or isolated area is the only bead or
isolated area associated with an isolated liquid volume such that
the amplified product is contained within such liquid volume. In
some embodiments, the liquid volume is an aqueous droplet within a
non-aqueous fluid. In some embodiments, the solid surface is a bead
and the droplet is part of a microemulsion. In some embodiments,
the liquid volume is a well in a plate. In some embodiments, the
solid support is a substantially planar substrate. In some
embodiments, a method of producing a solid support with multiple
copies of a nucleotide sequence covalently attached thereto by
performing the method described above, and further comprising
extending the (B) sequences to produce multiple polynucleotides
covalently attached to the solid support that are substantially
complementary to the amplified product and that comprise sequence
(A') near their 3' ends.
[0132] In some embodiments, a sequencing method comprises
performing the method described above and further comprises the
steps of removing the third primer extension product to render the
covalently attached polynucleotides single-stranded, and extending
a primer to sequence (A) to produce detectable oligonucleotide
fragments characteristic of the sequence of the polynucleotide
bound to the bead or isolated area. In some embodiments the
sequencing method comprises cleavable labeled terminators. In some
embodiments the sequencing method comprises pyrophosphate
detection. In some embodiments the sequencing method is an
isothermal sequencing method. In some embodiments the sequencing
method comprises cycle sequencing.
[0133] In some embodiments a method of performing bridge PCR
comprising performing the method described above and further
comprising the steps of exposing the amplified product to a solid
substrate comprising oligonucleotide sequences attached thereto
complementary to the A and B' sequences on the amplified product in
the presence of components necessary for polymerase chain reaction,
and thermal cycling the system to perform bridge PCR
amplification.
[0134] In some embodiments a method of performing rolling circle
amplification comprising performing the method described previously
and further comprising the steps of: (o) hybridizing the amplified
product to a nucleic acid sequence comprising regions complementary
to A and B' sequences in close proximity; (p) optionally extending
the gap with a DNA polymerase enzyme; (q) ligating to form a
circular nucleic acid comprising the amplified product, and
performing rolling circle amplification by extending a primer that
is complementary to a sequence in the circular nucleic acid.
[0135] In some embodiments the primer is complementary to sequence
(A), sequence (B'), or a sequence that was between sequences (A)
and (B') in the amplified product. In some embodiments the primer
is an oligonucleotide attached to a solid surface.
[0136] In some embodiments, a method of PCR amplification
comprising performing the method previously described and further
comprising the steps of amplifying the amplified product using
primers complementary to sequences (A) and (B), or using primers
complementary to sequences (A') and (B'). In some embodiments, a
method of strand displacement amplification (SDA) comprising
performing the method previously described wherein sequences (A)
and (B') in the amplified product are designed to be cleaved by a
restriction enzyme, and performing strand displacement
amplification on the amplified product.
[0137] One aspect of the invention for preparing DNA with defined
3' and 5' sequences comprises a method comprising: (a) denaturing a
double-stranded target DNA; (b) annealing to the target DNA and
extending with a DNA polymerase comprising strand displacement
activity, a first primer comprising a DNA segment and a 5' RNA
segment, wherein a 3' portion of the primer comprises a random
sequence, and a 5' portion of the of the primer comprises sequence
(A), which is not complementary to the target DNA; to form a first
primer extension product hybridized to the target DNA and
comprising sequence (A) at its 5' end; (c) separating the first
primer extension product from the target DNA; (d) annealing to the
first primer extension product and extending a second primer
comprising a 3' complementary DNA region that comprises a random
sequence, wherein the second primer is a tailed primer comprising a
5' sequence (B), to form a double-stranded product comprising a
first primer extension product and a second primer extension
product, whereby a double-stranded product with a DNA/RNA
heteroduplex at one end is generated; (e) cleaving the RNA in the
heteroduplex from the first primer extension product such that a
portion of the second primer extension product that is
complementary to sequence (A) is single stranded; (f) annealing to
the second primer extension product an oligonucleotide comprising a
3'-DNA segment that is complementary to sequence (A') and a 5' RNA
segment comprising sequence (C); (g) extending the oligonucleotide
along the second primer extension product to form an
oligonucleotide extension product comprising a sequence (B'),
complementary to sequence (B) on the second primer extension
product; (h) denaturing the double-stranded DNA product; (i)
attaching the single-stranded first primer extension product to
solid support by annealing sequence (B') to the bead or isolated
area comprising a sequence (B); and (j) extending sequence (B) on
the solid support to produce a third primer extension product,
hybridized to the oligonucleotide extension product, comprising a
3' sequence (A') and (C'), whereby a DNA/RNA heteroduplex at one
end is generated.
[0138] One aspect of the invention comprises a method for
amplifying a nucleic acid representative of a target DNA comprising
carrying out steps (a) through (j) above and further comprising the
steps of: (k) cleaving the RNA from the heteroduplex polynucleotide
product hybridized to the amplified product using RNase H to
produce a single-stranded portion of the second primer extension
product corresponding to sequence (C'); (l) annealing an
amplification primer to the single-stranded portion of the
amplified product complementary to sequence (C'), wherein the
amplification primer has a DNA portion and a 5' RNA portion; (m)
extending the amplification primer with an enzyme having strand
displacement activity to produce an amplified product hybridized to
the amplified product on the bead or isolated area; and (n)
repeating steps (k) to (m) to produce multiple copies of the second
polynucleotide product comprising sequences (A) and (B').
[0139] In some embodiments, the amplification is a clonal
amplification. In some embodiments, the solid support is a bead or
isolated area on a surface. In some embodiments, the bead or
isolated area is the only bead or isolated area within isolated
liquid volume such that the amplified product is contained within
such liquid volume. In some embodiments, the liquid volume is an
aqueous droplet within a non-aqueous fluid. In some embodiments,
the solid surface is a bead and the droplet is part of a
microemulsion. In some embodiments, the liquid volume is a well in
a plate. In some embodiments, the solid support is a substantially
planar substrate. In some embodiments, the bead or isolated area
comprises covalently attached multiple oligonucleotides comprising
the sequence (B) at their 3' ends, whereby upon the amplification
of step (m), multiple copies of amplified products comprising
sequence (B') at their 5' end are hybridized to the bead or
isolated area.
[0140] In some embodiments, a method of producing a bead or
isolated area with multiple copies of a nucleotide sequence
covalently attached thereto by performing the method previously
described and further comprising extending the (B) sequences to
produce a multiple polynucleotides covalently attached to the bead
or isolated area that are substantially complementary to the
amplified product and that comprise sequence (A') near their 5'
ends.
[0141] In some embodiments, a sequencing method comprising
performing the method previously described, further comprising the
steps of removing the amplified product to render the covalently
attached polynucleotides single-stranded, and extending a primer to
sequence (A') to produce detectable oligonucleotide fragments
characteristic of the sequence of the polynucleotide bound to the
bead or isolated area. In some embodiments, the sequencing method
comprises cleavable labeled terminators. In some embodiments, the
sequencing method comprises pyrophosphate detection. In some
embodiments, the sequencing method is an isothermal sequencing
method. In some embodiments, the sequencing method comprises cycle
sequencing.
[0142] In some embodiments, a method of performing bridge PCR
comprising performing the method previously described and further
comprising the steps of exposing the amplified products to a solid
substrate comprising oligonucleotide sequences attached thereto
complementary to the A and B' sequences on the amplified products
in the presence of components necessary for polymerase chain
reaction, and thermal cycling the system to perform bridge PCR
amplification.
[0143] In some embodiments, a method of performing rolling circle
amplification comprising performing the method previously described
and further comprising the steps of: (o) hybridizing the amplified
products to a target nucleic acid comprising regions complementary
to A and B' sequences in close proximity; (p) optionally extending
the gap with a polymerase enzyme; (q) ligating to form a circular
nucleic acid comprising the amplified product, and performing
rolling circle amplification by extending a primer that is
complementary to a sequence in the circular nucleic acid.
[0144] In some embodiments, the primer is complementary to sequence
(A), sequence (B'), or a sequence that was between sequences (A)
and (B') in the amplified product. In some embodiments, the primer
is an oligonucleotide attached to a solid surface.
[0145] In some embodiments, a method of PCR amplification
comprising performing the method previously described and further
comprising the steps of amplifying the amplified product using
primers complementary to sequences (A) and (B), or using primers
complementary to sequences (A') and (B').
[0146] In some embodiments, a method of strand displacement
amplification (SDA) comprising performing the method previously
described wherein sequences (A) and (B') in the amplified product
are designed to be cleaved by a restriction enzyme, and performing
strand displacement amplification on the amplified product.
INCORPORATION BY REFERENCE
[0147] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0148] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0149] FIG. 1 illustrates a method of producing a polynucleotide
bound to a solid support wherein the polynucleotide comprises
sequences related to a target nucleic acid and comprises a defined
sequence (A') at its 3' end.
[0150] FIG. 2 illustrates a method of producing a polynucleotide
comprising a ligand, sequences related to a target RNA, and a
defined sequence (A') at its 3' end. The Figure also illustrates
binding such polynucleotide to a solid surface.
[0151] FIG. 3 illustrates a method of producing a polynucleotide
comprising a ligand, sequences related to a target DNA and a
defined sequence (A') at its 3' end. The Figure also illustrates
binding such polynucleotide to a solid surface.
[0152] FIG. 4 shows a method of producing amplified product from a
nucleotide, bound to the bead through a ligand, wherein the
amplified product comprises sequences related to the target nucleic
acid and a defined sequence (B') at its 3' end.
[0153] FIG. 5A shows a method of producing a second primer
extension product comprising, from its 5' end, a ligand, a defined
sequence (B), a sequence representative of a target polynucleotide,
a sequence (A') and a sequence (C'). The Figure also illustrates
binding the second primer extension product to a solid surface.
[0154] FIG. 5B illustrates an isothermal amplification using a
composite primer utilizing the second primer extension product
produced as illustrated in FIG. 5A bound to the bead.
[0155] FIG. 6 illustrates the capture by the solid surface
comprising oligonucleotides of sequence (B) of amplification
product having defined 3' and 5' sequences by hybridization to
(B'), the 3' defined sequence of the amplified product. The Figure
also shows the extension of the sequence (B) bound to the solid
surface to produce a polynucleotide bound to the solid surface. The
Figure also illustrates the use of sequencing primers (A) to
sequence the bound polynucleotide.
[0156] FIG. 7 shows the capture by the solid surface comprising
oligonucleotides of sequence (A') of amplification product having
defined 3' and 5' sequences by hybridization to (A), the 5' defined
sequence of the amplified product.
[0157] FIG. 8 shows how the of amplification product having defined
3' and 5' sequences can be used for analysis of sequence D by
hybridization, gap ligation, and rolling circle amplification.
[0158] FIG. 9 illustrates an alternative method of producing a
polynucleotide comprising a ligand that can be bound to a solid
support that comprises sequences related to a target nucleic acid
and comprises a defined sequence (E') at its 3' end.
[0159] FIG. 10 shows how the polynucleotide produced as shown in
FIG. 9 can be used to produce amplified product.
[0160] FIG. 11 shows how the amplification illustrated in FIG. 10
can be carried out while the polynucleotide is bound to a solid
surface.
[0161] FIG. 12 shows how a chimeric RNA/DNA oligonucleotide (G/E)
can be used with the product of the method illustrated in FIG. 9 to
produce amplified product with defined sequences at its 3' and 5'
ends.
[0162] FIG. 13 illustrates an alternative method of producing a
polynucleotide bound to a solid support wherein the polynucleotide
comprises sequences related to a target nucleic acid and comprises
a defined sequence (A') at its 3' end.
[0163] FIG. 14 illustrates an alternative method of producing a
polynucleotide bound to a solid support wherein the polynucleotide
comprises sequence B, sequences related to a target RNA, and a
defined sequence (A') at its 3' end.
[0164] FIG. 15 shows a method of producing amplified product from a
polynucleotide, bound to the bead through sequence B, wherein the
amplified product comprises sequences complementary to the target
nucleic acid and a defined sequence (B') at its 3' end.
[0165] FIG. 16 shows an alternative method of producing a
polynucleotide bound to a solid support wherein the polynucleotide
comprises sequence B, sequences related to a target DNA and a
defined sequence (A') at its 3' end. The figure also illustrates
production of amplified product from the bound polynucleotide,
wherein the amplified product comprises defined sequence B' at its
3' end.
[0166] FIG. 17 shows an alternative method of producing a
polynucleotide having defined 3' and 5' sequences from a RNA target
and a chimeric oligonucleotide primer. A chimeric oligonucleotide
extension product is bound to a solid support through sequence B'
and is used to generate a bound polynucleotide which comprises from
its 5' end, a defined sequence (B), a sequence representative of a
target polynucleotide, a sequence (A') and a sequence (C').
[0167] FIG. 18 illustrates an isothermal amplification using a
composite primer and the bound polynucleotide product produced in
FIG. 17.
[0168] FIG. 19 shows an alternative method of producing a
polynucleotide having defined 3' and 5' sequences from a DNA target
and a chimeric oligonucleotide primer. A chimeric oligonucleotide
extension product is bound to a solid support through sequence B'
and is used to generate a bound polynucleotide which comprises from
its 5' end, a defined sequence (B), a sequence representative of a
target polynucleotide, a sequence (A') and a sequence (C').
DETAILED DESCRIPTION OF THE INVENTION
[0169] The invention provides methods, compositions, and kits
useful in the analysis of nucleic acids. Some aspects of the
invention relate to the preparation of polynucleotides comprising
ligands and binding such ligands to solid surfaces for analysis and
for archiving. The polynucleotides bound to the surface are
generally produced in a manner that allows for them to be readily
manipulated, for example, by incorporating defined 3' and/or 5'
regions into the polynucleotides. The bound polynucleotides can
then treated by the methods described herein to produce amplified
product. The methods of the invention provide for producing
amplified product with defined 3' and/or 5' ends. The amplified
products having defined 3' and 5' ends can be used for further
manipulation and analysis. They can be used, for example in
molecular inversion probe (MIP) analysis.
[0170] The polynucleotides and amplified product of the present
invention generally comprise sequences that are related to (i.e.
either equivalent to or complementary to) the target RNA or target
DNA from which they are derived. In some embodiments, the methods
of the invention can be used for the global isolation and
amplification of target RNA or DNA, e.g. messenger RNA (mRNA) or
genomic DNA. Thus, the methods can be used to produce a plurality
of polynucleotides and/or amplification products having sequence
related to the target RNA or DNA, and also having defined, e.g.
universal, sequences at their 3' and/or 5' ends. This plurality of
polynucleotides and or amplification products can be representative
of the target nucleic acid or subset of the target nucleic acid
such as to comprise a library of representative sequences.
[0171] One aspect of the invention relates to the clonal
amplification of nucleic acid sequences of interest. The
polynucleotides of the present invention attached to solid
substrates can be attached in a manner in which polynucleotides of
different specific sequences representative of the target nucleic
acid are isolated from one another, for example, each attached to a
different bead, or each attached to an isolated area on a surface.
In one embodiment, a plurality of polynucleotides, each comprising
a specific sequence is bound to a plurality of beads or isolated
areas such that only one copy of each polynucleotide is bound to
each bead or each isolated surface area. These isolated
polynucleotides can then be clonally amplified such that, for
example each bead or surface area is retained within an isolated
volume. This can be accomplished with beads, for example, by using
a microemulsion in which, on average, each droplet comprises one or
fewer beads per droplet. The methods of the present invention allow
for the archiving of the polynucleotides bound to the beads and the
subsequent analysis of the polynucleotides, for example, using
clonal amplification.
[0172] In one aspect of the invention, the polynucleotide bound to
the surface is produced in a manner that is amenable to the
sequencing of the polynucleotide, thus revealing information about
the target nucleic acid from which it is derived.
Method for Generating Polynucleotide Bound to a Solid Surface
[0173] One aspect of the invention is a method for attaching a
polynucleotide sequence that is representative of a sequence within
a nucleic acid target molecule to a solid surface. The
polynucleotide sequence that is produced is representative of the
sequence within a nucleic acid target molecule if it is either the
same as, or complementary to the sequence within the target nucleic
acid. Where the target nucleic acid is double stranded, the method
can produce sequences that are representative of both of the
strands. The polynucleotide can be, for example either DNA or
RNA.
[0174] The first step of the method comprises: (a) extending a
first primer comprising a DNA segment and a 5' RNA segment, wherein
a 3' portion of the primer, sequence (P), is complementary to a
target nucleic acid, and a 5' portion of the of the primer,
sequence (A), is not complementary to the target nucleic acid, to
form a first primer extension product hybridized to the target
nucleic acid. The first primer may be extended by a DNA polymerase
such as an RNA-dependent DNA polymerase for extending the first
primer along an RNA target nucleic acid, or a DNA-dependent DNA
polymerase for extending the first primer along a DNA target
nucleic acid. In some embodiments, the 3' portion of the primer
that is complementary to the target nucleic acid is a specific
sequence. For example, where a specific region of interest of a
target nucleic acid that is known or suspected to be upstream of a
specific sequence on the target nucleic acid, sequence (P) of the
composite primer can be designed to hybridize to this specific
sequence on the target nucleic acid such that extension of the
primer results in producing a first primer extension product that
is complementary to such upstream region. The specific sequence may
be common to a family of target RNA. A combination of primers with
various specific sequences at the 3' end can also be useful. The
specific sequence may be common to a family of target RNA. A
combination of primers with various specific sequences at the 3'
end can also be useful. In some embodiments, such as where the
target nucleic acid comprises mRNA, and the mRNA comprises a
plurality of sequences, each having a 3' poly-A segment; the
specific sequence (P) can comprise a sequence that can hybridize to
the poly-A region of the mRNA, thus allowing the extension of the
first primer to produce a plurality of first primer extension
products, each of which is complementary to the region of an mRNA
molecule adjacent to the poly-A region. In some embodiments, the
sequence (P) comprises a random sequence, such that the extension
of the first primer results in a plurality of first primer
extension products complementary to the sequences adjacent to the
sequence where each random species hybridizes. The use of a random
sequence such as sequence (P) at the 3' end of the primer can be
useful for performing a global amplification of a nucleic acid
target, generating a plurality of sequences which together can
represent, for example substantially the whole sequence of the
target nucleic acid. In some embodiments, the relative amounts of
the various sequences can be used to quantitate the relative amount
of a given sequence in a sample, for example to determine the level
of expression in an mRNA sample, or to determine gene copy number
in a DNA sample.
[0175] The first primer extension product comprises a 5' portion
comprising sequence (A). Sequence A comprises RNA. In some
embodiments, sequence (A) is RNA, and sequence (P) is DNA. In other
embodiments, sequence (A) may comprise some (DNA). In some
embodiments, sequence (P) may comprise some RNA. In some
embodiments, sequence (A) and sequence (P) are adjacent.
[0176] The method further comprises step (b) separating or removing
the first primer extension product from the target nucleic acid.
The first primer extension product can be separated from the target
nucleic acid by a variety of methods. In some cases the separation
can be affected by denaturing the complex comprising the first
primer extension product and the nucleic acid. Denaturation can be
performed, for example by heating the sample, or by adding a
denaturing agent, or using a combination of heating the sample and
adding denaturing agents. Other methods of separating the first
primer extension product from the target nucleic acid involve
selectively cleaving or degrading the target nucleic acid. Where
the target nucleic acid is RNA, the cleaving or degrading can be
accomplished with an enzyme that cleaves RNA from an RNA/DNA hybrid
such as RNase H, or chemically, for example with the addition of
alkali. In some embodiments, the target nucleic acid is completely
cleaved or degraded. In other embodiments, the target nucleic acid
is only partly cleaved or degraded. The amount of cleavage or
degradation required is that amount which allows the extension of
the second primer. In some embodiments, the cleavage or degradation
is carried out partially, and the fragments of the target nucleic
acid that remain can constitute the second primer for step (c).
[0177] The method further comprises step (c) extending a second
primer to produce a double stranded product comprising a second
primer extension product hybridized to the first primer extension
product, wherein the second primer comprises a 3' segment
complementary to a portion of the first primer extension product
and a ligand, whereby a portion of the 3' end of the second primer
extension product comprises a sequence (A') that is complementary
to the sequence (A) of the of the first primer. The extension of
the second primer is carried out with a DNA polymerase as described
herein. The second primer can comprise RNA, DNA, or can be a
composite primer comprising both RNA and DNA. The second primer can
be a tailed primer having a 3' portion which is complementary to
the first primer extension product, and a 5' portion, sequence (B),
which is not complementary to the first primer extension product.
In some embodiments, the second primer can comprise a specific
primer sequence that is designed to hybridize to a specific
sequence in the first primer extension product. In some embodiments
the second primer comprises a random primer sequence that randomly
binds to the first primer extension product. Extension of the
second primer comprising a random sequence produces a plurality of
second primer extension products. The use of a random sequence at
the 3' end of the primer is useful, for example, in performing
global amplification of a target RNA or target DNA, whereby a
plurality of second primer extension products are produced which is
representative of the sequence of the target nucleic acid. In some
embodiments, for example where the first primer is designed to
hybridize to a specific sequence on a target RNA, or a sequence
common to a family of RNA targets, random priming by the second
primer may ensure amplification of the entire selected target or
family of selected targets. In some cases, random primer by the
second primer in combination with extension by a polymerase having
substantial strand-displacement activity may ensure amplification
of the entire selected target, an entire transcriptome, an entire
genome, an entire family of selected targets, or a substantial
portion thereof. In this embodiment, the second primer extension
products comprise sequences which are the same or substantially the
same as the sequences in the target nucleic acid (sense copies),
for example, the second primer extension products or the amplicons
therefrom may comprise sequences that are 95%, 99%, 99.9%, 99.999%,
99.9999%, 99.999999% or more identical to the sequences in the
target nucleic acid or their complement. The second primer
comprises a ligand that is a member of a ligand-receptor pair. In
some embodiments, the ligand is attached to the primer at the 5'
end of the primer. In some embodiments, the ligand is a small
molecule, such as biotin or digoxigenin. In some embodiments, the
receptor is an antibody, and the ligand is a molecule or portion of
a molecule recognized by the antibody.
[0178] The second primer extension product is extended such that
the 3' portion of the second primer extension product comprises a
sequence (A') which is complementary to sequence (A) of the first
primer. Since sequence (A) on the first primer extension product
comprises RNA, both DNA dependent DNA polymerase activity and RNA
dependent DNA polymerase activity are used in step (c). The primer
extension results in a product that is at least partially double
stranded.
[0179] The method further comprises step (d) binding the ligand to
a receptor bound to a solid surface whereby the second primer
extension product is attached to the solid surface. The solid
surface may comprise a bead or a set of beads, a magnetic bead or a
set of beads, a substantially planar array, a well in a plate, a
series of wells in a plate, an isolated surface, or a set of
isolated surfaces. The ligand-receptor pair may comprise any pair
of binding agents that are capable of specifically associating. For
example, the ligand receptor pair may be avidin and biotin or
biotin and avidin respectively. Similarly, the ligand-receptor pair
may be any pair of proteins, peptides or small molecules that bind
to each other. Additionally, the ligand-receptor pair may comprise
nucleic acid sequences such a RNA, DNA, peptide nucleic acids or
their analogs that bind or hybridize specifically. Other examples
of receptors include aptamers, antibodies, affibodies, enzymes, or
any protein, peptide, macromolecule, or small molecule specifically
capable of binding to a ligand. Other examples of ligands include
any protein, peptide, macromolecule, or small molecule specifically
capable of binding to a receptor.
[0180] The receptor is a member of the ligand-receptor pair such
that binding of the ligand results in attaching the second primer
extension product to the solid surface. In some embodiments, the
second primer extension product is still hybridized to the first
primer extension product when it is attached to the solid surface.
In some embodiments, the first primer extension product is removed
from the second primer extension product such that a single
stranded polynucleotide is attached to the solid surface. The
method produces a nucleic acid that is bound to a solid surface
that has a specific sequence (A') at its 3' end. The specific, or
universal, sequence (A') can be a site for primer hybridization and
further analysis or amplification of the nucleic acid bound to the
bead. As described above, in some embodiments, the nucleic acid
bound to the bead also comprises sequence (B) at or near its 5'
end.
[0181] One aspect of the invention comprises amplification of the
nucleic acid bound to the bead. In some embodiments, the
amplification is carried out using isothermal amplification using a
composite RNA/DNA primer, RNase H, and a polymerase with strand
displacement activity. For this embodiment, the sequence (A') acts
as the site to which the composite RNA/DNA amplification primer
hybridizes, allowing for amplification. In some embodiments, for
example where random sequences at the 3' end of the first and/or
second primer are used, a plurality of different nucleic acids
bound to a solid surface is created in which each of the nucleic
acids has a specific sequence (A') at its 3' end (and in some
embodiments also a specific sequence (B) at its 5' end), and where
the different nucleic acids have different intervening sequences,
wherein the intervening sequences are identical to or substantially
identical to the sequences in the target nucleic acid. The set of
bound nucleic acids thus generated can be analyzed, for example, by
sequencing in order to provide information about the sequence of
the target nucleic acid.
[0182] Step (d) of binding the polynucleotides to the solid surface
through the ligand can be carried out such that only one nucleic
acid is bound to an isolated area of a surface or only one nucleic
acid is bound to a single bead. This isolated binding of nucleic
acids can be used for clonal amplification of the specific bound
nucleic acid in that area or on that bead. Such bound, isolated
nucleic acids can also be stored and archived for later analysis,
for example by sequencing. The bound, isolated nucleic acids can be
amplified, stored, and analyzed multiple times.
[0183] A schematic exemplary of an embodiment of the invention
relating to method for generating polynucleotide bound to a solid
surface is shown in FIG. 1. Step I shows the extension of a first
primer comprising a DNA segment and a 5' RNA segment, wherein a 3'
portion of the primer, sequence (P), is complementary to a target
nucleic acid and a 5' portion of the of the primer, sequence (A),
is not complementary to the target nucleic acid, to form a first
primer extension product hybridized to the target nucleic acid. The
target nucleic acid can be, for example, DNA or RNA. The target
nucleic acid as shown is single stranded. The target nucleic acid
may be single stranded in the sample, or can be double stranded,
and rendered single stranded, for example, but denaturation with
heat. The sequence (P) can represent a specific sequence, a
sequence that will hybridize to Poly-A, a sequence common to a
plurality of regions (consensus sequence), or a random sequence.
Step II illustrates the separating or removing of the first primer
extension product from the target nucleic acid. The separation or
removing can be accomplished for instance by denaturation of the
complex, or by selective degradation of the target nucleic acid.
For example, where the target nucleic acid is RNA, the
separation/removing/degradation can be accomplished by alkali, or
by enzymatic cleavage, for example by RNase H. Step III illustrates
extending a second primer to produce a double stranded product
comprising a second primer extension product hybridized to the
first primer extension product, wherein the second primer comprises
a 3' segment complementary to a portion of the first primer
extension product and a ligand, whereby a portion of the 3' end of
the second primer extension product comprises a sequence (A') that
is complementary to the sequence (A) of the of the first primer. In
the embodiment shown the second primer comprises a sequence (B)
that is 5' to the segment that is complementary to the first primer
extension product, and the ligand is attached 5' to the sequence
(B). This step requires both DNA dependent DNA polymerase activity
and RNA dependent DNA polymerase activity to product the second
primer extension product. Step V illustrates binding of the ligand
to a receptor bound to a solid surface whereby the second primer
extension product is attached to the solid surface. In the
embodiment shown, the second primer extension product is bound to
the solid substrate in double stranded form while still hybridized
to the first primer extension product. In some embodiments, the
second primer extension product is rendered single stranded before
binding of the ligand to the solid surface. The bound second primer
extension product comprises defined sequence (B) at its 5' end and
defined sequence (A') at its 3' end. The bound second primer
extension product can be stored or archived, and later used for
analysis and amplification, for example clonal amplification as
described herein.
Method for Generating a Polynucleotide Comprising a Ligand for
Binding to a Solid Surface from an RNA Target
[0184] The invention provides methods, compositions and kits for
copying, storing, and amplifying polynucleotides having sequences
related to target ribonucleic acid (RNA) sequences. The methods
provide for amplification of a single RNA species or pool of RNA
species. The methods are suitable for, for example, generation of
libraries, including cDNA libraries. The methods can generate
single stranded RNA or DNA products, which are readily suitable for
multiplex analysis by microarray technologies, as well as
electrophoresis-based technologies such as differential display,
and for sequencing.
[0185] The methods of the invention can copy, store, and amplify of
one or more species of RNA, such as a pool of RNA sequences, and is
most particularly suitable for the amplification of all RNA (such
as whole transcriptome or total RNA) sequences in a biological
sample. Thus, one of the major advantages of the methods of the
invention is the ability to copy, store, and amplify an entire pool
of sequences, which is essential for the ability to analyze the
gene expression profile in cells, such as the cells in a biological
sample of interest. The methods of the invention have the potential
of amplifying a multiplicity, a large multiplicity, and in some
embodiments all RNA (such as whole transcriptome or total RNA in a
sample) sequences in a sample.
[0186] Insofar as many mRNAs have a unique polyA 3'-end, the
amplification initiated from the 3'-end sequence of mRNAs is most
common for preparation of cDNA libraries and subsequent sequence
analysis for determination of gene expression profiling or other
applications. The methods of the invention are similarly suited for
preparation of libraries of amplified 3'-portions of mRNAs. The
sequence of the first primer used in the methods of invention can
be designed to be complementary to a multiplicity, or all, of the
mRNA species in the sample by using random sequences, according to
methods known in the art. The methods are also useful for whole
transcriptome amplification. The methods of the invention can be
used for the total RNA in samples such as viral RNA.
[0187] The method for generating a polynucleotide comprising a
ligand for binding to a solid surface from an RNA target comprise
the step of (a) extending a first primer comprising a DNA segment
and a 5' RNA segment, wherein a 3' portion of the primer is
complementary to a target RNA and a 5' portion, sequence (A), of
the of the primer is not complementary to the target RNA; to form a
first primer extension product hybridized to the target RNA,
forming an RNA/DNA hybrid. In some embodiments, the 3' portion of
the primer that is complementary to the target RNA is a specific
sequence. For example, where a specific region of interest of a
target RNA that is known or suspected to be upstream of a specific
sequence on the target RNA, the sequence that is complementary to
the target RNA of the first primer can be designed to hybridize to
this specific sequence on the target RNA such that extension of the
primer results in producing a first primer extension product that
is complementary to such upstream region. The specific sequence may
be common to a family of target RNA. A combination of primers with
various specific sequences at the 3' end can also be useful. The
specific sequence may be common to a family of target RNA. A
combination of primers with various specific sequences at the 3'
end can also be useful. In some embodiments, such as where the
target RNA comprises mRNA, and the mRNA comprises a plurality of
sequences, each having a 3' poly-A segment; the specific sequence
that is complementary to the target RNA can comprise a sequence
that will hybridize to the poly-A region of the mRNA, thus allowing
the extension of the first primer to produce a plurality of first
primer extension products, each of which is complementary to the
region of an mRNA molecule adjacent to the poly-A region. In some
embodiments, the sequence that is complementary to the target RNA
comprises a random sequence, such that the extension of the first
primer results in a plurality of first primer extension products
complementary to the sequences adjacent to the sequence where each
random species hybridizes. The use of a random sequence at the 3'
end of the primer can be useful for performing a global
amplification of a target RNA, generating a plurality of sequences
which together can represent, for example substantially the whole
sequence of the target RNA. In some embodiments, the relative
amounts of the various sequences can be used to quantitate the
relative amount of a given sequence in a sample, for example to
determine the level of expression in an mRNA sample. In some
embodiments more than one type of sequence that is complementary to
the target RNA can be used, for instance both a primer with a
random sequence and a primer, or combination of primers with a
specific sequence complementary to RNA can be used. In some
embodiments, multiple primers comprising different specific
sequences can be used.
[0188] The method further comprises the step of: (b) cleaving the
target RNA from the RNA/DNA hybrid. In some embodiments, the
cleaving of the target RNA from the RNA/DNA hybrid involves
selectively cleaving or degrading the target RNA. In some cases the
cleaving can be affected by denaturing the complex comprising the
first primer extension product and the nucleic acid. Denaturation
can be performed, for example by heating the sample, or by adding a
denaturing agent, or using a combination of heating the sample and
adding denaturing agents. The cleaving can be accomplished with an
enzyme that cleaves RNA from an RNA/DNA hybrid such as RNase H, or
a combination of RNase enzymes, or chemically. In some embodiments,
the target RNA is completely cleaved. In other embodiments, the
target RNA is only partly cleaved or degraded. The amount of
cleaving required is that amount which will allow the extension of
the second primer. In some embodiments, the cleaving is carried out
partially, and the fragments of the target RNA that remain can
constitute the second primer for step (c).
[0189] The method further comprises: step (c) extending a second
primer, comprising a ligand and a 3' segment complementary to a
portion of the first primer extension product, to produce a double
stranded product with a DNA/RNA heteroduplex at one end; wherein
the double stranded product comprises a second primer extension
product hybridized to the first primer extension product, and
whereby a portion of the 3' end of the second primer extension
product comprises a sequence (A') that is complementary to the
sequence (A) of the of the first primer. The extension of the
second primer is carried out with a DNA polymerase as described
herein. The second primer can comprise RNA, DNA, or can be a
composite primer comprising both RNA and DNA. The second primer can
be a tailed primer having a 3' portion which is complementary to
the first primer extension product, and a 5' portion, sequence (B),
which is not complementary to the first primer extension product.
In some embodiments, the second primer can comprise a specific
primer sequence that is designed to hybridize to a specific
sequence in the first primer extension product. In some embodiments
the second primer comprises a random primer sequence that randomly
binds to the first primer extension product. Extension of the
second primer comprising a random sequence produces a plurality of
second primer extension products. The use of a random sequence at
the 3' end of the primer is useful, for example, in performing
global amplification of a target RNA, whereby a plurality of second
primer extension products are produced which is representative of
the sequence of the target RNA. In some embodiments, for example
where the first primer is designed to hybridize to a specific
sequence on a target RNA, or a sequence common to a family of RNA
targets, random priming by the second primer ensures amplification
of the entire selected target or family of selected targets. In
this embodiment, the second primer extension products comprise
sequences which are the same or substantially the same as the
sequences in the target RNA (sense copies). The second primer
comprises a ligand that is a member of a ligand-receptor pair. In
some embodiments, the ligand is attached to the primer at the 5'
end of the primer. In some embodiments, the ligand is a small
molecule, such as biotin or digoxigenin. In some embodiments, the
receptor is an antibody, and the ligand is a molecule or portion of
a molecule recognized by the antibody.
[0190] The second primer extension product is extended such that
the 3' portion of the second primer extension product comprises a
sequence (A') which is complementary to sequence (A) of the first
primer. Since sequence (A) on the first primer extension product
comprises RNA, both DNA dependent DNA polymerase activity and RNA
dependent DNA polymerase activity are used in step (c). The primer
extension results in a product that is at least partially double
stranded. The method produces a nucleic acid that comprises a
ligand allowing it to be bound to a solid surface and that has a
specific sequence (A') at its 3' end. The specific, or universal,
sequence (A') can be a site for primer hybridization and further
analysis or amplification of the nucleic acid bound to the bead. As
described above, in some embodiments, the nucleic acid attached to
the ligand also comprises sequence (B) at or near its 5' end.
[0191] In some embodiments, the sample comprising the target RNA is
in a sample that also comprises DNA. In such cases, it can be
advantageous to add a selective DNA dependent DNA polymerase
inhibitor such as actinomycin such that it is present during step
(a) to selectively inhibit the production of extension product
complementary to the DNA during step (a). The presence of a DNA
dependent DNA polymerase inhibitor such as actinomycin is
particularly advantageous when a first primer comprising a random
sequence is used, as the inhibitor allows for the selective
creation of first primer extension products to RNA without the need
of separating the RNA from the DNA. This is also advantageous when
the priming is carried out at specific target sequences since the
sequence may be the same on the DNA when the DNA and RNA in the
sample represent total nucleic acid from the same biological
entity, for example, human tissue, animal tissue, and the like. The
use of DNA dependent DNA polymerase inhibitors such as actinomycin
is described in copending applications.
[0192] In some embodiments, the method further comprises step (d)
binding the ligand to a receptor bound to a solid surface whereby
the second primer extension product is attached to the solid
surface. The receptor bound to the solid surface is a member of the
ligand-receptor pair such that binding of the ligand results in
attaching the second primer extension product to the solid surface.
In some embodiments, the second primer extension product is still
hybridized to the first primer extension product when it is
attached to the solid surface. In some embodiments, the first
primer extension product is removed from the second primer
extension product such that a single stranded polynucleotide is
attached to the solid surface. The method produces a nucleic acid
that is bound to a solid surface that has a specific sequence (A')
at its 3' end. The specific, or universal, sequence (A') can be a
site for primer hybridization and further analysis or amplification
of the nucleic acid bound to the bead. As described above, in some
embodiments, the nucleic acid bound to the bead also comprises
sequence (B) at or near its 5' end. One aspect of the invention
comprises amplification of the nucleic acid bound to the bead. In
some embodiments, the amplification is carried out using isothermal
amplification using a composite RNA/DNA primer, RNase H, and a
polymerase with strand displacement activity. For this embodiment,
the sequence (A') acts as the site to which the composite RNA/DNA
amplification primer hybridizes, allowing for amplification.
[0193] In some embodiments, the amplification of the second primer
extension product comprises the steps of: (e) cleaving the RNA in
the heteroduplex from the first primer extension product such that
a portion of the second primer extension product that is
complementary to sequence (A) is single stranded; (f) annealing an
amplification primer to the single stranded portion of the second
primer extension product complementary to sequence (A), wherein the
amplification primer has a DNA portion and a 5' RNA portion; (g)
extending the amplification primer with a DNA polymerase having
strand displacement activity to produce an amplified product
hybridized to the second primer extension product; (h) cleaving the
RNA from the amplified product hybridized to the second primer
extension product; and (i) repeating steps (f) to (h) to produce
multiple copies of amplified product. Where this method of
amplification is used, and a sequence (B) is incorporated into the
second primer extension product as described above, amplification
product is generated which comprises sequence (B') complementary to
sequence B at or near its 3' end.
[0194] In some embodiments, for example where random sequences at
the 3' end of the first and/or second primer are used, a plurality
of different nucleic acids bound to a solid surface is created in
which each of the nucleic acids has a specific sequence (A') at its
3' end (and in some embodiments also a specific sequence (B) at its
5' end), and where the different nucleic acids have different
intervening sequences, wherein the intervening sequences are
identical to or substantially identical to the sequences in the
target RNA. The set of bound nucleic acids thus generated can be
analyzed, for example, by sequencing in order to provide
information about the sequence of the target RNA.
[0195] The solid surface can be any of a variety of surfaces, some
described in more detail below. The solid surface can be, for
example a planar surface, for example, a planar array. In some
embodiments the solid surface comprises a plurality of beads. In
some embodiments the beads are magnetic.
[0196] The step of binding the polynucleotides to the solid surface
through the ligand, step (d), can be carried out such that only one
nucleic acid is bound to an isolated area of a surface or only one
nucleic acid is bound to a single bead. This isolated binding of
nucleic acids can be used for clonal amplification of the specific
bound nucleic acid in that area or on that bead. Such bound,
isolated nucleic acids can also be stored and archived for later
analysis, for example by sequencing. The bound, isolated nucleic
acids can be amplified, stored, and analyzed multiple times.
[0197] In some embodiments, the method further comprises treating
the solid surface with reagents to produce multiple copies of an
amplification product that are substantially complementary the
second primer extension product. This step comprises carrying out
an amplification reaction wherein the bound nucleic acid acts as a
template for the amplification. Generally, the amplification is
carried out using the sequence (A') on the second primer extension
product for the hybridization of primer. In some embodiments, the
amplification produces single stranded amplified product, In some
embodiments, the amplification provides double stranded product. In
some embodiments, the second primer comprises a specific sequence
(B), which becomes incorporated into the second primer extension
product. In some embodiments the amplification is an isothermal
amplification reaction comprising a composite RNA/DNA primer, RNase
H, and a DNA polymerase with strand displacement activity. In some
embodiments, the amplification is carried out using polymerase
chain reaction, (PCR). For example where the second primer
extension product comprises both as sequence (B) at or near its 5'
end and a sequence (A') at or near its 3' end, a set of primers,
one designed to hybridize to all or a portion of the sequence (A')
and the other designed to hybridize to sequence (B'), the
complement of sequence (B), can be used to carry out a PCR reaction
to exponentially produce double stranded amplified product.
[0198] In some embodiments the amplification is carried out such
that the amplified product is not attached to the substrate, but is
freely dissolved in the solution. In other embodiments, the
amplification is carried out such that the amplified product
remains bound to the substrate, for example by performing solid
phase PCR such as bridge PCR. In yet other embodiments, an
amplified product is generated that may float freely in solution,
but which comprises a sequence, for example sequence (A) or
sequence (B'), that allows it to be captured to another solid
surface or other portion of the solid surface by hybridization to a
complementary sequence bound to such surface (e.g. sequence (A') or
sequence (B). In some embodiments, the amplified product is a
single-stranded product and, because it is generated at the solid
surface, the amplified product readily captured by complementary
sequences, e.g. sequence (B), bound to the surface.
[0199] In one aspect of the invention, a plurality of beads is
used, and the methods described above are carried out such that on
average, one or fewer second primer extension product molecules are
bound per bead. The beads are dispersed into an aqueous solution,
and a plurality of microreactors, e.g. droplets, are produced such
that on average one or fewer beads is contained within each of the
plurality of microreactors. The amplification of the second primer
extension products bound to the beads is then carried out such that
the clonal amplification of each of the plurality of second primer
extension products in the separate microreactors is achieved. This
clonal amplification in microreactors can be performed on a sample
of target RNA, such as whole transcriptome or total RNA, wherein
the plurality of second primer extension products comprise
sequences that correspond to most, to substantially all, or to all
of the sequences in the target RNA. In some embodiments, the
amplified products are captured by bead having attached thereto a
plurality of oligonucleotides comprising complementary sequences
bound to such surface (e.g. sequence (A') or sequence (B)), which
are complementary to sequence (A) or sequence (B') on the amplified
product.
[0200] In some embodiments, the plurality of beads, produced as
described above, with each bead comprising a single second primer
extension product can comprise a library. These libraries can be
stored, then later clonally amplified. In some embodiments, a
library of beads can comprise a plurality of beads wherein each
bead had multiple copies of a single amplification product
generated from a second primer extension product. These libraries
can be analyzed, for example by sequencing. The libraries can be
stored, and later analyzed. In some embodiments the libraries can
be stored, then analyzed multiple times.
[0201] A schematic exemplary of an embodiment of the invention
relating to method for generating a polynucleotide comprising a
ligand for binding to a solid surface from an RNA target is shown
in FIG. 2. FIG. 2 shows a target RNA and a chimeric RNA/DNA first
primer. The primer is first annealed to the target RNA. Step I
illustrates extending a first primer comprising a DNA segment and a
5' RNA segment, wherein a 3' portion of the primer is complementary
to a target RNA and a 5' portion, sequence (A), of the of the
primer is not complementary to the target RNA; to form a first
primer extension product hybridized to the target RNA, forming an
RNA/DNA hybrid. The sequence complementary to a target RNA can be a
specific sequence, a sequence that will hybridize to Poly-A, a
sequence common to a plurality of regions (consensus sequence), or
a random sequence. Step II represents cleaving the target RNA from
the RNA/DNA hybrid. Cleaving can be accomplished thermally,
chemically, or enzymatically, e.g. with RNase H. The second primer
comprising a ligand is then annealed to the first primer extension
product. Step III(a) and step III(b) illustrate extending a second
primer, comprising a ligand and a 3' segment complementary to a
portion of the first primer extension product, to produce a double
stranded product with a DNA/RNA heteroduplex at one end; wherein
the double stranded product comprises a second primer extension
product hybridized to the first primer extension product, and
whereby a portion of the 3' end of the second primer extension
product comprises a sequence (A') that is complementary to the
sequence (A) of the of the first primer. Step III(a) shows step III
for a second primer that does not comprise sequence (B). Step
III(b) shows step III for a second primer that comprises a sequence
(B) that is 5' of the segment complementary to a portion of the
first primer extension product. In each case the second primer is
shown with a ligand attached to the 5' end. The second primer
extension product comprising a ligand and a defined sequence (A) at
its 3' end is useful for storage, archiving and analysis as it has
a ligand capable of binding to a solid surface. Such second primer
extension product also comprises a sequence that is representative
of (identical to or substantially identical to) a sequence in the
target RNA, so analysis of this product provides information about
the target RNA. Step IV shows the binding the ligand to a solid
surface, whereby the second primer extension product becomes bound
to the solid surface.
Method for Generating a Polynucleotide Comprising a Ligand for
Binding to a Solid Surface from a DNA Target
[0202] The methods of the present invention can be used to analyze
the DNA (e.g. genomic DNA) samples that are important for many
studies. The methods can be used for high-throughput genomic
analysis, and can be used for forensic and paleoarcheology work
which can be severely limited by nucleic acid sample size. The
methods can be used, for example, for the genotyping of multiple
loci in the study of complex diseases. The methods can also be used
for the determination of genomic instability in various
pathological conditions such as cancer, which is most precisely
carried out in well defined cell populations, such as that obtained
by laser capture micro-dissection or cell sorting. The DNA
amplification technologies described herein provide global
amplification of very small polynucleotide samples, for example,
from one or a very few cells.
[0203] One aspect of the invention is a method for generating from
a DNA target a polynucleotide comprising a ligand for binding to a
solid surface.
[0204] The method comprises the steps of: (a) denaturing a
double-stranded target DNA. Double stranded DNA can be denatured,
for example by heating, or by the addition of denaturing
agents.
[0205] The method further comprises step (b) annealing to the
target DNA and extending with a DNA polymerase comprising strand
displacement activity, a first primer comprising a DNA segment and
a 5' RNA segment, wherein a 3' portion of the primer comprises a
random sequence, and a 5' portion of the of the primer comprises
sequence (A), which is not complementary to the target DNA; to form
a plurality of first primer extension products, each with sequence
(A) at its 5' end. The enzyme that carries out step (b) is
generally a DNA polymerase. In some cases a mixture of DNA
polymerases can be used. The sequence that is complementary to the
target DNA comprises a random sequence, such that the extension of
the first primer results in a plurality of first primer extension
products complementary to the sequences adjacent to the sequence
where each random species hybridizes. The use of a random sequence
at the 3' end of the primer as sequence can be useful for
performing a global amplification of a DNA target, generating a
plurality of sequences which together can represent, for example
substantially the whole sequence of the target DNA. In some
embodiments, the relative amounts of the various sequences can be
used to quantitate the relative amount of a given sequence in a
sample, for example to determine the number of gene copies in a DNA
sample, or obtaining sequence information. In some embodiments, the
extension of one first primer, will result in the release of a
downstream first primer extension product. This can occur
throughout the target DNA resulting in the release of multiple
first primer extension products from the target DNA. This process
can occur simultaneously on both of the strands of the
double-stranded DNA target, thus creating first primer extension
products complementary to sequences in both strands.
[0206] In some embodiments, the first primer extension step is
carried out with a DNA polymerase capable of extension at elevated
temperature that is not compatible with subsequent hybridization of
the random sequence to the displaced primer-extension product. For
example, Bst DNA polymerase can be used which is active at elevated
temperature. The reaction can be carried out stepwise, first with
an incubation at a lower temperature such as about 25.degree. C.,
followed by an incubation at higher temperature such as about
50.degree. C. In some embodiments, the first incubation is carried
out below about 30.degree. C., and the second incubation is carried
out above about 40.degree. C. In some embodiments, a DNA polymerase
which is active at temperatures above about 45.degree. C. is used
to extend the first primer. Mixtures of DNA polymerases can also be
useful.
[0207] The method further comprises step (c) extending a second
primer comprising a ligand and a 3' DNA region that comprises a
random sequence, wherein the primer is optionally a tailed primer
comprising a nucleic acid sequence (B) that is 5' of the random
sequence, to form a plurality of double-stranded products each
comprising a first primer extension product and a second primer
extension product whereby the second primer extension product
comprises a ligand. This step may be carried out with or without
prior denaturation. If carried out without denaturation, generally,
only the single stranded displaced first primer extension product
will hybridize to the second primer. Generally the second primer
does not comprise RNA. The extension of the second primer is
carried out with a DNA polymerase as described herein. The second
primer can be a tailed primer having a 3' portion which is
complementary to the first primer extension product, and a 5'
portion, sequence (B), which is not complementary to the first
primer extension product. The second primer comprises a random
primer sequence that randomly binds to the first primer extension
product. Extension of the second primer comprising a random
sequence produces a plurality of second primer extension products.
The use of a random sequence at the 3' end of the primer is useful,
for example, in performing global amplification of a target DNA,
whereby a plurality of second primer extension products are
produced which is representative of the sequence of the target DNA.
In some embodiments, for example where the first primer is designed
to hybridize to a specific sequence on a target RNA, or a sequence
common to a family of RNA targets, random priming by the second
primer ensures amplification of the entire selected target or
family of selected targets. In this embodiment, the second primer
extension products comprise sequences which are the same or
substantially the same as the sequences in the target DNA. The
second primer comprises a ligand that is a member of a
ligand-receptor pair. In some embodiments, the ligand is attached
to the primer at the 5' end of the primer. In some embodiments, the
ligand is a small molecule, such as biotin or digoxigenin. In some
embodiments, the receptor is an antibody, and the ligand is a
molecule or portion of a molecule recognized by the antibody.
[0208] The second primer extension product is extended such that
the 3' portion of the second primer extension product comprises a
sequence (A') which is complementary to sequence (A) of the first
composite primer. Since sequence (A) on the first primer extension
product comprises RNA, both DNA dependent DNA polymerase activity
and RNA dependent DNA polymerase activity are used in step (c). The
primer extension results in a product that is at least partially
double stranded. The method produces a nucleic acid that comprises
a ligand allowing it to be bound to a solid surface and that has a
specific sequence (A') at its 3' end. The specific, or universal,
sequence (A') can be a site for primer hybridization and further
analysis or amplification of the nucleic acid bound to the bead. As
described above, in some embodiments, the nucleic acid attached to
the ligand also comprises sequence (B) at or near its 5' end.
[0209] In some embodiments, The method further comprises step (d)
binding the ligand to a receptor bound to a solid surface whereby
the second primer extension product is attached to the solid
surface. The receptor bound to the solid surface is a member of the
ligand-receptor pair such that binding of the ligand results in
attaching the second primer extension product to the solid surface.
In some embodiments, the second primer extension product is still
hybridized to the first primer extension product when it is
attached to the solid surface. In some embodiments, the first
primer extension product is removed from the second primer
extension product such that a single stranded polynucleotide is
attached to the solid surface. The method produces a nucleic acid
that is bound to a solid surface that has a specific sequence (A')
at its 3' end. The specific, or universal, sequence (A') can be a
site for primer hybridization and further analysis or amplification
of the nucleic acid bound to the bead. As described above, in some
embodiments, the nucleic acid bound to the bead also comprises
sequence (B) at or near its 5' end. One aspect of the invention
comprises amplification of the nucleic acid bound to the bead. In
some embodiments, the amplification is carried out using isothermal
amplification using a composite RNA/DNA primer, RNase H, and a
polymerase with strand displacement activity. For this embodiment,
the sequence (A') acts as the site to which the composite RNA/DNA
amplification primer hybridizes, allowing for amplification.
[0210] In some embodiments, the amplification of the second primer
extension product comprises the steps of: (e) cleaving the RNA in
the heteroduplex from the first primer extension product such that
a portion of the second primer extension product that is
complementary to sequence (A) is single stranded; (f) annealing an
amplification primer to the single stranded portion of the second
primer extension product complementary to sequence (A), wherein the
amplification primer has a DNA portion and a 5' RNA portion; (g)
extending the amplification primer with a DNA polymerase having
strand displacement activity to produce an amplified product
hybridized to the second primer extension product; (h) cleaving the
RNA from the amplified product hybridized to the second primer
extension product; and (i) repeating steps (f) to (h) to produce
multiple copies of amplified product. Where this method of
amplification is used, and a sequence (B) is incorporated into the
second primer extension product as described above, amplification
product is generated which comprises sequence (B') complementary to
sequence B at or near its 3' end.
[0211] In some embodiments, a plurality of different nucleic acids
bound to a solid surface is created in which each of the nucleic
acids has a specific sequence (A') at its 3' end (and in some
embodiments also a specific sequence (B) at its 5' end), and where
the different nucleic acids have different intervening sequences,
wherein the intervening sequences are identical to or substantially
identical to the sequences in the target DNA. The set of bound
nucleic acids thus generated can be analyzed, for example, by
sequencing in order to provide information about the sequence of
the target DNA.
[0212] The solid surface can be any of a variety of surfaces, some
described in more detail below. The solid surface can be, for
example a planar surface, for example, a planar array. In some
embodiments the solid surface comprises a plurality of beads. In
some embodiments the beads are magnetic.
[0213] The step of binding the polynucleotides to the solid surface
through the ligand, step (d), can be carried out such that only one
nucleic acid is bound to an isolated area of a surface or only one
nucleic acid is bound to a single bead. This isolated binding of
nucleic acids can be used for clonal amplification of the specific
bound nucleic acid in that area or on that bead. Such bound,
isolated nucleic acids can also be stored and archived for later
analysis, for example by sequencing. The bound, isolated nucleic
acids can be amplified, stored, and analyzed multiple times.
[0214] In some embodiments, the method further comprises treating
the solid surface with reagents to produce multiple copies of an
amplification product that are substantially complementary the
second primer extension product. This step comprises carrying out
an amplification reaction wherein the bound nucleic acid acts as a
template for the amplification. Generally, the amplification is
carried out using the sequence (A') on the second primer extension
product for the hybridization of primer. In some embodiments, the
amplification produces single stranded amplified product, In some
embodiments, the amplification provides double stranded product. In
some embodiments, the second primer comprises a specific sequence
(B), which becomes incorporated into the second primer extension
product. In some embodiments the amplification is an isothermal
amplification reaction comprising a composite RNA/DNA primer, RNase
H, and a DNA polymerase with strand displacement activity. In some
embodiments, the amplification is carried out using polymerase
chain reaction, (PCR). For example where the second primer
extension product comprises both as sequence (B) at or near its 5'
end and a sequence (A') at or near its 3' end, a set of primers,
one designed to hybridize to all or a portion of the sequence (A')
and the other designed to hybridize to sequence (B'), the
complement of sequence (B), can be used to carry out a PCR reaction
to exponentially produce double stranded amplified product.
[0215] In some embodiments the amplification is carried out such
that the amplified product is not attached to the substrate, but is
freely dissolved in the solution. In other embodiments, the
amplification is carried out such that the amplified product
remains bound to the substrate, for example by performing solid
phase PCR such as bridge PCR. In yet other embodiments, an
amplified product is generated that may float freely in solution,
but which comprises a sequence, for example sequence (A) or
sequence (B'), that allows it to be captured to another solid
surface or other portion of the solid surface by hybridization to a
complementary sequence bound to such surface (e.g. sequence (A') or
sequence (B)). In some embodiments, the amplified product is a
single-stranded product and, because it is generated at the solid
surface, the amplified product readily captured by complementary
sequences, e.g. sequence (B), bound to the surface.
[0216] In one aspect of the invention, a plurality of beads is
used, and the methods described above are carried out such that on
average, one or fewer second primer extension product molecules are
bound per bead. The beads are dispersed into an aqueous solution,
and a plurality of microreactors, e.g. droplets, are produced such
that on average one or fewer beads is contained within each of the
plurality of microreactors. The amplification of the second primer
extension products bound to the beads is then carried out such that
the clonal amplification of each of the plurality of second primer
extension products in the separate microreactors is achieved. This
clonal amplification in microreactors can be performed on a sample
of target DNA, such as genomic DNA, wherein the plurality of second
primer extension products comprise sequences that correspond to
most, to substantially all, or to all of the sequences in the
target DNA. In some embodiments, the amplified products are
captured by bead having attached thereto a plurality of
oligonucleotides comprising complementary sequences bound to such
surface (e.g. sequence (A') or sequence (B)), which are
complementary to sequence (A) or sequence (B') on the amplified
product.
[0217] In some embodiments, the plurality of beads, produced as
described above, with each bead comprising a single second primer
extension product can comprise a library. These libraries can be
stored, then later clonally amplified. In some embodiments, a
library of beads can comprise a plurality of beads wherein each
bead had multiple copies of a single amplification product
generated from a second primer extension product. These libraries
can be analyzed, for example by sequencing. The libraries can be
stored, and later analyzed. In some embodiments the libraries can
be stored, then analyzed multiple times.
[0218] A schematic exemplary of an embodiment of the invention
relating to method for generating a polynucleotide comprising a
ligand for binding to a solid surface from a DNA target is shown in
FIG. 3. Step I represents denaturing a double-stranded target DNA,
for example by raising the temperature. Steps II and III illustrate
annealing to the target DNA and extending with a DNA polymerase
comprising strand displacement activity, a first primer comprising
a DNA segment and a 5' RNA segment, wherein a 3' portion of the
primer comprises a random sequence, and a 5' portion of the of the
primer comprises sequence (A), which is not complementary to the
target DNA; to form a plurality of first primer extension products,
each with sequence (A) at its 5' end. The enzyme that carries out
step (b) is generally a DNA polymerase. In some cases a mixture of
DNA polymerases can be used. In some embodiments, a DNA polymerase
with strand displacement activity is used such that a growing first
primer extension product can displace a downstream first primer
extension product, producing a plurality of first primer extension
products, representing different regions of the sequence of the
target DNA are produced. Step IV illustrates extending a second
primer comprising a ligand and a 3' DNA region that comprises a
random sequence, wherein the primer is optionally a tailed primer
comprising a nucleic acid sequence (B) that is 5' of the random
sequence, to form a plurality of double-stranded products each
comprising a first primer extension product and a second primer
extension product whereby the second primer extension product
comprises a ligand. Step IV(b) shows step IV in which the second
primer comprises a segment (B) that is 5' of the 3' DNA region that
comprises a random sequence. Step IV(a) shows step IV where the
second primer does not comprise a (B) sequence. The second primer
extension products comprising a ligand and a defined sequence (A)
at its 3' end are useful for storage, archiving and analysis as
they have a ligand capable of binding to a solid surface. Such
second primer extension products also comprises sequences that are
representative of (identical to or substantially identical to) a
sequence in the target DNA, so analysis of these products provides
information about the target DNA. Step V shows the binding the
ligand to a solid surface, whereby the plurality of second primer
extension products become bound to the solid surface.
Method for Archiving and Clonal Expansion
[0219] An aspect of the invention is a method for archiving and
clonal expansion of a nucleotide sequence.
[0220] The method comprises the steps of: (a) obtaining a plurality
of partially double-stranded DNA products comprising a first
polynucleotide and a second polynucleotide, wherein the second
polynucleotide comprises a sequence (A') at its 3' end and a
ligand, and the sequence (A') portion of the second polynucleotide
is single-stranded, wherein optionally the second polynucleotide
comprises a sequence (B) at or near its 5' end. The plurality of
partially double-stranded DNA products comprising a first
polynucleotide and a second polynucleotide, wherein the second
polynucleotide comprises a sequence (A') at its 3' end and a
ligand, and the sequence (A') portion of the second polynucleotide
is single-stranded, wherein optionally the second polynucleotide
comprises a sequence (B) at or near its 5' end may be obtained, for
example, by the methods described above.
[0221] The method further comprises step (b) attaching the
partially double stranded DNA products to a plurality of beads or a
plurality of isolated areas on a surface by binding the ligands to
the bead or isolated area. The receptor bound to the solid surface
is a member of the ligand-receptor pair such that binding of the
ligand results in attaching the second polynucleotide to the solid
surface. In some embodiments, the second polynucleotide is still
hybridized to the first polynucleotide when it is attached to the
solid surface. In some embodiments, the first polynucleotide is
removed from the second polynucleotide such that a single stranded
polynucleotide is attached to the solid surface. The method
produces a polynucleotide that is bound to a solid surface that has
a specific sequence (A') at its 3' end. The specific, or universal,
sequence (A') can be a site for primer hybridization and further
analysis or amplification of the polynucleotide bound to the bead.
As described above, in some embodiments, the polynucleotide bound
to the bead also comprises sequence (B) at or near its 5' end. One
aspect of the invention comprises amplification of the
polynucleotide bound to the bead. In some embodiments, a plurality
of different polynucleotides bound to a solid surface is created in
which each of the polynucleotides has a specific sequence (A') at
its 3' end (and in some embodiments also a specific sequence (B) at
its 5' end), and where the different polynucleotides have different
intervening sequences, wherein the intervening sequences are
identical to or substantially identical to the sequences in the
target nucleic acid. The set of bound polynucleotides thus
generated can be analyzed, for example, by sequencing in order to
provide information about the sequence of the target nucleic
acid.
[0222] The solid surface can be any of a variety of surfaces, some
described in more detail below. The solid surface can be, for
example a planar surface, for example, a planar array. In some
embodiments the solid surface comprises a plurality of beads. In
some embodiments the beads are magnetic.
[0223] The step of binding the polynucleotides to the solid surface
through the ligand, step (b), can be carried out such that only one
polynucleotide is bound to an isolated area of a surface or only
one polynucleotide is bound to a single bead. This isolated binding
of polynucleotides can be used for clonal amplification of the
specific bound polynucleotide in that area or on that bead. Such
bound, isolated polynucleotides can also be stored and archived for
later analysis, for example by sequencing. The bound, isolated
polynucleotides can be amplified, stored, and analyzed multiple
times.
[0224] The method further comprises the steps of: (c) annealing an
amplification primer to the single stranded portion of the second
polynucleotide complementary to sequence (A'), wherein the
amplification primer has a DNA portion and a 5' RNA portion; (d)
extending the amplification primer with an enzyme having strand
displacement activity to produce a plurality of amplified products
hybridized to the second polynucleotide products; (e) cleaving the
RNA from the amplified product hybridized to the second
polynucleotides using RNase H; and (f) repeating steps (c) to (e)
to produce multiple copies of amplified products.
[0225] In some embodiments, the DNA products are attached to beads
or isolated areas of the solid surface, and on average, one DNA
product is attached to one or fewer beads or isolated areas. The
plurality of beads or isolated areas can be stored for later
analysis, and in some cases can later be amplified with steps (c)
through (f). In some embodiments, the amplification is a clonal
amplification carried out in multiple isolated volumes wherein, on
average, one isolated volume has one or fewer beads or isolated
areas.
[0226] A schematic exemplary of an embodiment of the invention
relating to amplification of polynucleotides bound to a solid
surface is shown in FIG. 4. The polynucleotides bound to the solid
surface can be generated, for example from RNA or from DNA by the
methods described above. A polynucleotides bound to the solid
surface can be a second primer extension product comprising a
sequence (A') at its 3' end. In some embodiments, as in those shown
in FIG. 4, the polynucleotides bound to the surface are hybridized
to a first primer extension product comprising and RNA sequence
(A). For clarity, the polynucleotides are referred to as second
primer extension products. It will be understood, however, by those
of ordinary skill in the art that polynucleotides of the same
structure as shown here can be used in the present amplification
methods. Where the polynucleotides are single stranded, it would be
understood by those skilled in the art that the amplification can
be carried out without the first RNase H step (Step I). In FIG. 4,
the steps labeled (b), e.g. Step I(b), represents the method for
the case in which the second primer comprises a sequence (B) 5' to
the 3' sequence that is complementary to the first primer extension
product, while steps labeled (a), e.g. Step I(a) represent the
method in which the second primer, and therefore the second primer
extension product, does not include sequence (B).
[0227] In FIG. 4, Step I illustrates cleaving the RNA from the
first primer extension products such that a portion of the second
primer extension product that is complementary to sequence (A) is
single stranded. As shown, the cleavage is performed using RNase H.
Chemical and thermal means can alternatively be employed. Step II
shows annealing an amplification primer to the single stranded
portion of the second primer extension products complementary to
sequence (A), wherein the amplification primer has a DNA portion
and a 5' RNA portion. Step III illustrates extending the
amplification primer with an enzyme having strand displacement
activity to produce an amplified product hybridized to the second
primer extension product. Step IV illustrates the step of cleaving
the RNA from the amplified product hybridized to the second primer
extension product. The product of step IV can be the starting point
for another round of amplification in that Step II, the
hybridization of and amplification primer, can occur, followed by
the subsequent steps. Thus, steps II-IV can be repeated to produce
multiple copies of amplified product. Note that for case (b) the
amplified product that is produced comprises the sequence (B'),
complementary to sequence (B) at its 3' end.
[0228] In some embodiments, the amplification illustrated in FIG. 4
can be carried out on beads. In some embodiments, the beads have
only one bound second primer extension product. In some embodiments
such beads are individually in isolated volumes or microreactors
allowing for clonal amplification.
Method for Generating a Polynucleotide Having a Defined 3' and 5'
Sequences from an RNA Target
[0229] One aspect of the invention is a method for generating a
polynucleotide having a defined 3' and 5' sequences from an RNA
target. The method utilizes a composite RNA/DNA oligonucleotide in
order to extend the second primer extension product such that the
second primer extension product comprises a sequence (C') at its 3'
end than can be used as a site for isothermal amplification in a
manner such that the sequence (A) is present at or near the 5' end
of the amplified product produced in this amplification.
[0230] The method comprises the steps: (a) extending a first primer
comprising a DNA segment and a 5' RNA segment, wherein a 3' portion
of the primer is complementary to a target RNA and a 5' portion,
sequence (A), of the of the primer is not complementary to the
target RNA; to form a first primer extension product hybridized to
the target RNA, forming an RNA/DNA hybrid. In some embodiments, the
3' portion of the primer that is complementary to the target RNA is
a specific sequence. For example, where a specific region of
interest of a target RNA that is known or suspected to be upstream
of a specific sequence on the target RNA, the sequence that is
complementary to the target RNA of the first primer can be designed
to hybridize to this specific sequence on the target RNA such that
extension of the primer results in producing a first primer
extension product that is complementary to such upstream region.
The specific sequence may be common to a family of target RNA. A
combination of primers with various specific sequences at the 3'
end can also be useful. In some embodiments, such as where the
target RNA comprises mRNA, and the mRNA comprises a plurality of
sequences, each having a 3' poly-A segment; the specific sequence
that is complementary to the target RNA can comprise a sequence
that will hybridize to the poly-A region of the mRNA, thus allowing
the extension of the first primer to produce a plurality of first
primer extension products, each of which is complementary to the
region of an mRNA molecule adjacent to the poly-A region. In some
embodiments, the sequence that is complementary to the target RNA
comprises a random sequence, such that the extension of the first
primer results in a plurality of first primer extension products
complementary to the sequences adjacent to the sequence where each
random species hybridizes. The use of a random sequence at the 3'
end of the primer can be useful for performing a global
amplification of a RNA target, generating a plurality of sequences
which together can represent, for example substantially the whole
sequence of the target RNA. In some embodiments, the relative
amounts of the various sequences can be used to quantitate the
relative amount of a given sequence in a sample, for example to
determine the level of expression in an mRNA sample. In some
embodiments more than one type of sequence that is complementary to
the target RNA can be used, for instance both a primer with a
random sequence and a primer, or combination of primers with a
specific sequence complementary to RNA can be used. In some
embodiments, multiple primers comprising different specific
sequences can be used.
[0231] The method further comprises step (b) cleaving the target
RNA from the RNA/DNA hybrid. In some embodiments, the cleaving of
the target RNA from the RNA/DNA hybrid involves selectively
cleaving or degrading the target RNA. In some cases the cleaving
can be affected by denaturing the complex comprising the first
primer extension product and the nucleic acid. Denaturation can be
performed, for example by heating the sample, or by adding a
denaturing agent, or using a combination of heating the sample and
adding denaturing agents. The cleaving can be accomplished with an
enzyme that cleaves RNA from an RNA/DNA hybrid such as RNase H, or
a combination of RNase enzymes, or chemically. In some embodiments,
the target RNA is completely cleaved. In other embodiments, the
target RNA is only partly cleaved or degraded. The amount of
cleaving required is that amount which will allow the extension of
the second primer. In some embodiments, the cleaving is carried out
partially, and the fragments of the target RNA that remain can
constitute the second primer for step (c).
[0232] The method further comprises step (c) extending a second
primer, comprising a ligand and a 3' segment complementary to a
portion of the first primer extension product, to produce a double
stranded product with a DNA/RNA heteroduplex at one end; wherein
the double stranded product comprises a second primer extension
product hybridized to the first primer extension product, and
wherein a portion of the 3' end of the second primer extension
product comprises a sequence (A') that is complementary to the
sequence (A) of the of the first primer. The extension of the
second primer is carried out with a DNA polymerase as described
herein. The second primer can comprise RNA, DNA, or can be a
composite primer comprising both RNA and DNA. The second primer can
be a tailed primer having a 3' portion which is complementary to
the first primer extension product, and a 5' portion, sequence (B),
which is not complementary to the first primer extension product.
In some embodiments, the second primer can comprise a specific
primer sequence that is designed to hybridize to a specific
sequence in the first primer extension product. In some embodiments
the second primer comprises a random primer sequence that randomly
binds to the first primer extension product. Extension of the
second primer comprising a random sequence produces a plurality of
second primer extension products. The use of a random sequence at
the 3' end of the primer is useful, for example, in performing
global amplification of a target RNA, whereby a plurality of second
primer extension products are produced which is representative of
the sequence of the target RNA. In some embodiments, for example
where the first primer is designed to hybridize to a specific
sequence on a target RNA, or a sequence common to a family of RNA
targets, random priming by the second primer ensures amplification
of the entire selected target or family of selected targets. In
this embodiment, the second primer extension products comprise
sequences which are the same or substantially the same as the
sequences in the target RNA (sense copies). The second primer
comprises a ligand that is a member of a ligand-receptor pair. In
some embodiments, the ligand is attached to the primer at the 5'
end of the primer. In some embodiments, the ligand is a small
molecule, such as biotin or digoxigenin. In some embodiments, the
receptor is an antibody, and the ligand is a molecule or portion of
a molecule recognized by the antibody.
[0233] The second primer extension product is extended such that
the 3' portion of the second primer extension product comprises a
sequence (A') which is complementary to sequence (A) of the first
primer. Since sequence (A) on the first primer extension product
comprises RNA, both DNA dependent DNA polymerase activity and RNA
dependent DNA polymerase activity are used in step (c). The primer
extension results in a product that is at least partially double
stranded. The product further comprises a DNA-RNA heteroduplex
region. The method produces a nucleic acid that comprises a ligand
allowing it to be bound to a solid surface and that has a specific
sequence (A') at its 3' end. The specific, or universal, sequence
(A') can be a site for primer hybridization and further analysis or
amplification of the nucleic acid bound to the bead. As described
above, in some embodiments, the nucleic acid attached to the ligand
also comprises sequence (B) at or near its 5' end.
[0234] In some embodiments, the sample comprising the target RNA is
in a sample that also comprises DNA. In such cases, it can be
advantageous to add a selective DNA dependent DNA polymerase
inhibitor such as actinomycin such that it is present during step
(a) to selectively inhibit the production of extension product
complementary to the DNA during step (a). The presence of a DNA
dependent DNA polymerase inhibitor such as actinomycin is
particularly advantageous when a first primer comprising a random
sequence is used, as the inhibitor allows for the selective
creation of first primer extension products to RNA without the need
of separating the RNA from the DNA. This is also advantageous when
the priming is carried out at specific target sequences since the
sequence may be the same on the DNA when the DNA and RNA in the
sample represent total nucleic acid from the same biological
entity, for example, human tissue, animal tissue, and the like. The
use of DNA dependent DNA polymerase inhibitors such as actinomycin
is described in copending application.
[0235] The method further comprises step (d) cleaving the RNA in
the heteroduplex from the first primer extension product such that
a portion of the second primer extension product that is
complementary to sequence (A) is single stranded. The cleaving of
RNA can be performed, for example by treatment with RNase H, which
will selectively cleave the RNA portion of the DNA/RNA partial
heteroduplex formed in step (c).
[0236] The method further comprises step (e) annealing to the
second primer extension product an oligonucleotide comprising a
3'-DNA segment that is complementary to sequence (A') and a 5' RNA
segment comprising sequence (C). The oligonucleotide comprises at
least one DNA and at least one RNA portion. In some embodiments the
5' DNA segment is complementary to all of sequence (A'), in other
embodiments, the 5' DNA segment is complementary to portion of
sequence (A'). In some embodiments, 5' RNA segment comprising
sequence (C) is partly complementary to sequence (A').
[0237] The method optionally comprises step: (f) extending the
oligonucleotide to form an oligonucleotide extension product
hybridized to the second primer extension product. In some
embodiments, the oligonucleotide is optionally extended from its 3'
end to produce an oligonucleotide extension product hybridized to
the second primer extension product and displacing the DNA portion
of the first primer extension product. In some embodiments, the
second primer comprises a sequence (B), such that the
oligonucleotide extension product will comprise a sequence (B') at
or near its 3' end that is complementary to sequence (B).
[0238] The method further comprises step (g) extending the second
primer extension product to create a heteroduplex such that the
second primer comprises a DNA sequence (C') that is complementary
to sequence (C). The DNA sequence (C') is created by a DNA
polymerase that has RNA dependent DNA polymerase activity. This
step creates an RNA/DNA heteroduplex region that can be used for
further manipulation of the second primer extension product.
[0239] The method further comprises step (h) cleaving the RNA from
the heteroduplex created in step (g) to produce a single-stranded
portion of the second primer extension product corresponding to
sequence (C'). The RNA can be cleaved from the heteroduplex, for
example, by RNase H.
[0240] In some embodiments, The method further comprises step (i)
binding the ligand on the second primer extension product to a
solid surface. The binding can be performed at various stages or
steps during the procedure depending, for example, on whether it is
advantageous to carry out the subsequent steps on a solid surface.
The binding step is generally performed after step (c). In some
embodiments, the binding the ligand to the solid surface is
performed before step (h). In some embodiments, the binding the
ligand to the solid surface is performed after step (h). The
receptor bound to the solid surface is a member of the
ligand-receptor pair such that binding of the ligand results in
attaching the second primer extension product to the solid surface.
In some embodiments, the second primer extension product is still
hybridized to the first primer extension product when it is
attached to the solid surface. In some embodiments, the first
primer extension product is removed from the second primer
extension product such that a single stranded polynucleotide is
attached to the solid surface. The method produces a nucleic acid
that is bound to a solid surface that has a specific sequence (A')
and (C') at its 3' end. The specific sequence (C') can be a site
for primer hybridization and further analysis or amplification of
the nucleic acid bound to the bead. As described above, in some
embodiments, the nucleic acid bound to the bead also comprises
sequence (B) at or near its 5' end. One aspect of the invention
comprises amplification of the nucleic acid bound to the bead. In
some embodiments, the amplification is carried out using isothermal
amplification using a composite RNA/DNA primer, RNase H, and a
polymerase with strand displacement activity. For this embodiment,
the sequence (C') acts as the site to which the composite RNA/DNA
amplification primer hybridizes, allowing for amplification. When
the sequence (C') acts as a site to which a composite amplification
primer binds, the amplified product that is produced has the
sequence (A) (and a portion of sequence (C) at its 5' end. Where
the second primer comprises the sequence (B), the amplified product
also has the sequence (B'), complementary to (B) at or near its 3'
end. Thus the method produced amplified product with defined
sequences at or near both its 3' and 5' ends.
[0241] In some embodiments, for example where random sequences at
the 3' end of the first and/or second primer are used, a plurality
of different nucleic acids bound to a solid surface is created in
which each of the nucleic acids has a specific sequence (A') and
(C') at its 3' end (and in some embodiments also a specific
sequence (B) at its 5' end), and where the different nucleic acids
have different intervening sequences, wherein the intervening
sequences are identical to or substantially identical to the
sequences in the target RNA. The set of bound nucleic acids thus
generated can be analyzed, for example, by sequencing in order to
provide information about the sequence of the target RNA.
[0242] The solid surface can be any of a variety of surfaces, some
described in more detail below. The solid surface can be, for
example a planar surface, for example, a planar array. In some
embodiments the solid surface comprises a plurality of beads. In
some embodiments the beads are magnetic.
[0243] The step of binding the polynucleotides to the solid surface
through the ligand, step (i), can be carried out such that only one
nucleic acid is bound to an isolated area of a surface or only one
nucleic acid is bound to a single bead. This isolated binding of
nucleic acids can be used for clonal amplification of the specific
bound nucleic acid in that area or on that bead. Such bound,
isolated nucleic acids can also be stored and archived for later
analysis, for example by sequencing. The bound, isolated nucleic
acids can be amplified, stored, and analyzed multiple times.
[0244] In some embodiments, the method further comprises treating
the solid surface with reagents to produce multiple copies of an
amplification product that are substantially complementary the
second primer extension product. This step comprises carrying out
an amplification reaction wherein the bound nucleic acid acts as a
template for the amplification. Generally, the amplification is
carried out using the sequence (C') on the second primer extension
product for the hybridization of primer. In some embodiments, the
amplification produces single stranded amplified product, In some
embodiments, the amplification provides double stranded product. In
some embodiments, the second primer comprises a specific sequence
(B), which becomes incorporated into the second primer extension
product. In some embodiments the amplification is an isothermal
amplification reaction comprising a composite RNA/DNA primer, RNase
H, and a DNA polymerase with strand displacement activity. In some
embodiments, the amplification is carried out using polymerase
chain reaction, (PCR). For example where the second primer
extension product comprises both as sequence (B) at or near its 5'
end and a sequence (C') at or near its 3' end, a set of primers,
one designed to hybridize to all or a portion of the sequence (C')
and the other designed to hybridize to sequence (B'), the
complement of sequence (B), can be used to carry out a PCR reaction
to exponentially produce double stranded amplified product.
[0245] In some embodiments, the amplification is performed by a
method comprising the following steps: (j) annealing an
amplification primer, wherein the amplification primer has a DNA
portion and a 5' RNA portion, to the single stranded portion of the
second primer extension product complementary to sequence (C'); (k)
extending the amplification primer with an enzyme having strand
displacement activity to produce an amplified product; (l) cleaving
the RNA from the amplified product; and (m) repeating steps (j) to
(l) to produce multiple copies of amplified product wherein the 5'
portion of the amplified product has a sequence complementary to
sequence (A'). Where the second primer further comprises a segment
(B) that is not complementary to the first primer extension product
sequence, this amplification method, utilizing sequence (C'),
allows for the production of an amplified product comprises a
sequence (B') at or near its 3' end that is substantially
complementary to sequence (B), and a sequence (A) near its 5' end
that is complementary to sequence (A'), thus producing an amplified
polynucleotide product with defined 3' and 5' ends.
[0246] In some embodiments the amplification is carried out such
that the amplified product is not attached to the substrate, but is
freely dissolved in the solution. In other embodiments, the
amplification is carried out such that the amplified product
remains bound to the substrate, for example by performing solid
phase PCR such as bridge PCR. In yet other embodiments, an
amplified product is generated that may float freely in solution,
but which comprises a sequence, for example sequence (A) or
sequence (B'), that allows it to be captured to another solid
surface or other portion of the solid surface by hybridization to a
complementary sequence bound to such surface (e.g. sequence (A') or
sequence (B). In some embodiments, the amplified product is a
single-stranded product and, because it is generated at the solid
surface, the amplified product readily captured by complementary
sequences, e.g. sequence (B), bound to the surface.
[0247] In one aspect of the invention, a plurality of beads is
used, and the methods described above are carried out such that on
average, one or fewer second primer extension product molecules are
bound per bead. The beads are dispersed into an aqueous solution,
and a plurality of microreactors, e.g. droplets, are produced such
that on average one or fewer beads is contained within each of the
plurality of microreactors. The amplification of the second primer
extension products bound to the beads is then carried out such that
the clonal amplification of each of the plurality of second primer
extension products in the separate microreactors is achieved. This
clonal amplification in microreactors can be performed on a sample
of target RNA, such as whole transcriptome or total RNA, wherein
the plurality of second primer extension products comprise
sequences that correspond to most, to substantially all, or to all
of the sequences in the target RNA. In some embodiments, the
amplified products are captured by bead having attached thereto a
plurality of oligonucleotides comprising complementary sequences
bound to such surface (e.g. sequence (A') or sequence (B)), which
are complementary to sequence (A) or sequence (B') on the amplified
product.
[0248] In some embodiments, the plurality of beads, produced as
described above, with each bead comprising a single second primer
extension product can comprise a library. These libraries can be
stored, then later clonally amplified. In some embodiments, a
library of beads can comprise a plurality of beads wherein each
bead had multiple copies of a single amplification product
generated from a second primer extension product. These libraries
can be analyzed, for example by sequencing. The libraries can be
stored, and later analyzed. In some embodiments the libraries can
be stored, then analyzed multiple times.
[0249] In some embodiments, a bead or isolated area of the solid
surface comprises covalently attached thereto multiple
oligonucleotides comprising the sequence (B) at their 3' ends,
whereby upon the amplification of step (m) multiple copies of
amplified product comprising sequence (B') at their 5' end are
hybridized to the bead or isolated area. For example, where beads
are used, a plurality of beads in a plurality of microreactors
wherein, the plurality of beads has, on average one or fewer second
primer extension products bound to it and there are, on average,
one or fewer beads in each microreactor, a clonal amplification of
the plurality of second primer extension products can be carried
out, and the amplified products in each of the microreactors will
bind to the bead through the sequence (B') on the amplified product
to the sequence (B) on the beads. This approach produces a
plurality of beads, each with multiple copies of a different
sequence bound to it. Where these sequences are representative of
the target RNA, the plurality of beads can constitute a library
representative of such RNA.
[0250] After the amplified products are bound to the beads by
hybridization, the (B) sequences on the beads can be extended along
the amplified product by a DNA polymerase or mixture of polymerases
to produce a multiple polynucleotides covalently attached to the
bead or isolated area that are substantially complementary to the
amplified product and also comprise sequence (A') near their 5'
ends. Where the (B) sequences are covalently attached to the beads,
this method provides for the production of beads with
polynucleotides complementary to amplified product covalently
attached to the beads. Covalently attached polynucleotides such as
those produce here are more robust than nucleotides that are
attached only by hybridization to the beads. Thus, the covalently
attached polynucleotides can be more stable and can be used with
analysis methods and sequencing methods that have harsher
conditions which would result in the displacement of
polynucleotides bound only by hybridization.
[0251] In some embodiments, the amplified product is removed from
the covalently bound polynucleotide to render the polynucleotide
single stranded. Such single stranded covalently bound
polynucleotides comprise a specific sequence at their 3' ends
comprising sequence (A') and a portion of sequence (C'). This
specific sequence at the 3' end of the covalently bound
polynucleotide can act as a hybridization site for a primer
complementary to sequence (A') that can act as a primer to carry
out sequencing by any of a variety of sequencing methods, for
example, those described herein.
[0252] The sequencing methods can comprise the use of cleavable
labeled terminators. The sequencing method can comprise
pyrophosphate detection. The sequencing method can comprise an
isothermal sequencing method, for example using chimeric primers,
RNase H, and a polymerase with strand displacement activity. The
sequencing method can also comprise cycle sequencing.
[0253] In some embodiments, the sequencing methods comprise
sequencing by ligation. Sequencing by ligation involves using a DNA
ligase. Although commonly represented as joining two pairs of ends
at once, as in the ligation of restriction enzyme fragments, ligase
can also join the ends on only one of the two strands (for example,
when the other strand either already continuous or lacks a terminal
phosphate necessary for ligation). DNA ligase is sensitive to the
structure of DNA and has very low efficiency when there are
mismatches between the bases of the two strands. Sequencing by
ligation relies upon the sensitivity of DNA ligase for base-pairing
mismatches.
[0254] The target molecule to be sequenced is a single strand of
unknown DNA sequence, flanked on at least one end by a known
sequence. A short "anchor" strand is brought in to bind the known
sequence. A mixed pool of probe oligonucleotides is then brought in
(eight or nine bases long), labeled (typically with fluorescent
dyes or other detection means) according to the position that will
be sequenced. These molecules hybridize to the target DNA sequence,
next to the anchor sequence, and DNA ligase preferentially joins
the molecule to the anchor when its bases match the unknown DNA
sequence. Based on the fluorescence or other signal produced by the
molecule, one can infer the identity of the nucleotide at this
position in the unknown sequence. The oligonucleotide probes may
also be constructed with cleavable linkages which can be cleaved
after identifying the label. This will both remove the label and
regenerate a 5' phosphate on the end of the ligated probe,
preparing the system for another round of ligation.
[0255] This cycle can be repeated several times to read longer
sequences. This sequences every Nth base, where N is the length of
the probe left behind after cleavage. To sequence the skipped
positions, the anchor and ligated oligonucleotides may be stripped
off the target DNA sequence, and another round of sequencing by
ligation started with an anchor one or more bases shorter. A
simpler, albeit more limited, technique is to do repeated rounds of
a single ligation where the label corresponds to different position
in the probe, followed by stripping the anchor and ligated probe.
Sequencing by ligation can proceed in either direction (either
5'-3' or 3'-5') depending on which end of the probe
oligonucleotides are blocked by the label. The 3'-5' direction is
more efficient for doing multiple cycles of ligation. Note that
this is the opposite direction to polymerase based sequencing
methods.
[0256] One feature unique to sequencing by ligation is the
possibility of labeling the probe oligonucleotides according to
various combinations of bases at more than one position. This has
error detection capabilities not available to polymerase-based
sequencing methods. The Applied Biosystems SOLiD sequencing system
uses 2-base encoding to improve its error rates.
[0257] In some embodiments the methods of the invention provide for
performing bridge PCR comprising making amplified product as
described above with defined 3' and 5' ends, and further comprising
the steps of exposing the amplified product to a solid substrate
comprising oligonucleotide sequences attached thereto complementary
to the defined 3' and 5' sequences, for example, A and B'
sequences, on the amplified product in the presence of components
necessary for polymerase chain reaction, and thermal cycling the
system to perform bridge PCR amplification.
[0258] In some embodiments the methods of the invention provide for
making amplified product as described above with defined 3' and 5'
ends and further performing rolling circle amplification comprising
performing the steps of: (n) hybridizing the amplified product to
an oligonucleotide comprising regions complementary to A and B'
sequences in close proximity; (o) optionally extending the gap with
a polymerase enzyme; (p) ligating to form a circular nucleic acid
comprising the amplified product, and performing rolling circle
amplification by extending a primer that is complementary to a
sequence in the circular nucleic acid. In some embodiments, the
rolling circle amplification uses primers complementary to sequence
(A), sequence (B'), or a sequence that was between sequences (A)
and (B') in the amplified product. In some cases, such a primer can
be an oligonucleotide attached to a solid surface, thus resulting
in amplified product bound to the surface
[0259] In some embodiments the methods of the invention provide for
performing PCR comprising making amplified product as described
above with defined 3' and 5' ends, further comprising the steps of
amplifying the amplified product using primers complementary to
sequences (A) and (B), or using primers complementary to sequences
(A') and (B').
[0260] In some embodiments the methods of the invention provide for
performing strand displacement amplification (SDA) comprising
making amplified product as described above with defined 3' and 5'
ends, wherein the defined 3' and 5' ends, for example, sequences
(A) and (B'), in the amplified product are designed to be cleaved
by a restriction enzyme, and performing strand displacement
amplification on the amplified product.
Method for Generating a Polynucleotide Having a Defined 3' and 5'
Sequences from a DNA Target
[0261] One aspect of the invention is a method for generating a
polynucleotide having a defined 3' and 5' sequences from a DNA
target. The method utilizes a composite RNA/DNA oligonucleotide in
order to extend the second primer such that the second primer
extension products comprise a sequence (C') at its 3' end than can
be used as a site for isothermal amplification in a manner such
that the sequence (A) is present at or near the 5' end of the
amplified product produced in this amplification, and where a
second primer comprising sequence (B) is used, amplified products
with defined sequences at both the 3' and 5' ends can be
produced.
[0262] The method comprises the step: (a) denaturing a
double-stranded target DNA. Double stranded DNA can be denatured,
for example by heating, or by the addition of denaturing
agents.
[0263] The method further comprises step (b) annealing to the
target DNA and extending with a DNA polymerase comprising strand
displacement activity, a first primer comprising a DNA segment and
a 5' RNA segment, wherein a 3' portion of the primer comprises a
random sequence, and a 5' portion of the of the primer comprises
sequence (A), which is not complementary to the target DNA; to form
a plurality of first primer extension products, each with sequence
(A) at its 5' end. The sequence that is complementary to the target
DNA comprises a random sequence, such that the extension of the
first primer results in a plurality of first primer extension
products complementary to the sequences adjacent to the sequence
where each random species hybridizes. The use of a random sequence
at the 3' end of the primer can be useful for performing a global
amplification of a target DNA, generating a plurality of sequences
which together can represent, for example substantially the whole
sequence of the target DNA. In some embodiments, the relative
amounts of the various sequences can be used to quantitate the
relative amount of a given sequence in a sample, for example to
determine the number of gene copies in a DNA sample, or obtaining
sequence information. In some embodiments, the extension of one
first primer, will result in the release of a downstream first
primer extension product. This can occur throughout the target DNA
resulting in the release of multiple first primer extension
products from the target DNA. This process can occur simultaneously
on both of the strands of the double-stranded DNA target, thus
creating first primer extension products complementary to sequences
in both strands.
[0264] In some embodiments, the first primer extension step is
carried out with a DNA polymerase capable of extension at elevated
temperature that is not compatible with subsequent hybridization of
the random sequence to the displaced primer-extension product. For
example, Bst DNA polymerase can be used which is active at elevated
temperature. The reaction can be carried out stepwise, first with
an incubation at a lower temperature such as about 25.degree. C.,
followed by an incubation at higher temperature such as about
50.degree. C. In some embodiments, the first incubation is carried
out below about 30.degree. C., and the second incubation is carried
out above about 40.degree. C. In some embodiments, a DNA polymerase
which is active at temperatures above about 45.degree. C. is used
to extend the first primer. Mixtures of DNA polymerases can also be
useful.
[0265] The method further comprises step (c), extending a second
primer, comprising a ligand and a 3' segment complementary to a
portion of the first primer extension product, to produce a double
stranded product with a DNA/RNA heteroduplex at one end; wherein
the double stranded product comprises a second primer extension
product hybridized to the first primer extension product, and
wherein a portion of the 3' end of the second primer extension
product comprises a sequence (A') that is complementary to the
sequence (A) of the of the first primer. In this step, a second
primer comprising a ligand and a 3' DNA region that comprises a
random sequence is used. The primer is optionally a tailed primer
comprising a nucleic acid sequence (B) that is 5' of the random
sequence, to form a plurality of double-stranded products each
comprising a first primer extension product and a second primer
extension product whereby the second primer extension products
comprise a ligand. This step may be carried out with or without
prior denaturation. If carried out without denaturation, generally,
only the single stranded displaced first primer extension products
will hybridize to the second primer. Generally the second primer
does not comprise RNA. The extension of the second primer is
carried out with a DNA polymerase as described herein. The second
primer can be a tailed primer having a 3' portion which is
complementary to the first primer extension products, and a 5'
portion, sequence (B), which is not complementary to the first
primer extension products. The second primer comprises a random
primer sequence that randomly binds to the first primer extension
products. Extension of the second primer comprising a random
sequence produces a plurality of second primer extension products.
The use of a random sequence at the 3' end of the primer is useful,
for example, in performing global amplification of a target DNA,
whereby a plurality of second primer extension products are
produced which is representative of the sequence of the target DNA.
In some embodiments, for example where the first primer is designed
to hybridize to a specific sequence on a target RNA, or a sequence
common to a family of RNA targets, random priming by the second
primer ensures amplification of the entire selected target or
family of selected targets. In this embodiment, the second primer
extension products comprise sequences which are the same or
substantially the same as the sequences in the target DNA. The
second primer comprises a ligand that is a member of a
ligand-receptor pair. In some embodiments, the ligand is attached
to the primer at the 5' end of the primer. In some embodiments, the
ligand is a small molecule, such as biotin or digoxigenin. In some
embodiments, the receptor is an antibody, and the ligand is a
molecule or portion of a molecule recognized by the antibody.
[0266] The second primer extension products are extended such that
the 3' portion of the second primer extension product comprises a
sequence (A') which is complementary to sequence (A) of the first
composite primer. Since sequence (A) on the first primer extension
products comprise RNA, both DNA dependent DNA polymerase activity
and RNA dependent DNA polymerase activity are used in step (c). The
primer extension results in a products that are at least partially
double stranded. The method produces a nucleic acid that comprises
a ligand allowing it to be bound to a solid surface and that has a
specific sequence (A') at its 3' end. The specific, or universal,
sequence (A') can be a site for primer hybridization and further
analysis or amplification of the nucleic acid bound to the bead. As
described above, in some embodiments, the nucleic acid attached to
the ligand also comprises sequence (B) at or near its 5' end.
[0267] The method further comprises step (d) cleaving the RNA from
the first primer extension products such that a portion of the
second primer extension products that are complementary to sequence
(A) is single stranded. The cleaving of RNA can be performed, for
example by treatment with RNase H, which will selectively cleave
the RNA portion of the DNA/RNA partial heteroduplex formed in step
(c).
[0268] The method further comprises step (e) annealing to the
second primer extension products an oligonucleotide comprising a
3'-DNA segment that is complementary to sequence (A') and a 5' RNA
segment comprising sequence (C). The oligonucleotide comprises at
least one DNA and at least one RNA portion. In some embodiments the
5' DNA segment is complementary to all of sequence (A'), in other
embodiments, the 5' DNA segment is complementary to portion of
sequence (A'). In some embodiments, 5' RNA segment comprising
sequence (C) is partly complementary to sequence (A').
[0269] The method optionally comprises step: (f) extending the
oligonucleotide to form a plurality of oligonucleotide extension
products hybridized to the second primer extension products. In
some embodiments, the oligonucleotide is optionally extended from
its 3' end to produce a plurality of oligonucleotide extension
products hybridized to the second primer extension product and
displacing the DNA portion of the first primer extension product.
In some embodiments, the second primer comprises a sequence (B),
such that the oligonucleotide extension products will comprise a
sequence (B') at or near their 3' ends that are complementary to
sequence (B).
[0270] The method further comprises step (g) extending the second
primer extension products to create a heteroduplex such that the
second primer comprises a DNA sequence (C') that is complementary
to sequence (C). The DNA sequence (C') is created by a DNA
polymerase that has RNA dependent DNA polymerase activity. This
step creates an RNA/DNA heteroduplex region that can be used for
further manipulation of the second primer extension products.
[0271] The method further comprises step (h) cleaving the RNA from
the heteroduplex created in step (g) to produce a single-stranded
portion of the second primer extension products corresponding to
sequence (C'). The RNA can be cleaved from the heteroduplex, for
example, by RNase H.
[0272] In some embodiments, The method further comprises step (i)
binding the ligand on the second primer extension products to a
solid surface. The binding step can be performed at various stages
or steps during the procedure depending, for example, on whether it
is advantageous to carry out the subsequent steps on a solid
surface. The binding step is generally performed after step (c). In
some embodiments, the binding the ligand to the solid surface is
performed before step (h). In some embodiments, the binding the
ligand to the solid surface is performed after step (h). The
receptor bound to the solid surface is a member of the
ligand-receptor pair such that binding of the ligand results in
attaching the second primer extension products to the solid
surface. In some embodiments, the second primer extension products
are still hybridized to the first primer extension products when
they are attached to the solid surface. In some embodiments, the
first primer extension products are removed from the second primer
extension products such that a single stranded polynucleotide is
attached to the solid surface. The method produces polynucleotides
that are bound to a solid surface that have a specific sequence
(A') and (C') at their 3' ends. The specific sequence (C') can be a
site for primer hybridization and further analysis or amplification
of the nucleic acid bound to the bead. As described above, in some
embodiments, the nucleic acid bound to the bead also comprises
sequence (B) at or near its 5' end. One aspect of the invention
comprises amplification of the nucleic acid bound to the bead. In
some embodiments, the amplification is carried out using isothermal
amplification using a composite RNA/DNA primer, RNase H, and a
polymerase with strand displacement activity. For this embodiment,
the sequence (C') acts as the site to which the composite RNA/DNA
amplification primer hybridizes, allowing for amplification. When
the sequence (C') acts as a site to which a composite amplification
primer binds, the amplified products that are produced have the
sequence (A) (and a portion of sequence (C) at their 5' ends)).
Where the second primer comprises the sequence (B), the amplified
products also have the sequence (B'), complementary to (B) at or
near their 3' ends. Thus the method produced amplified products
with defined sequences at or near both its 3' and 5' ends.
[0273] Due to the use of random priming, a plurality of different
nucleic acids bound to a solid surface is created in which each of
the nucleic acids has a specific sequence (A') and (C') at its 3'
end (and in some embodiments also a specific sequence (B) at its 5'
end), and where the different nucleic acids have different
intervening sequences, wherein the intervening sequences are
identical to or substantially identical to the sequences in the
target DNA. The set of bound nucleic acids thus generated can be
analyzed, for example, by sequencing in order to provide
information about the sequence of the target DNA.
[0274] The solid surface can be any of a variety of surfaces, some
described in more detail below. The solid surface can be, for
example a planar surface, for example, a planar array. In some
embodiments the solid surface comprises a plurality of beads. In
some embodiments the beads are magnetic.
[0275] The step of binding the polynucleotides to the solid surface
through the ligand, step (i), can be carried out such that only one
nucleic acid is bound to an isolated area of a surface or only one
nucleic acid is bound to a single bead. This isolated binding of
nucleic acids can be used for clonal amplification of the specific
bound nucleic acid in that area or on that bead. Such bound,
isolated nucleic acids can also be stored and archived for later
analysis, for example by sequencing. The bound, isolated nucleic
acids can be amplified, stored, and analyzed multiple times. This
allows, for example, for the analysis of an individuals genes, or a
portion of the individual genes at one time, then allows for
archiving the genetic material at a later date by the same or by
different tests.
[0276] In some embodiments, the method further comprises treating
the solid surface with reagents to produce multiple copies of
amplification products that are substantially complementary to the
second primer extension products. This step comprises carrying out
an amplification reaction wherein the bound nucleic acid acts as a
template for the amplification. Generally, the amplification is
carried out using the sequence (C') on the second primer extension
products as for primer hybridization. In some embodiments, the
amplification produces single stranded amplified products. In some
embodiments, the amplification provides double stranded products.
In some embodiments, the second primer comprises a specific
sequence (B), which becomes incorporated into the second primer
extension products. In some embodiments the amplification is an
isothermal amplification reaction comprising a composite RNA/DNA
primer, RNase H, and a DNA polymerase with strand displacement
activity. In some embodiments, the amplification is carried out
using polymerase chain reaction, (PCR). For example where the
second primer extension products comprises both as sequence (B) at
or near its 5' end and a sequence (C') at or near its 3' end, a set
of primers, one designed to hybridize to all or a portion of the
sequence (C') and the other designed to hybridize to sequence (B'),
the complement of sequence (B), can be used to carry out a PCR
reaction to exponentially produce double stranded amplified
products.
[0277] In some embodiments, the amplification is performed by a
method comprising the following steps: (j) annealing an
amplification primer, wherein the amplification primer has a DNA
portion and a 5' RNA portion, to the single stranded portion of the
second primer extension products complementary to sequence (C');
(k) extending the amplification primer with an enzyme having strand
displacement activity to produce an amplified products; (l)
cleaving the RNA from the amplified products; and (m) repeating
steps (j) to (l) to produce multiple copies of amplified products
wherein the 5' portion of the amplified products have a sequence
complementary to sequence (A'). Where the second primer further
comprises a segment (B) that is not complementary to the first
primer extension products sequence, this amplification method,
utilizing sequence (C'), allows for the production of amplified
products comprises a sequence (B') at or near their 3' ends that is
substantially complementary to sequence (B), and a sequence (A)
near their 5' end that are complementary to sequence (A'), thus
producing an amplified polynucleotide products with defined 3' and
5' ends.
[0278] In some embodiments the amplification is carried out such
that the amplified products are not attached to the substrate, but
is freely dissolved in the solution. In other embodiments, the
amplification is carried out such that the amplified products
remains bound to the substrate, for example by performing solid
phase PCR such as bridge PCR. In yet other embodiments, amplified
products are generated that may float freely in solution, but which
comprise a sequence, for example sequence (A) or sequence (B'),
that allows them to be captured to another solid surface or other
portion of the solid surface by hybridization to a complementary
sequence bound to such surface (e.g. sequence (A') or sequence (B).
In some embodiments, the amplified product is a single-stranded
product and, because it is generated at the solid surface, the
amplified product readily captured by complementary sequences, e.g.
sequence (B), bound to the surface.
[0279] In one aspect of the invention, a plurality of beads is
used, and the methods described above are carried out such that on
average, one or fewer second primer extension product molecules are
bound per bead. The beads are dispersed into an aqueous solution,
and a plurality of microreactors, e.g. droplets, are produced such
that on average one or fewer beads is contained within each of the
plurality of microreactors. The amplification of the second primer
extension products bound to the beads is then carried out such that
the clonal amplification of each of the plurality of second primer
extension products in the separate microreactors is achieved. This
clonal amplification in microreactors can be performed on a sample
of target DNA, such as genomic DNA, wherein the plurality of second
primer extension products comprise sequences that correspond to
most, to substantially all, or to all of the sequences in the
target DNA. In some embodiments, the amplified products are
captured by bead having attached thereto a plurality of
oligonucleotides comprising complementary sequences bound to such
surface (e.g. sequence (A') or sequence (B)), which are
complementary to sequence (A) or sequence (B') on the amplified
products.
[0280] In some embodiments, the plurality of beads, produced as
described above, with each bead comprising a single second primer
extension product can comprise a library. The library can
represent, for example, the genomic DNA of an individual, or the
genomic DNA from a group of cells or from a single cell. These
libraries can be stored, then later clonally amplified. In some
embodiments, a library of beads can comprise a plurality of beads
wherein each bead had multiple copies of a single amplification
product generated from a second primer extension product. These
libraries can be analyzed, for example by sequencing. The libraries
can be stored, and later analyzed. In some embodiments the
libraries can be stored, then analyzed multiple times.
[0281] In some embodiments, a bead or isolated area of the solid
surface comprises covalently attached thereto multiple
oligonucleotides comprising the sequence (B) at their 3' ends,
whereby upon the amplification of step (m) multiple copies of
amplified product comprising sequence (B') at their 5' end are
hybridized to the bead or isolated area. For example, where beads
are used, a plurality of beads in a plurality of microreactors
wherein, the plurality of beads has, on average one or fewer second
primer extension products bound to it and there are, on average,
one or fewer beads in each microreactor, a clonal amplification of
the plurality of second primer extension products can be carried
out, and the amplified products in each of the microreactors will
bind to the bead through the sequence (B') on the amplified product
to the sequence (B) on the beads. This approach produces a
plurality of beads, each with multiple copies of a different
sequence bound to it. Where these sequences are representative of
the target DNA, the plurality of beads can constitute a library
representative of such DNA.
[0282] After the amplified products are bound to the beads by
hybridization, the (B) sequences on the beads can be extended to
produce a multiple polynucleotides covalently attached to the bead
or isolated area that are substantially complementary to the
amplified product and also comprise sequence (A') near their 5'
ends. Where the (B) sequences are covalently attached to the beads,
this method provides for the production of beads with
polynucleotides complementary to amplified product covalently
attached to the beads. Covalently attached polynucleotides such as
those produce here are more robust than nucleotides that are
attached only by hybridization to the beads. Thus, the covalently
attached polynucleotides can be more stable and can be used with
analysis methods and sequencing methods that have harsher
conditions which would result in the displacement of
polynucleotides bound only by hybridization.
[0283] In some embodiments, the amplified product is removed from
the covalently bound polynucleotide to render the polynucleotide
single stranded. Such single stranded covalently bound
polynucleotides comprise a specific sequence at their 3' ends
comprising sequence (A') and a portion of sequence (C'). This
specific sequence at the 3' end of the covalently bound
polynucleotide can act as a hybridization site for a primer
complementary to sequence (A) that can act as a primer to carry out
sequencing by any of a variety of sequencing methods, for example,
those described herein. The single stranded covalently bound
polynucleotides derived from DNA can be sequenced as described
above for the single stranded covalently bound polynucleotides
derived from DNA, such as pyrosequencing, cycle sequencing,
isothermal sequencing and other methods such as those described
below.
[0284] The sequencing methods can comprise the use of cleavable
labeled terminators. The sequencing method can comprise
pyrophosphate detection. The sequencing method can comprise an
isothermal sequencing method, for example using chimeric primers,
RNase H, and a polymerase with strand displacement activity. The
sequencing method can also comprise cycle sequencing.
[0285] In some embodiments the methods of the invention provide for
performing bridge PCR comprising making amplified product as
described above with defined 3' and 5' ends, and further comprising
the steps of exposing the amplified product to a solid substrate
comprising oligonucleotide sequences attached thereto complementary
to the defined 3' and 5' sequences, for example, A and B'
sequences, on the amplified product in the presence of components
necessary for polymerase chain reaction, and thermal cycling the
system to perform bridge PCR amplification. The bridge PCR can be
carried out such that isolated lawns of amplified product are
obtained wherein each lawn comprises polynucleotides having
sequences representative of a portion of the sequence of the target
DNA.
[0286] In some embodiments the methods of the invention provide for
making amplified product as described above with defined 3' and 5'
ends and further performing rolling circle amplification comprising
performing the steps of: (n) hybridizing the amplified product to a
target DNA comprising regions complementary to A and B' sequences
in close proximity; (o) optionally extending the gap with a
polymerase enzyme; (p) ligating to form a circular nucleic acid
comprising the amplified product, and performing rolling circle
amplification by extending a primer that is complementary to a
sequence in the circular nucleic acid. In some embodiments, the
rolling circle amplification uses primers complementary to sequence
(A), sequence (B'), or a sequence that was between sequences (A)
and (B') in the amplified product. In some cases, such a primer can
be an oligonucleotide attached to a solid surface, thus resulting
in amplified product bound to the surface
[0287] In some embodiments the methods of the invention provide for
performing PCR comprising making amplified product as described
above with defined 3' and 5' ends, further comprising the steps of
amplifying the amplified product using primers complementary to
sequences (A) and (B), or using primers complementary to sequences
(A') and (B').
[0288] In some embodiments the methods of the invention provide for
performing strand displacement amplification (SDA) comprising
making amplified product as described above with defined 3' and 5'
ends, wherein the defined 3' and 5' ends, for example, sequences
(A) and (B'), in the amplified product are designed to be cleaved
by a restriction enzyme, and performing strand displacement
amplification on the amplified product.
[0289] A schematic exemplary of an embodiment of the invention
relating to generating a polynucleotide having a defined 3' and 5'
sequences is shown in FIGS. 5A and 5B. The starting material in 5A
can be generated as described in the methods herein. The starting
material can be generated, for example, from the methods above from
a target RNA or a target DNA. While the starting material is
described as, for example first primer extension product, it will
be understood that a complex of the same structure as shown and
described can be used in the method described here.
[0290] In FIG. 5A, step I shows the step of: cleaving the RNA from
the first primer extension product in the DNA-RNA heteroduplex such
that a portion of the second primer extension product that is
complementary to sequence (A) is single stranded. As shown, the
cleavage is performed using RNase H. Chemical and thermal means can
alternatively be employed. Step II illustrates annealing to the
second primer extension product an oligonucleotide comprising a
3'-DNA segment that is complementary to sequence (A') and a 5' RNA
segment comprising sequence (C). Step III illustrates extending the
second primer extension product to create a DNA-RNA heteroduplex
such that the second primer comprises a DNA sequence (C') that is
complementary to sequence (C). Step IV illustrates the optional
step of extending the oligonucleotide to form an oligonucleotide
extension product hybridized to the second primer extension
product. Step V illustrates binding the ligand to a solid surface
resulting in binding of the second primer extension product to the
solid surface. While the step of binding the ligand to the solid
surface is shown at step V is to be understood that the binding
step can be performed at many steps in the process. For example, in
this case, the binding step could alternately have been carried out
before step I, or could be carried out after amplification.
[0291] In FIG. 5B, step VI illustrates cleaving the RNA from the
DNA-RNA heteroduplex created in step III to produce a
single-stranded portion of the second primer extension product
corresponding to sequence (C'). Step VII illustrates annealing an
amplification primer, wherein the amplification primer has a DNA
portion and a 5' RNA portion, to the single stranded portion of the
second primer extension product complementary to sequence (C').
Step VIII illustrates extending the amplification primer with a DNA
polymerase having strand displacement activity to produce an
amplified product. Step IX illustrates the step of cleaving the RNA
from the extended hybridized amplification primer in the DNA-RNA
heteroduplex. The product of step IX is now capable of hybridizing
another amplification primer, allowing for steps VII to IX to occur
again, resulting in the generation of another amplified product.
These steps can thus be repeated to produce multiple copies of
amplified product wherein the 5' portion of the amplified product
has a sequence complementary to sequence (A'). In the embodiment
illustrated in FIGS. 5A and 5B the second primer comprised a
sequence (B), and as such, the amplified product shown in FIG. 5B
comprises a defined sequences on both the 5' and 3' ends.
[0292] A schematic exemplary of an embodiment of the invention
relating to the capture of amplification product on a solid surface
is shown in FIG. 6. In some embodiments, the amplified product can
be captured onto a surface by hybridization to the defined
sequences on the 5' and/or 3' ends of the amplified product. This
approach can be useful for example, where a clonal amplification of
a given sequence or plurality of sequences that are representative
of a target RNA or target DNA produces multiple copies of a
specific sequence having defined 5' and 3' ends. The clonal
amplification can be carried out in an isolated volume, or on in an
isolated area on a surface. In some embodiments, the clonal
amplification is carried out with beads in droplets of an inverse
emulsion, wherein each droplet has on average one or fewer beads.
In some embodiments, it is useful to capture the amplified product
on a solid surface, for example a bead. FIG. 6 illustrates how a
solid surface, for example the surface of a bead can have multiple
oligonucleotides attached thereto. In the embodiment illustrated,
the amplified product has a defined sequence (B') at its 3' end and
a defined sequence (A) near its 5' end. The bead has multiple
oligonucleotides attached to its surface comprising the sequence
(B), which is complementary to the sequence (B') at the 3' end of
an amplified product. The bead can capture the amplified product as
shown by hybridization under the right conditions. The capture of
amplified product is illustrated in step I. This type of bead can
be used as a library, and can be used for sequencing, for example,
by extending from sequence (B). FIG. 6 also illustrates in step II
that the (B) sequences on the bead can be extended along the
amplified product to produce a polynucleotide attached to the bead
which has sequence (A') at or near its 3' end and sequence (B) at
its 5' end. Such a polynucleotide can be made single stranded, for
example by heat or chemical denaturation, and washing as
illustrated in step III. The bead with single stranded
polynucleotide which has sequence (A') at or near its 3' end and
sequence (B) at its 5' can be more stable than a bead to which a
polynucleotide is held by hybridization alone. The polynucleotide
attached to the bead can be covalently bound. The beads made in
this fashion can be used to create libraries with long storage
life. The single stranded product of step III can be used for
archiving, storage, and a variety of manipulations made possible by
the defined sequences at the 5' and 3' ends. For example, as shown
in FIG. 6, a sequencing primer (A) can be hybridized to sequence
(A') to allow the sequencing of the polynucleotide attached to the
bead by methods such as those described herein.
[0293] FIG. 7 illustrates that the amplified product with a defined
sequence (B') at its 3' end and a defined sequence (A) near its 5'
end can also be captured onto surfaces such as beads by
hybridization to an oligonucleotide attached to the solid surface
that comprises the sequence (A'). In some cases, the capture is by
specific nucleic acid hybridization of sequence (A) of the
amplified product to the complementary sequence (A'). In other
cases, the oligonucleotide attached to the surface of the solid
surface may not be a perfect complement of at least a portion of
the 5' end of the amplified product. For example, the
oligonucleotide may differ by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
12, 15, 20, or more nucleotides from the perfect complement of
sequence (A). The oligonucleotide may be attached to the solid
surface by any method known in the art. For example, the
oligonucleotide may be attached via non-covalent interaction such
as by ionic, hydrogen, or hydrophobic interaction or a combination
thereof. In some cases, the oligonucleotide may be attached by one
or more covalent bonds between the oligonucleotide and the solid
surface. In some cases, the oligonucleotide may be attached by
interaction between a receptor ligand pair such as for example an
avidin molecule on the surface and a biotin molecule on the
oligonucleotide, or any receptor ligand pair known in the art.
[0294] FIG. 8 illustrates that the amplified products with defined
3' and 5' sequences can be used for gap ligation to create a
circular polynucleotide which can then be amplified and
characterized using rolling circle amplification. In FIG. 8, the
amplified product with a defined sequence (B') at its 3' end and a
defined sequence (A) near its 5' end is hybridized to a sequence D
with sequences complementary to sequence (A) and sequence (B')
(with B' hybridizes upstream from (A), wherein the hybridization of
sequences (A) and (B') leave a gap. The gap is closed with a ligase
and alternatively with a polymerase for larger gaps. The circular
polynucleotide, e.g. DNA, can then be amplified and characterized
using rolling circle amplification. Primers for rolling circle
amplification can hybridize (3) to sequence (A), (2) to sequence
(B'), or (1) to the sequence between (A) and (B') on the amplified
product. These methods can be used for the accurate determination
of mutations such as single nucleotide polymorphisms (SNPs).
Alternative Method for Generating a Polynucleotide Having a Defined
3' and 5' Sequences from an RNA Target
[0295] One aspect of the present invention is an alternative method
for generating a polynucleotide having a defined 3' and 5'
sequences from an RNA target. Unlike the methods described in
detail above, this method generally does not use a composite
RNA/DNA primer as the first primer. Here, a tailed primer, usually
not comprising RNA, composed, for example, of DNA is used to create
the first primer extension product. In this method, the second
primer comprises a composite RNA/DNA primer, and the third primer
comprises a ligand for binding the third primer extension product
to a solid surface.
[0296] The method comprises step: (a) extending a first primer
comprising a 3' portion complementary to a target RNA and a 5'
portion, sequence (D), not complementary to the target RNA, to form
a first primer extension product hybridized to the target RNA,
forming an RNA/DNA hybrid. The first primer generally does not
comprise RNA, and may be all DNA. The first primer is a tailed
primer comprising a 5' portion, sequence (D) which is generally not
complementary to the target RNA, and does not hybridize to the
target RNA. In some embodiments, the 3' portion of the primer that
is complementary to the target RNA is a specific sequence. For
example, where a specific region of interest of a target RNA that
is known or suspected to be upstream of a specific sequence on the
target RNA, the sequence that is complementary to the target RNA of
the first primer can be designed to hybridize to this specific
sequence on the target RNA such that extension of the primer
results in producing a first primer extension product that is
complementary to such upstream region. The specific sequence may be
common to a family of target RNA. A combination of primers with
various specific sequences at the 3' end can also be useful. In
some embodiments, such as where the target RNA comprises mRNA, and
the mRNA comprises a plurality of sequences, each having a 3'
poly-A segment; the specific sequence that is complementary to the
target RNA can comprise a sequence that will hybridize to the
poly-A region of the mRNA, thus allowing the extension of the first
primer to produce a plurality of first primer extension products,
each of which is complementary to the region of an mRNA molecule
adjacent to the poly-A region. In some embodiments, the sequence
that is complementary to the target RNA comprises a random
sequence, such that the extension of the first primer results in a
plurality of first primer extension products complementary to the
sequences adjacent to the sequence where each random species
hybridizes. The use of a random sequence at the 3' end of the
primer can be useful for performing a global amplification of a RNA
target, generating a plurality of sequences which together can
represent, for example substantially the whole sequence of the
target RNA. In some embodiments, the relative amounts of the
various sequences can be used to quantitate the relative amount of
a given sequence in a sample, for example to determine the level of
expression in an mRNA sample. In some embodiments more than one
type of sequence that is complementary to the target RNA can be
used, for instance both a primer with a random sequence and a
primer, or combination of primers with a specific sequence
complementary to RNA can be used. In some embodiments, multiple
primers comprising different specific sequences can be used.
[0297] The method further comprises step (b) cleaving the target
RNA from the RNA/DNA hybrid. In some cases the cleaving can be
accomplished by denaturing the complex comprising the first primer
extension product and the nucleic acid. Denaturation can be
performed, for example by heating the sample, or by adding a
denaturing agent, or using a combination of heating the sample and
adding denaturing agents. The cleaving can be accomplished with an
enzyme that cleaves RNA from an RNA/DNA hybrid such as RNase H, or
a combination of RNase enzymes, or chemically. In some embodiments,
the target RNA is completely cleaved. In other embodiments, the
target RNA is only partly cleaved or degraded. The amount of
cleaving required is that amount which will allow the extension of
the second primer.
[0298] The method further comprises step (c) extending a second
primer comprising a DNA segment and a 5' RNA segment, wherein a 3'
portion of the primer is complementary to the first primer
extension product and a 5' portion, sequence (E), of the of the
second primer is not complementary to the first primer extension
product, to produce a double-stranded DNA product comprising the
first primer extension product hybridized to a second primer
extension product, whereby the second primer extension product has
a sequence (D') that is complementary to sequence (D) at its 3'
end. The extension of the second primer is carried out with a DNA
polymerase as described herein. The second primer is a composite
RNA/DNA primer having a 3' portion which is complementary to the
first primer extension product, and a 5' portion, sequence (E),
which is not complementary to the first primer extension product.
In some embodiments, the second primer can comprise a specific
primer sequence that is designed to hybridize to a specific
sequence in the first primer extension product. In some embodiments
the second primer comprises a random primer sequence that randomly
binds to the first primer extension product. Extension of the
second primer comprising a random sequence produces a plurality of
second primer extension products. The use of a random sequence at
the 3' end of the primer is useful, for example, in performing
global amplification of a target RNA, whereby a plurality of second
primer extension products are produced which is representative of
the sequence of the target RNA. In some embodiments, for example
where the first primer is designed to hybridize to a specific
sequence on a target RNA, or a sequence common to a family of RNA
targets, random priming by the second primer ensures amplification
of the entire selected target or family of selected targets. In
this embodiment, the second primer extension products comprise
sequences which are the same or substantially the same as the
sequences in the target RNA (sense copies).
[0299] The second primer extension product is extended such that
the 3' portion of the second primer extension product comprises a
sequence (D') which is complementary to sequence (D) of the first
primer. The primer extension results in a product that is at least
partially double stranded.
[0300] In some embodiments, the sample comprising the target RNA is
in a sample that also comprises DNA. In such cases, it can be
advantageous to add a selective DNA dependent DNA polymerase
inhibitor such as actinomycin such that it is present during step
(a) to selectively inhibit the production of extension product
complementary to the DNA during step (a). The presence of a DNA
dependent DNA polymerase inhibitor such as actinomycin is
particularly advantageous when a first primer comprising a random
sequence is used, as the inhibitor allows for the selective
creation of first primer extension products to RNA without the need
of separating the RNA from the DNA. This is also advantageous when
the priming is carried out at specific target sequences since the
sequence may be the same on the DNA when the DNA and RNA in the
sample represent total nucleic acid from the same biological
entity, for example, human tissue, animal tissue, and the like. The
use of DNA dependent DNA polymerase inhibitors such as actinomycin
is described in copending application.
[0301] The method further comprises step (d) denaturing the
double-stranded DNA product. Double stranded DNA can be denatured,
for example by heating, or by the addition of denaturing
agents.
[0302] The method further comprises step (e) annealing to the
second primer extension product and extending a third primer
comprising, from its 5' end, a ligand, optionally a sequence (F),
and a sequence (D), wherein sequence (D) is complementary to
sequence (D') on the second primer extension product to produce a
double-stranded DNA product comprising the second primer extension
product hybridized to a third primer extension product, whereby the
third primer extension product comprises a sequence (E') at its 3'
end complementary to sequence (E) in a DNA-RNA heteroduplex. The
third primer comprises a ligand that is a member of a
ligand-receptor pair. In some embodiments, the ligand is attached
to the primer at the 5' end of the primer. In some embodiments, the
ligand is a small molecule, such as biotin or digoxigenin. In some
embodiments, the receptor is an antibody, and the ligand is a
molecule or portion of a molecule recognized by the antibody. The
method produces a nucleic acid that comprises a ligand allowing it
to be bound to a solid surface and that has a specific sequence
(E') at its 3' end. The specific sequence (E') can be a site for
primer hybridization and further analysis or amplification of the
nucleic acid bound to the bead. As described above, the nucleic
acid attached to the ligand also comprises sequence (D') at or near
its 5' end, and in some embodiments comprises the sequence
(F').
[0303] In some embodiments, the method further comprises the step
of binding the ligand to a solid surface, whereby the third primer
extension product is bound to the solid surface.
[0304] In some embodiments, the method further comprises the steps
of: (f) cleaving the RNA portion of the second primer extension
product in the DNA-RNA heteroduplex, whereby sequence (E') of the
third primer extension product is single stranded, (g) annealing an
oligonucleotide comprising a 3' DNA segment (E) that is
complementary to sequence (E') and a 5' RNA segment comprising
sequence (G), (h) extending the third primer extension product to
produce a sequence (G') at its 3' end complementary to sequence
(G), and (i) cleaving the RNA from the heteroduplex created in step
(h) to produce a single-stranded portion of the third primer
extension product corresponding to sequence (G'). The cleaving of
RNA in steps (f) and (i) can be performed, for example by treatment
with RNase H, which will selectively cleave the RNA portion of the
DNA/RNA partial heteroduplex formed in step (e). The
oligonucleotide comprises at least one DNA and at least one RNA
portion. In some embodiments the 3' DNA segment is complementary to
all of sequence (E'), in other embodiments, the 3' DNA segment is
complementary to portion of sequence (E'). In some embodiments, 5'
RNA segment comprising sequence (G) is partly complementary to
sequence (E'). In some embodiments, the oligonucleotide is
optionally extended from its 3' end to produce an oligonucleotide
extension product hybridized to the third primer extension product
and displacing the DNA portion of the second primer extension
product. In some embodiments, the third primer comprises a sequence
(F), such that the oligonucleotide extension product will comprise
a sequence (F') at or near its 3' end that is complementary to
sequence (F). The DNA sequence (G') is created by a DNA polymerase
that has RNA dependent DNA polymerase activity. This step creates
an RNA/DNA heteroduplex region that can be used for further
manipulation of the second primer extension product.
[0305] In some embodiments the method further comprises binding the
ligand to a solid surface, whereby the third primer extension
product comprising sequence (G') is bound to the solid surface. The
solid surface can be any of a variety of surfaces, some described
in more detail below. The solid surface can be, for example a
planar surface, for example, a planar array. In some embodiments
the solid surface comprises a plurality of beads. In some
embodiments the beads are magnetic. The receptor bound to the solid
surface is a member of the ligand-receptor pair such that binding
of the ligand results in attaching the second primer extension
product to the solid surface. In some embodiments, the second
primer extension product is still hybridized to the first primer
extension product when it is attached to the solid surface. In some
embodiments, the second primer extension product is removed from
the third primer extension product such that a single stranded
polynucleotide is attached to the solid surface. The method
produces a nucleic acid that is bound to a solid surface that has a
specific sequence (E') and (G') at its 3' end. The specific
sequence (G') can be a site for primer hybridization and further
analysis or amplification of the nucleic acid bound to the bead. As
described above, in some embodiments, the nucleic acid bound to the
bead also comprises sequence (F) at or near its 5' end. One aspect
of the invention comprises amplification of the nucleic acid bound
to the bead. In some embodiments, the amplification is carried out
using isothermal amplification using a composite RNA/DNA primer,
RNase H, and a polymerase with strand displacement activity. For
this embodiment, the sequence (G') acts as the site to which the
composite RNA/DNA amplification primer hybridizes, allowing for
amplification. When the sequence (G') acts as a site to which a
composite amplification primer binds, the amplified product that is
produced has the sequence (E) (and a portion of sequence (G) at its
5' end. The amplified product has a sequence (D') at or near its 3'
end. Where the third primer comprises the sequence (F), the
amplified product also has the sequence (F'), complementary to (F)
at or near its 3' end. Thus the method produced amplified product
with defined sequences at or near both its 3' and 5' ends.
[0306] In some embodiments, for example where random sequences at
the 3' end of the first and/or second primer are used, a plurality
of different nucleic acids bound to a solid surface is created in
which each of the nucleic acids has a specific sequence (E') and
(G') at its 3' end, the sequence (D) at or near its 5' end (and in
some embodiments also a specific sequence (F) at its 5' end), and
where the different nucleic acids have different intervening
sequences, wherein the intervening sequences are identical to or
substantially identical to the sequences in the target RNA. The set
of bound nucleic acids thus generated can be analyzed, for example,
by sequencing in order to provide information about the sequence of
the target RNA.
[0307] In some embodiments, the method further comprises treating
the solid surface with reagents to produce multiple copies of an
amplification product that are substantially complementary the
second primer extension product. This step comprises carrying out
an amplification reaction wherein the bound nucleic acid acts as a
template for the amplification. Generally, the amplification is
carried out using the sequence (G') on the third primer extension
product for the hybridization of primer. In some embodiments, the
amplification produces single stranded amplified product, In some
embodiments, the amplification provides double stranded product.
The third primer extension product comprises the specific sequence
(D). In some embodiments, the third primer comprises a specific
sequence (F), which thus becomes incorporated into the third primer
extension product. In some embodiments the amplification is an
isothermal amplification reaction comprising a composite RNA/DNA
primer, RNase H, and a DNA polymerase with strand displacement
activity. In some embodiments, the amplification is carried out
using polymerase chain reaction, (PCR). For example where the third
primer extension product comprises both as sequence (F) at or near
its 5' end and a sequence (G') at or near its 3' end, a set of
primers, one designed to hybridize to all or a portion of the
sequence (G') and the other designed to hybridize to sequence (F'),
complementary to sequence (F), and/or to sequence (D') to
exponentially produce double stranded amplified product.
[0308] One aspect of the invention is a method of amplifying a
sequence representative of a sequence within a target RNA
comprising the above steps and further comprising: (j) annealing an
amplification primer, wherein the amplification primer has a DNA
portion and a 5' RNA portion, to the single stranded portion of the
third primer extension product complementary to sequence (G'); (k)
extending the amplification primer with an enzyme having strand
displacement activity to produce an amplified product; (l) cleaving
the RNA from the amplified product; and (m) repeating steps (j) to
(l) to produce multiple copies of amplified product wherein the 5'
portion of the amplified product has a sequence (E) complementary
to sequence (E') and the 3' end of the amplified product has
sequence (D') complementary to sequence (D) and optionally sequence
(F') complementary to sequence (F).
[0309] The step of binding the polynucleotides to the solid surface
through the ligand can be carried out such that only one nucleic
acid is bound to an isolated area of a surface or only one nucleic
acid is bound to a single bead. This isolated binding of nucleic
acids can be used for clonal amplification of the specific bound
nucleic acid in that area or on that bead. Such bound, isolated
nucleic acids can also be stored and archived for later analysis,
for example by sequencing. The bound, isolated nucleic acids can be
amplified, stored, and analyzed multiple times.
[0310] In some embodiments the amplification is carried out such
that the amplified product is not attached to the substrate, but is
freely dissolved in the solution. In other embodiments, the
amplification is carried out such that the amplified product
remains bound to the substrate, for example by performing solid
phase PCR such as bridge PCR. In yet other embodiments, an
amplified product is generated that may float freely in solution,
but which comprises a sequence, for example sequence (E) or
sequence (D'), that allows it to be captured to another solid
surface or other portion of the solid surface by hybridization to a
complementary sequence bound to such surface (e.g. sequence (E') or
sequence (D). In some embodiments, the amplified product is a
single-stranded product and, because it is generated at the solid
surface, the amplified product readily captured by complementary
sequences, e.g. sequence (B), bound to the surface.
[0311] In one aspect of the invention, a plurality of beads is
used, and the methods described above are carried out such that on
average, one or fewer third primer extension product molecules are
bound per bead. The beads are dispersed into an aqueous solution,
and a plurality of microreactors, e.g. droplets, are produced such
that on average one or fewer beads is contained within each of the
plurality of microreactors. The amplification of the third primer
extension products bound to the beads is then carried out such that
the clonal amplification of a plurality of third primer extension
products is achieved. This clonal amplification in microreactors
can be performed on a sample of target RNA, such as whole
transcriptome or total RNA, wherein the plurality of third primer
extension products comprise sequences that correspond to most, to
substantially all, or to all of the sequences in the target RNA. In
some embodiments, the amplified products are captured by bead
having attached thereto a plurality of oligonucleotides comprising
complementary sequences bound to such surface (e.g. sequence (E')
or sequence (D)), which are complementary to sequence (E) or
sequence (D') on the amplified product.
[0312] In some embodiments, the plurality of beads, produced as
described above, with each bead comprising a single third primer
extension product can comprise a library. These libraries can be
stored, then later clonally amplified. In some embodiments, a
library of beads can comprise a plurality of beads wherein each
bead had multiple copies of a single amplification product
generated from a third primer extension product. These libraries
can be analyzed, for example by sequencing. The libraries can be
stored, and later analyzed. In some embodiments the libraries can
be stored, then analyzed multiple times.
[0313] In some embodiments, a bead or isolated area of the solid
surface comprises covalently attached thereto multiple
oligonucleotides comprising the sequence (D) (or F) at their 3'
ends, whereby upon the amplification of step (m) multiple copies of
amplified product comprising sequence (D') (or F') at their 5' end
are hybridized to the bead or isolated area. For example, where
beads are used, a plurality of beads in a plurality of
microreactors wherein, the plurality of beads has, on average one
or fewer third primer extension products bound to it and there are,
on average, one or fewer beads in each microreactor, a clonal
amplification of the plurality of third primer extension products
can be carried out, and the amplified products in each of the
microreactors will bind to the bead through the sequence (D')
(and/or F') on the amplified product to the sequence (D) (and/or F)
on the beads. This approach produces a plurality of beads, each
with multiple copies of a different sequence bound to it. Where
these sequences are representative of the target RNA, the plurality
of beads can constitute a library representative of such RNA.
[0314] After the amplified products are bound to the beads by
hybridization, the (D) sequences on the beads can be extended to
produce a multiple polynucleotides covalently attached to the bead
or isolated area that are substantially complementary to the
amplified product and also comprise sequence (E') near their 5'
ends. Where the (D) (and or F) sequences are covalently attached to
the beads, this method provides for the production of beads with
polynucleotides complementary to amplified product covalently
attached to the beads. Covalently attached polynucleotides such as
those produce here are more robust than nucleotides that are
attached only by hybridization to the beads. Thus, the covalently
attached polynucleotides can be more stable and can be used with
analysis methods and sequencing methods that have harsher
conditions which would result in the displacement of
polynucleotides bound only by hybridization.
[0315] In some embodiments, the amplified product is removed from
the covalently bound polynucleotide to render the polynucleotide
single stranded. Such single stranded covalently bound
polynucleotides comprise a specific sequence at their 3' ends
comprising sequence (E') and a portion of sequence (G'). This
specific sequence at the 3' end of the covalently bound
polynucleotide can act as a hybridization site for a primer
complementary to sequence (E) that can act as a primer to carry out
sequencing by any of a variety of sequencing methods, for example,
those described herein.
[0316] The sequencing methods can comprise the use of cleavable
labeled terminators. The sequencing method can comprise
pyrophosphate detection. The sequencing method can comprise an
isothermal sequencing method, for example using chimeric primers,
RNase H, and a polymerase with strand displacement activity. The
sequencing method can also comprise cycle sequencing.
[0317] The amplified products with defined 3' and 5' ends can be
used in the methods described herein for the amplified products
produced by the other methods. They can be used, for example, for
bridge PCR, rolling circle amplification, and strand displacement
amplification.
[0318] A schematic exemplary of an embodiment of the invention
relating to an alternative method for generating a polynucleotide
having a defined 3' and 5' sequences from an RNA target is shown in
FIG. 9. Step I illustrates the step of extending a DNA first primer
comprising a 3' portion complementary to a target RNA and a 5'
portion, sequence (D), not complementary to the target RNA, to form
a first primer extension product hybridized to the target RNA,
forming an RNA/DNA hybrid. Step II illustrates cleaving the target
RNA from the RNA/DNA hybrid, and hybridizing the second primer.
Step III illustrates extending a second primer comprising a DNA
segment and a 5' RNA segment, wherein a 3' portion of the primer is
complementary to the first primer extension product and a 5'
portion, sequence (E), of the of the second primer is not
complementary to the first primer extension product, to produce a
double-stranded DNA product comprising the first primer extension
product hybridized to a second primer extension product, whereby
the second primer extension product has a sequence (D') that is
complementary to sequence (D) at its 3' end. Step IV illustrates
denaturing the double-stranded DNA product. Step V illustrates
annealing to the second primer extension product and extending a
third primer comprising, from its 5' end, a ligand, optionally a
sequence (F), and a sequence (D), wherein sequence (D) is
complementary to sequence (D') on the second primer extension
product to produce a double-stranded DNA product comprising the
second primer extension product hybridized to a third primer
extension product, whereby the third primer extension product
comprises a sequence (E') at its 3' end complementary to sequence
(E). The products of steps V and VI may be useful for SPIA
amplification of a target RNA sequence or its complement.
[0319] Step VI illustrates the step of cleaving the RNA portion of
the second primer extension product in the DNA-RNA heteroduplex,
whereby sequence (E') of the third primer extension product is
single stranded.
[0320] FIG. 10 illustrates the isothermal amplification starting
from the partially double-stranded polynucleotide produced either
from RNA described above, or from DNA as described below. Step VII
comprises annealing an amplification primer comprising 5' RNA and
3' DNA segments. Step VIII shows the extension of the amplification
primer by a DNA polymerase with strand displacement activity. Step
IX shows the cleavage of the RNA portion of the amplification
primer. Step X illustrates the continued extension of the
amplification primer by a DNA polymerase with strand displacement
activity to produce an amplified product comprising sequence (D')
and sequence (F') at its 3' end. The product of step X can
hybridize to another amplification primer, and steps VII through X
can be repeated to produce multiple copies of amplified product.
While the ligand is unbound in this embodiment, it is to be
understood that the ligand can be bound to the surface at various
steps in the process. For example, it can be advantageous to have
the species bound during the amplification step. FIG. 11
illustrates the process shown in FIG. 10 where the ligand and third
primer extension product are bound to the support during
amplification.
Alternative Method for Generating a Polynucleotide Having a Defined
3' and 5' Sequences from a DNA Target
[0321] One aspect of the invention is an alternative method for
generating a polynucleotide having a defined 3' and 5' sequences
from a DNA target. This method generally does not use a composite
RNA/DNA primer as the first primer. Here, a tailed primer, usually
not comprising RNA, composed, for example, of DNA is used to create
a plurality of first primer extension products. In this method, the
second primer comprises a composite RNA/DNA primer, and the third
primer comprises a ligand for binding the third primer extension
products to a solid surface.
[0322] The method comprises step: (a) denaturing a double-stranded
target DNA. Double stranded DNA can be denatured, for example by
heating, or by the addition of denaturing agents.
[0323] The method further comprise step: (b) annealing to the
target DNA and extending a first primer comprising a 3' portion
comprising a random sequence and a 5' portion, sequence (D), which
is not complementary to the target DNA, to form a plurality of
first primer extension products, each comprising sequence (D) at
its 3' end. The first primer generally does not comprise RNA, and
may be all DNA. The first primer is a tailed primer comprising a 5'
portion, sequence (D) which is generally not complementary to the
target DNA, and does not hybridize to the target DNA. The sequence
that is complementary to the target DNA comprises a random
sequence, such that the extension of the first primer results in a
plurality of first primer extension products complementary to the
sequences adjacent to the sequence where each random species
hybridizes. The use of a random sequence at the 3' end of the
primer can be useful for performing a global amplification of a DNA
target, generating a plurality of sequences which together can
represent, for example substantially the whole sequence of the
target DNA. In some embodiments, the relative amounts of the
various sequences can be used to quantitate the relative amount of
a given sequence in a sample, for example to determine the number
of gene copies in a target DNA sample. In some embodiments more
than one type of sequence that is complementary to the target DNA
can be used, for instance both a primer with a random sequence and
a primer with a sequence complementary to DNA can be used. In some
embodiments, multiple primers comprising different specific
sequences can be used.
[0324] The method further comprises step (c): extending a second
primer comprising a DNA segment and a 5' RNA segment, wherein a 3'
portion comprises a random sequence, and a 5' portion, sequence
(E), of the of the second primer is not complementary to the first
primer extension products, to produce a plurality of
double-stranded DNA products comprising a first primer extension
product hybridized to a second primer extension product, whereby
the second primer extension products have a sequence (D') that is
complementary to sequence (D) at their 3' ends. The extension of
the second primer is carried out with a DNA polymerase as described
herein. The second primer is a composite RNA/DNA primer having a 3'
portion which is complementary to the first primer extension
product, and a 5' portion, sequence (E), which is not complementary
to the first primer extension product. The second primer comprises
a random primer sequence that randomly binds to the first primer
extension product. Extension of the second primer comprising a
random sequence produces a plurality of second primer extension
products. The use of a random sequence at the 3' end of the primer
is useful, for example, in performing global amplification of a
target DNA, whereby a plurality of second primer extension products
are produced which is representative of the sequence of the target
DNA. In some embodiments, for example where the first primer is
designed to hybridize to a specific sequence on a target RNA, or a
sequence common to a family of RNA targets, random priming by the
second primer ensures amplification of the entire selected target
or family of selected targets. In this embodiment, the second
primer extension products comprise sequences which are the same or
substantially the same as the sequences in the target DNA (sense
copies).
[0325] The second primer extension products are extended such that
the 3' portion of the second primer extension products comprises a
sequence (D') which is complementary to sequence (D) of the first
primer. The primer extension results in products that are at least
partially double stranded.
[0326] The method further comprises step (d) denaturing the
double-stranded DNA product. Double stranded DNA can be denatured,
for example by heating, or by the addition of denaturing
agents.
[0327] The method further comprises step (e) annealing to the
second primer extension product and extending a third primer
comprising, from its 5' end, a ligand, optionally a sequence (F),
and a sequence (D), wherein sequence (D) is complementary to
sequence (D') on the second primer extension products to produce
double-stranded DNA products comprising second primer extension
products hybridized to a third primer extension products, whereby
the third primer extension products comprise a sequence (E') at its
3' end complementary to sequence (E). The third primer comprises a
ligand that is a member of a ligand-receptor pair. In some
embodiments, the ligand is attached to the primer at the 5' end of
the primer. In some embodiments, the ligand is a small molecule,
such as biotin or digoxigenin. In some embodiments, the receptor is
an antibody, and the ligand is a molecule or portion of a molecule
recognized by the antibody. The method produces nucleic acids that
comprise a ligand allowing them to be bound to a solid surface and
that has a specific sequence (E') at its 3' end. The specific
sequence (E') can be a site for primer hybridization and further
analysis or amplification of the nucleic acid bound to the bead. As
described above, the nucleic acid attached to the ligand also
comprises sequence (D') at or near its 5' end, and in some
embodiments comprises the sequence (F').
[0328] In some embodiments, the method further comprises the step
of binding the ligand to a solid surface, whereby the third primer
extension product is bound to the solid surface.
[0329] In some embodiments, the method further comprises the steps
of: (f) cleaving the RNA portion of the second primer extension
products in the DNA-RNA heteroduplex, whereby sequence (E') of the
third primer extension products is single stranded, (g) annealing
an oligonucleotide comprising a 3' DNA segment (E) that is
complementary to sequence (E') and a 5' RNA segment comprising
sequence (G), (h) extending the third primer extension products to
produce a sequence (G') at their 3' end complementary to sequence
(G), and (i) cleaving the RNA from the heteroduplex created in step
(h) to produce a single-stranded portion of the third primer
extension products corresponding to sequence (G'). The cleaving of
RNA in steps (f) and (i) can be performed, for example by treatment
with RNase H, which will selectively cleave the RNA portion of the
DNA/RNA partial heteroduplex formed in step (e). The
oligonucleotide comprises at least one DNA and at least one RNA
portion. In some embodiments the 3' DNA segment is complementary to
all of sequence (E'), in other embodiments, the 3' DNA segment is
complementary to portion of sequence (E'). In some embodiments, 5'
RNA segment comprising sequence (G) is partly complementary to
sequence (E'). In some embodiments, the oligonucleotide is
optionally extended from its 3' end to produce a plurality of
oligonucleotide extension products hybridized to the third primer
extension products and displacing the DNA portion of the second
primer extension products. In some embodiments, the third primer
comprises a sequence (F), such that the oligonucleotide extension
products will comprise a sequence (F') at or near their 3' end that
is complementary to sequence (F). The DNA sequence (G') is created
by a DNA polymerase that has RNA dependent DNA polymerase activity.
This step creates an RNA/DNA heteroduplex region that can be used
for further manipulation of the second primer extension
products.
[0330] In some embodiments the method further comprises binding the
ligand to a solid surface, whereby the third primer extension
products comprising sequence (G') are bound to the solid surface.
The solid surface can be any of a variety of surfaces, some
described in more detail below. The solid surface can be, for
example a planar surface, for example, a planar array. In some
embodiments the solid surface comprises a plurality of beads. In
some embodiments the beads are magnetic. The receptor bound to the
solid surface is a member of the ligand-receptor pair such that
binding of the ligand results in attaching the second primer
extension products to the solid surface. In some embodiments, the
second primer extension products are still hybridized to the first
primer extension product when they are attached to the solid
surface. In some embodiments, the second primer extension products
are removed from the third primer extension products such that
single stranded polynucleotides are attached to the solid surface.
The method produces nucleic acids that are bound to a solid surface
having a defined sequence (E') and (G') at their 3' ends. The
specific sequence (G') can be a site for primer hybridization and
further analysis or amplification of the nucleic acids bound to the
beads. As described above, in some embodiments, the nucleic acid
bound to the bead also comprises sequence (F) at or near its 5'
end. One aspect of the invention comprises amplification of the
nucleic acids bound to the bead. In some embodiments, the
amplification is carried out using isothermal amplification using a
composite RNA/DNA primer, RNase H, and a polymerase with strand
displacement activity. For this embodiment, the sequence (G') acts
as the site to which the composite RNA/DNA amplification primer
hybridizes, allowing for amplification. When the sequence (G') acts
as a site to which a composite amplification primer binds, the
amplified products that are produced have the sequence (E) (and a
portion of sequence (G) at their 5' ends. The amplified products
have a sequence (D') at or near their 3' ends. Where the third
primer comprises the sequence (F), the amplified products also have
the sequence (F'), complementary to (F) at or near their 3' ends.
Thus the method produces amplified product with defined sequences
at or near both its 3' and 5' ends.
[0331] In some embodiments, for example where random sequences at
the 3' end of the first and/or second primer are used, a plurality
of different nucleic acids bound to a solid surface is created in
which each of the nucleic acids has a specific sequence (E') and
(G') at its 3' end, the sequence (D) at or near its 5' end (and in
some embodiments also a specific sequence (F) at its 5' end), and
where the different nucleic acids have different intervening
sequences, wherein the intervening sequences are identical to or
substantially identical to the sequences in the target DNA. The set
of bound nucleic acids thus generated can be analyzed, for example,
by sequencing in order to provide information about the sequence of
the target DNA.
[0332] In some embodiments, the method further comprises treating
the solid surface with reagents to produce multiple copies of
amplification products that are substantially complementary the
second primer extension products. This step comprises carrying out
an amplification reaction wherein the bound nucleic acid acts as a
template for the amplification. Generally, the amplification is
carried out using the sequence (G') on the third primer extension
product for the hybridization of primer. In some embodiments, the
amplification produces single stranded amplified products, In some
embodiments, the amplification provides double stranded products.
The third primer extension products comprise the specific sequence
(D'). In some embodiments, the third primer comprises a specific
sequence (F), which thus becomes incorporated into the third primer
extension products. In some embodiments the amplification is an
isothermal amplification reaction comprising a composite RNA/DNA
primer, RNase H, and a DNA polymerase with strand displacement
activity. In some embodiments, the amplification is carried out
using polymerase chain reaction, (PCR). For example where the third
primer extension products comprise both as sequence (F) at or near
its 5' end and a sequence (G') at or near its 3' end, a set of
primers, one designed to hybridize to all or a portion of the
sequence (G') and the other designed to hybridize to sequence (F'),
the complement of sequence (B), or sequence (D), can be used to
carry out a PCR reaction to exponentially produce double stranded
amplified products.
[0333] One aspect of the invention is a method of amplifying a
sequence representative of a sequence within a target DNA
comprising the above steps and further comprising: (j) annealing an
amplification primer, wherein the amplification primer has a DNA
portion and a 5' RNA portion, to the single stranded portion of the
third primer extension products complementary to sequence (G'); (k)
extending the amplification primer with an enzyme having strand
displacement activity to produce an amplified products; (l)
cleaving the RNA from the amplified products; and (m) repeating
steps (j) to (l) to produce multiple copies of amplified products
wherein the 5' portion of the amplified products have a sequence
(E) complementary to sequence (E') and the 3' end of the amplified
products have sequence (D') complementary to sequence (D) and
optionally sequence (F') complementary to sequence (F).
[0334] The step of binding the polynucleotides to the solid surface
through the ligand can be carried out such that only one nucleic
acid is bound to an isolated area of a surface or only one nucleic
acid is bound to a single bead. This isolated binding of nucleic
acids can be used for clonal amplification of the specific bound
nucleic acid in that area or on that bead. Such bound, isolated
nucleic acids can also be stored and archived for later analysis,
for example by sequencing. The bound, isolated nucleic acids can be
amplified, stored, and analyzed multiple times.
[0335] In some embodiments the amplification is carried out such
that the amplified products are not attached to the substrate, but
is freely dissolved in the solution. In other embodiments, the
amplification is carried out such that the amplified products
remains bound to the substrate, for example by performing solid
phase PCR such as bridge PCR. In yet other embodiments, amplified
products are generated that may float freely in solution, but which
comprise a sequence, for example sequence (E) or sequence (D'),
that allows them to be captured to another solid surface or other
portion of the solid surface by hybridization to a complementary
sequence bound to such surface (e.g. sequence (E') or sequence (D).
In some embodiments, the amplified product is a single-stranded
product and, because it is generated at the solid surface, the
amplified product readily captured by complementary sequences, e.g.
sequence (B), bound to the surface.
[0336] In one aspect of the invention, a plurality of beads is
used, and the methods described above are carried out such that on
average, one or fewer third primer extension product molecules are
bound per bead. The beads are dispersed into an aqueous solution,
and a plurality of microreactors, e.g. droplets, are produced such
that on average one or fewer beads is contained within each of the
plurality of microreactors. The amplification of the third primer
extension products bound to the beads is then carried out such that
the clonal amplification of a plurality of third primer extension
products is achieved. This clonal amplification in microreactors
can be performed on a sample of target DNA such as genomic, wherein
the plurality of third primer extension products comprise sequences
that correspond to most, to substantially all, or to all of the
sequences in the target DNA. In some embodiments, the amplified
products are captured by bead having attached thereto a plurality
of oligonucleotides comprising complementary sequences bound to
such surface (e.g. sequence (E') or sequence (D)), which are
complementary to sequence (E) or sequence (D') on the amplified
product.
[0337] In some embodiments, the plurality of beads, produced as
described above, with each bead comprising a single third primer
extension product can comprise a library. These libraries can be
stored, then later clonally amplified. In some embodiments, a
library of beads can comprise a plurality of beads wherein each
bead had multiple copies of a single amplification product
generated from a third primer extension product. These libraries
can be analyzed, for example by sequencing. The libraries can be
stored, and later analyzed. In some embodiments the libraries can
be stored, then analyzed multiple times.
[0338] In some embodiments, a bead or isolated area of the solid
surface comprises covalently attached thereto multiple
oligonucleotides comprising the sequence (D) (or F) at their 3'
ends, whereby upon the amplification of step (m) multiple copies of
amplified product comprising sequence (D') (or F') at their 5' end
are hybridized to the bead or isolated area. For example, where
beads are used, a plurality of beads in a plurality of
microreactors wherein, the plurality of beads has, on average one
or fewer third primer extension products bound to it and there are,
on average, one or fewer beads in each microreactor, a clonal
amplification of the plurality of third primer extension products
can be carried out, and the amplified products in each of the
microreactors will bind to the bead through the sequence (D')
(and/or F') on the amplified product to the sequence (D) (and/or F)
on the beads. This approach produces a plurality of beads, each
with multiple copies of a different sequence bound to it. Where
these sequences are representative of the target DNA, the plurality
of beads can constitute a library representative of such DNA.
[0339] After the amplified products are bound to the beads by
hybridization, the (D) sequences on the beads can be extended to
produce a multiple polynucleotides covalently attached to the bead
or isolated area that are substantially complementary to the
amplified product and also comprise sequence (E') near their 5'
ends. Where the (D) (and or F) sequences are covalently attached to
the beads, this method provides for the production of beads with
polynucleotides complementary to amplified product covalently
attached to the beads. Covalently attached polynucleotides such as
those produce here are more robust than nucleotides that are
attached only by hybridization to the beads. Thus, the covalently
attached polynucleotides can be more stable and can be used with
analysis methods and sequencing methods that have harsher
conditions which would result in the displacement of
polynucleotides bound only by hybridization.
[0340] In some embodiments, the amplified product is removed from
the covalently bound polynucleotide to render the polynucleotide
single stranded. Such single stranded covalently bound
polynucleotides comprise a specific sequence at their 3' ends
comprising sequence (E') and a portion of sequence (G'). This
specific sequence at the 3' end of the covalently bound
polynucleotide can act as a hybridization site for a primer
complementary to sequence (E) that can act as a primer to carry out
sequencing by any of a variety of sequencing methods, for example,
those described herein.
[0341] The sequencing can be used to reveal information about the
target DNA, for example the genomic DNA. The sequencing methods can
comprise the use of cleavable labeled terminators. The sequencing
method can comprise pyrophosphate detection. The sequencing method
can comprise an isothermal sequencing method, for example using
chimeric primers, RNase H, and a polymerase with strand
displacement activity. The sequencing method can also comprise
cycle sequencing.
[0342] The amplified products with defined 3' and 5' ends can be
used in the methods described herein for the amplified products
produced by the other methods. They can be used, for example, for
bridge PCR, rolling circle amplification, and strand displacement
amplification.
[0343] A schematic exemplary of an embodiment of the invention
relating to an alternative method for generating a polynucleotide
having a defined 3' and 5' sequences from an RNA target is shown in
FIG. 12. The steps shown in FIG. 12 are applicable to product that
is generated from the alternative method for either RNA or from DNA
as described above. Step I comprises the steps of annealing an
oligonucleotide comprising a 3' DNA segment (E) that is
complementary to sequence (E') and a 5' RNA segment comprising
sequence (G). Step II comprises extending the third primer
extension product to produce a sequence (G') at its 3' end
complementary to sequence (G). Step III comprises cleaving the RNA
from the heteroduplex created in step II to produce a
single-stranded portion of the third primer extension product
corresponding to sequence (G').
[0344] Step IV comprises annealing an amplification primer, wherein
the amplification primer has a DNA portion and a 5' RNA portion, to
the single stranded portion of the third primer extension product
complementary to sequence (G'). Step V comprises extending the
amplification primer with an enzyme having strand displacement
activity to produce an amplified product. Step VI comprises
cleaving the RNA from the amplified product. The product of step VI
can hybridize to another amplification primer, thus allowing steps
IV to VI to be repeated to produce multiple copies of amplified
product wherein the 5' portion of the amplified product has a
sequence (E) complementary to sequence (E') and the 3' end of the
amplified product has sequence (D') complementary to sequence (D)
and optionally sequence (F') complementary to sequence (F).
Alternative Method for Generating a Polynucleotide Bound to a Solid
Surface
[0345] One aspect of the invention is a method for attaching a
polynucleotide sequence that is representative of a sequence within
a nucleic acid target molecule to a solid surface. The terms solid
surface and solid support are used interchangeably herein. The
polynucleotide sequence that is produced is representative of the
sequence within a nucleic acid target molecule if it is either the
same as, or complementary to the sequence within the target nucleic
acid. Where the target nucleic acid is double stranded, the method
can produce sequences that are representative of both of the
strands. The polynucleotide can be, for example either DNA or
RNA.
[0346] The first step of the method comprises step: (a) extending a
first primer comprising a DNA segment and a 5' RNA segment, wherein
a 3' portion of the primer, sequence (P), is complementary to a
target nucleic acid and a 5' portion of the of the primer, sequence
(A), is not complementary to the target nucleic acid, to form a
first primer extension product hybridized to the target nucleic
acid. In some embodiments, the 3' portion of the primer that is
complementary to the target nucleic acid is a specific sequence.
For example, where a specific region of interest of a target
nucleic acid that is known or suspected to be upstream of a
specific sequence on the target nucleic acid, sequence (P) of the
composite primer can be designed to hybridize to this specific
sequence on the target nucleic acid such that extension of the
primer results in producing a first primer extension product that
is complementary to such upstream region. The specific sequence may
be common to a family of target RNA. A combination of primers with
various specific sequences at the 3' end can also be useful. In
some embodiments, such as where the target nucleic acid comprises
mRNA, and the mRNA comprises a plurality of sequences, each having
a 3' poly-A segment; the specific sequence (P) can comprise a
sequence that will hybridize to the poly-A region of the mRNA, thus
allowing the extension of the first primer to produce a plurality
of first primer extension products, each of which is complementary
to the region of an mRNA molecule adjacent to the poly-A region. In
some embodiments, the sequence (P) comprises a random sequence,
such that the extension of the first primer results in a plurality
of first primer extension products complementary to the sequences
adjacent to the sequence where each random species hybridizes. The
use of a random sequence such as sequence (P) at the 3' end of the
primer can be useful for performing a global amplification of a
nucleic acid target, generating a plurality of sequences which
together can represent, for example substantially the whole
sequence of the target nucleic acid. In some embodiments, the
relative amounts of the various sequences can be used to quantitate
the relative amount of a given sequence in a sample, for example to
determine the level of expression in an mRNA sample, or to
determine gene copy number in a DNA sample.
[0347] The first primer extension product comprises a 5' portion
comprising sequence (A). Sequence A comprises RNA. In some
embodiments, sequence (A) is RNA, and sequence (P) is DNA. In other
embodiments, sequence (A) will comprise some DNA. In some
embodiments, sequence (P) will comprise some RNA. In some
embodiments, sequence (A) and sequence (P) are adjacent.
[0348] The method further comprises step: (b) separating or
removing the first primer extension product from the target nucleic
acid. The first primer extension product can be separated from the
target nucleic acid by a variety of methods. In some cases the
separation can be affected by denaturing the complex comprising the
first primer extension product and the nucleic acid. Denaturation
can be performed, for example by heating the sample, or by adding a
denaturing agent, or using a combination of heating the sample and
adding denaturing agents. Other methods of separating the first
primer extension product from the target nucleic acid involve
selectively cleaving or degrading the target nucleic acid. Where
the target nucleic acid is RNA, the cleaving or degrading can be
accomplished by denaturing or heating the sample to degrade RNA or
with an enzyme that cleaves RNA from an RNA/DNA hybrid such as
RNase H, or chemically. In some embodiments, the target nucleic
acid is completely cleaved or degraded. In other embodiments, the
target nucleic acid is only partly cleaved or degraded. The amount
of cleavage or degradation required is that amount which will allow
the extension of the second primer. In some embodiments, the
cleavage or degradation is carried out partially, and the fragments
of the target nucleic acid that remain can constitute the second
primer for step (c).
[0349] The method further comprises step: (c) extending a second
primer to produce a double-stranded product comprising a second
primer extension product hybridized to the first primer extension
product, wherein the second primer comprises a 3' segment
complementary to a portion of the first primer extension product
and 5' segment non-complementary sequence (B) to the first primer
extension product, whereby a portion of the 3' end of the second
primer extension product comprises a sequence (A') that is
complementary to the sequence (A) of the of the first primer and a
portion of the 5' end of the second primer extension product
comprises non-complementary sequence (B).
[0350] The extension of the second primer is carried out with a DNA
polymerase as described herein. The second primer can comprise RNA,
DNA, or can be a composite primer comprising both RNA and DNA. The
second primer is generally a tailed primer having a 3' portion
which is complementary to the first primer extension product, and a
5' portion, sequence (B), which is not complementary to the first
primer extension product. In some embodiments, the second primer
can comprise a specific primer sequence that is designed to
hybridize to a specific sequence in the first primer extension
product. In some embodiments the second primer comprises a random
primer sequence that randomly binds to the first primer extension
product. Extension of the second primer comprising a random
sequence produces a plurality of second primer extension products.
The use of a random sequence at the 3' end of the primer is useful,
for example, in performing global amplification of a target RNA or
target DNA, whereby a plurality of second primer extension products
are produced which is representative of the sequence of the target
nucleic acid. In some embodiments, for example where the first
primer is designed to hybridize to a specific sequence on a target
RNA, or a sequence common to a family of RNA targets, random
priming by the second primer ensures amplification of the entire
selected target or family of selected targets. In this embodiment,
the second primer extension products comprise sequences which are
the same or substantially the same as the sequences in the target
nucleic acid (sense copies). The second primer comprises a sequence
(B) that is homologous to a sequence (B) on a solid support.
[0351] The second primer extension product is extended such that
the 3' portion of the second primer extension product comprises a
sequence (A') which is complementary to sequence (A) of the first
primer. Since sequence (A) on the first primer extension product
comprises RNA, both DNA dependent DNA polymerase activity and RNA
dependent DNA polymerase activity are used in step (c). The primer
extension results in a product that is at least partially double
stranded since sequence (B) is not hybridized to the first primer
extension product.
[0352] The method further comprises step: (d) adding an exonuclease
to the double-stranded DNA/RNA hybrid, whereby single stranded 3'
nucleotides are removed from the first primer extension product.
Non-limiting examples of an exonuclease include single-strand
specific 3'-exonucleases such as exonuclease 1. The exonuclease
should remove all of the single-stranded 3' nucleotides which are
not hybridized to sequence (B). In some embodiments, the
exonuclease may remove additional 3' nucleotides which are
hybridized to the second primer extension product. In other
embodiments, a polymerase comprising exonuclease activity may be
used. Non-limiting examples include a T4 polymerase comprising 3'
exonuclease activity.
[0353] The method further comprises step: (e) extending the first
primer extension product to produce a sequence (B'), complementary
to sequence (B) on the second primer extension product. The
extension of the first primer extension product is carried out with
a DNA polymerase as described herein and is generally carried out
with a DNA-dependent DNA polymerase if the second primer extension
product contains only DNA or with RNA-dependent DNA polymerase if
the second primer extension product contains a RNA sequence. The
primer extension results in a product that is double stranded and
comprises sequences (A) and (B') on the first primer extension
product and sequences (B) and (A') on the second primer extension
product.
[0354] The method further comprises step: (f) denaturing the first
and second primer extension products. The first primer extension
product can be separated from the second primer extension product
by denaturation. Denaturation can be performed, for example by
heating the sample, or by adding a denaturing agent, or using a
combination of heating the sample and adding denaturing agents.
[0355] The method further comprises step: (g) binding the sequence
(B') of the first primer extension product to a third primer
comprising sequence (B) bound to a solid surface, whereby the first
primer extension product is attached to the solid surface. The
third primer comprises an oligonucleotide with sequence (B) that is
complementary to the sequence (B') of the first primer extension
product and results in attaching the single stranded first primer
extension product to the solid surface. The method produces a
nucleic acid that is bound to a solid surface that has a specific
sequence (A) at its 5' end. As described above, in some
embodiments, the nucleic acid bound to the bead also comprises
sequence (B') at or near its 3' end.
[0356] Step (g) of binding the polynucleotides to the solid surface
sequence (B) can be carried out such that only one nucleic acid is
bound to an isolated area of a surface or only one nucleic acid is
bound to a single bead. This isolated binding of nucleic acids can
be used for clonal amplification of the specific bound nucleic acid
in that area or on that bead. Such bound, isolated nucleic acids
can also be stored and archived for later analysis, for example by
sequencing. The bound, isolated nucleic acids can be amplified,
stored, and analyzed multiple times.
[0357] The method further comprises step: (h) extending the
sequence (B) of the third primer to produce a double-stranded
product comprising a third primer extension product hybridized to
the first primer extension product, wherein the 5' end of the third
primer comprises a sequence (B) complementary to the sequence (B')
of the first primer extension product, whereby a portion of the 3'
end of the third primer extension product comprises a sequence (A')
that is complementary to the sequence (A) of the of the first
primer.
[0358] The extension of the third primer is carried out with a DNA
polymerase as described herein. The primer extension results in a
product that is double stranded and comprises sequences (A) and
(B') on the first primer extension product and sequences (B) and
(A') on the third primer extension product.
[0359] The specific, or universal, sequence (A') can be a site for
primer hybridization and further analysis or amplification of the
nucleic acid bound to the bead. One aspect of the invention
comprises amplification of the nucleic acid bound to the bead. In
some embodiments, the amplification is carried out using isothermal
amplification using a composite RNA/DNA primer, RNase H, and a
polymerase with strand displacement activity. For this embodiment,
the sequence (A') acts as the site to which the composite RNA/DNA
amplification primer hybridizes, allowing for amplification. In
some embodiments, for example where random sequences at the 3' end
of the first and/or second primer are used, a plurality of
different nucleic acids bound to a solid surface is created in
which each of the nucleic acids has a specific sequence (A') at its
3' end (and in some embodiments also a specific sequence (B) at its
5' end), and where the different nucleic acids have different
intervening sequences, wherein the intervening sequences are
identical to or substantially identical to the sequences in the
target nucleic acid. The set of bound nucleic acids thus generated
can be analyzed, for example, by sequencing in order to provide
information about the sequence of the target nucleic acid.
[0360] A schematic exemplary of an embodiment of the invention
relating to an alternative method for generating polynucleotide
bound to a solid surface is shown in FIG. 13. The figure shows a
target nucleic acid (RNA or DNA) and a chimeric RNA/DNA first
primer. The primer is first annealed to the target nucleic. Step Ia
(RNA target) and Ib (DNA target) illustrates extending a first
primer comprising a DNA segment and a 5' RNA segment, wherein a 3'
portion of the primer is complementary to a target nucleic acid and
a 5' portion, sequence (A), of the of the primer is not
complementary to the target nucleic acid, to form a first primer
extension product hybridized to the target nucleic acid, forming an
RNA/DNA hybrid. The sequence complementary to a target nucleic acid
can be a specific sequence, a sequence that will hybridize to
Poly-A, a sequence common to a plurality of regions (consensus
sequence), or a random sequence. Step IIa (RNA target) and IIb (DNA
target) represent separating the target nucleic acid from the
RNA/DNA hybrid. Separation of the target nucleic acid can be
accomplished thermally, chemically, or enzymatically, e.g. with
RNase H. The second primer comprising a 5' sequence (B) is then
annealed to the first primer extension product. Step IIIa and IIIb
illustrate extending a second primer, comprising a 5' sequence (B)
and a 3' segment complementary to a portion of the first primer
extension product, to produce a double stranded product with a
DNA/RNA heteroduplex at one end; wherein the double stranded
product comprises a second primer extension product hybridized to
the first primer extension product, and whereby a portion of the 3'
end of the second primer extension product comprises a sequence
(A') that is complementary to the sequence (A) of the of the first
primer. This embodiment provides for attachment of the first primer
extension product to the solid surface by creating a sequence (B'),
allowing attachment to a solid surface comprising sequences (B)
attached thereto. Step IV shows the removal of 3' nucleotides from
the 3' region of the first primer extension product that is not
hybridized to the second primer extension product. This step may be
done using exonucleases or using DNA polymerase comprising
exonuclease activity. Step V shows extension of the first primer
extension product by DNA polymerase to generate a sequence (B'),
complementary to the sequence (B) of the second primer extension
product. Step VIII shows the denaturation of the first and second
primer extension product by methods described previously. The first
primer extension product comprising a sequence (B') and a defined
sequence (A) at its 3' end is useful for storage, archiving and
analysis as it has a sequence (B') capable of binding to a solid
surface. Such first primer extension product also comprises a
sequence that is representative of (identical to or substantially
identical to) a sequence in the target RNA, so analysis of this
product provides information about the target RNA. Step VII shows
the binding of sequence (B') of the first primer extension product
to a sequence (B) on a solid surface, whereby the first primer
extension product becomes bound to the solid surface. Step VIII
shows extension of the immobilized sequence (B) oligonucleotide on
the solid support using DNA and RNA-dependent DNA polymerase,
resulting in another DNA/RNA heteroduplex.
Alternative Method for Generating a Polynucleotide for Binding to a
Solid Surface from an RNA Target
[0361] The invention provides methods, compositions and kits for
copying, storing, and amplifying polynucleotides having sequences
related to target ribonucleic acid (RNA) sequences. The methods
provide for amplification of a single RNA species or pool of RNA
species. The methods are suitable for, for example, generation of
libraries, including cDNA libraries. The methods can generate
single stranded RNA or DNA products, which are readily suitable for
multiplex analysis by microarray technologies, as well as
electrophoresis-based technologies such as differential display,
and for sequencing.
[0362] The methods of the invention can copy, store, and amplify
one or more species of RNA, such as a pool of RNA sequences, and is
most particularly suitable for the amplification of all RNA (such
as whole transcriptome or total RNA) sequences in a biological
sample. Thus, one of the major advantages of the methods of the
invention is the ability to copy, store, and amplify an entire pool
of sequences, which is essential for the ability to analyze the
gene expression profile in cells, such as the cells in a biological
sample of interest. The methods of the invention have the potential
of amplifying a multiplicity, a large multiplicity, and in some
embodiments all RNA (such as whole transcriptome or total RNA in a
sample) sequences in a sample.
[0363] Insofar as many mRNAs have a unique polyA 3'-end, the
amplification initiated from the 3'-end sequence of mRNAs is most
common for preparation of cDNA libraries and subsequent sequence
analysis for determination of gene expression profiling or other
applications. The methods of the invention are similarly suited for
preparation of libraries of amplified 3'-portions of mRNAs. The
sequence of the first primer used in the methods of invention can
be designed to be complementary to a multiplicity, or all, of the
mRNA species in the sample by using random sequences, according to
methods known in the art. The methods are also useful for whole
transcriptome amplification. The methods of the invention can be
used for the total RNA in samples such as viral RNA.
[0364] An aspect of the invention is a method for generating a
polynucleotide comprising a sequence (B') for binding to a solid
surface from a RNA target comprising the step of: (a) extending a
first primer comprising a DNA segment and a 5' RNA segment, wherein
a 3' portion of the primer is complementary to a target RNA and a
5' portion, sequence (A), of the of the primer is not complementary
to the target RNA; to form a first primer extension product
hybridized to the target RNA, forming an RNA/DNA hybrid. This
extension is generally performed with an enzyme comprising
RNA-dependent DNA polymerase activity.
[0365] In some embodiments, the 3' portion of the primer that is
complementary to the target RNA is a specific sequence. For
example, where a specific region of interest of a target RNA that
is known or suspected to be upstream of a specific sequence on the
target RNA, the sequence that is complementary to the target RNA of
the first primer can be designed to hybridize to this specific
sequence on the target RNA such that extension of the primer
results in producing a first primer extension product that is
complementary to such upstream region. The specific sequence may be
common to a family of target RNA. A combination of primers with
various specific sequences at the 3' end can also be useful. In
some embodiments, such as where the target RNA comprises mRNA, and
the mRNA comprises a plurality of sequences, each having a 3'
poly-A segment; the specific sequence that is complementary to the
target RNA can comprise a sequence that will hybridize to the
poly-A region of the mRNA, thus allowing the extension of the first
primer to produce a plurality of first primer extension products,
each of which is complementary to the region of an mRNA molecule
adjacent to the poly-A region. In some embodiments, the sequence
that is complementary to the target RNA comprises a random
sequence, such that the extension of the first primer results in a
plurality of first primer extension products complementary to the
sequences adjacent to the sequence where each random species
hybridizes. The use of a random sequence at the 3' end of the
primer can be useful for performing a global amplification of a
target RNA, generating a plurality of sequences which together can
represent, for example substantially the whole sequence of the
target RNA. In some embodiments, the relative amounts of the
various sequences can be used to quantitate the relative amount of
a given sequence in a sample, for example to determine the level of
expression in an mRNA sample. In some embodiments more than one
type of sequence that is complementary to the target RNA can be
used, for instance both a primer with a random sequence and a
primer, or combination of primers with a specific sequence
complementary to RNA can be used. In some embodiments, multiple
primers comprising different specific sequences can be used.
[0366] The method further comprises the step of: (b) removing the
target RNA from the RNA/DNA hybrid. In some embodiments, the
removal of the target RNA from the RNA/DNA hybrid involves
selectively cleaving or degrading the target RNA. In some cases,
the complex comprising the first primer extension product and the
nucleic acid can be heated in reaction conditions comprising Mg,
which leads to cleaving the RNA including the RNA in the RNA/DNA
hybrid. The RNA can also be removed from the RNA/DNA hybrid by
denaturation, performed, for example by heating the sample (thermal
methods), or by adding denaturing agents or using a combination of
heating the sample and adding denaturing agents. The cleaving can
be accomplished with an enzyme that cleaves RNA from an RNA/DNA
hybrid such as RNase H, or a combination of RNase enzymes, or
chemically. In some embodiments, the target RNA is completely
cleaved. In other embodiments, the target RNA is only partly
cleaved or degraded. The amount of cleaving required is that amount
which will allow the extension of the second primer.
[0367] The method further comprises the step of: (c) extending a
second primer, comprising a 3' segment complementary to a portion
of the first primer extension product and a 5' segment
non-complementary to the first primer extension product comprising
sequence (B), to produce a double-stranded DNA product with a
DNA/RNA heteroduplex at one end, wherein the double-stranded
product comprises a second primer extension product hybridized to
the first primer extension product and wherein a portion of the 3'
end of the second primer extension product comprises a sequence
(A') that is complementary to the sequence (A) of the of the first
primer.
[0368] The extension of the second primer is carried out with a DNA
polymerase as described herein. In some embodiments, a DNA
polymerase comprising both DNA and RNA dependent DNA polymerase
activities is used here. In other embodiments, both a RNA dependent
DNA polymerase and a DNA dependent DNA polymerase are used. The
second primer can comprise RNA, DNA, or can be a composite primer
comprising both RNA and DNA. In some embodiments, the second primer
can comprise a specific primer sequence that is designed to
hybridize to a specific sequence in the first primer extension
product. In some embodiments the second primer comprises a random
primer sequence that randomly binds to the first primer extension
product. Extension of the second primer comprising a random
sequence produces a plurality of second primer extension products.
The use of a random sequence at the 3' end of the primer is useful,
for example, in performing global amplification of a target RNA,
whereby a plurality of second primer extension products are
produced which is representative of the sequence of the target RNA.
In some embodiments, for example where the first primer is designed
to hybridize to a specific sequence on a target RNA, or a sequence
common to a family of RNA targets, random priming by the second
primer ensures amplification of the entire selected target or
family of selected targets. In this embodiment, the second primer
extension products comprise sequences which are the same or
substantially the same as the sequences in the target RNA (sense
copies). The second primer comprises a sequence (B) that is
homologous to a sequence (B) on a solid support.
[0369] The second primer extension product is extended such that
the 3' portion of the second primer extension product comprises a
sequence (A') which is complementary to sequence (A) of the first
primer. Since sequence (A) on the first primer extension product
comprises RNA, both DNA dependent DNA polymerase activity and RNA
dependent DNA polymerase activity are used in step (c). The primer
extension results in a product that is at least partially double
stranded since sequence (B) on the second primer does not hybridize
to the first primer extension product.
[0370] The method further comprises the step of: (d) adding an
exonuclease to the double-stranded DNA product, whereby single
stranded 3' nucleotides are removed from the 3' region of the first
primer extension product that is not hybridized to the second
primer extension product. Non-limiting examples of an exonuclease
include single-strand specific 3'-exonucleases such as exonuclease
1. The exonuclease should remove all of the single-stranded 3'
nucleotides which are not hybridized to sequence (B). In some
embodiments, the exonuclease may remove additional 3' nucleotides
which are hybridized to the second primer extension product. In
other embodiments, a polymerase comprising exonuclease activity may
be used. Non-limiting examples include a T4 polymerase comprising
3' exonuclease activity.
[0371] The method further comprises the step of: (e) extending the
first primer extension product to produce a sequence (B'),
complementary to sequence (B) on the second primer extension
product. The extension of the first primer extension product is
carried out with a DNA polymerase as described herein and is
generally carried out with a DNA-dependent DNA polymerase if the
second primer extension product contains only DNA or with
RNA-dependent DNA polymerase if the second primer extension product
contains a RNA sequence. The primer extension results in a product
that is double stranded and comprises sequences (A) and (B') on the
first primer extension product and sequences (B) and (A') on the
second primer extension product.
[0372] The method further comprises the step of: (f) denaturing the
double-stranded DNA product. The first primer extension product can
be separated from the second primer extension product by
denaturation. Denaturation can be performed, for example by heating
the sample, or by adding a denaturing agent, or using a combination
of heating the sample and adding denaturing agents.
[0373] The method further comprises the step of: (g) attaching the
single-stranded first primer extension product to a solid support
by annealing sequence (B') to the solid support comprising an
oligonucleotide attached thereto, comprising a sequence (B). The
oligonucleotide or third primer comprises an oligonucleotide
sequence (B) that is complementary to the sequence (B') of the
first primer extension product and results in attaching the single
stranded first primer extension product to the solid surface. The
method produces a nucleic acid that is bound to a solid surface
that has a specific sequence (A) at its 5' end. As described above,
in some embodiments, the nucleic acid bound to the bead also
comprises sequence (B') at or near its 3' end.
[0374] Step (g) of binding the polynucleotides to the solid surface
sequence (B) can be carried out such that only one nucleic acid is
bound to an isolated area of a surface or only one nucleic acid is
bound to a single bead. This isolated binding of nucleic acids can
be used for clonal amplification of the specific bound nucleic acid
in that area or on that bead. Such bound, isolated nucleic acids
can also be stored and archived for later analysis, for example by
sequencing. The bound, isolated nucleic acids can be amplified,
stored, and analyzed multiple times.
[0375] The method further comprises the step of: (h) extending
sequence (B) on the solid support to produce a third primer
extension product, hybridized to the second extension product,
wherein the third primer extension product comprises a 3' sequence
(A'), whereby a DNA/RNA heteroduplex at one end is generated. The
extension of the third primer is carried out with a DNA polymerase
as described herein. In some embodiments, a DNA polymerase
comprising both DNA and RNA dependent DNA polymerase activities is
used here. In other embodiments, both a RNA dependent DNA
polymerase and a DNA dependent DNA polymerase are used. The primer
extension results in a product that is double stranded and
comprises sequences (A) and (B') on the first primer extension
product and sequences (B) and (A') on the third primer extension
product.
[0376] The method produces a nucleic acid that is bound to a solid
surface that has a specific sequence (A') at its 3' end. The
specific, or universal, sequence (A') can be a site for primer
hybridization and further analysis or amplification of the nucleic
acid bound to the bead. One aspect of the invention comprises
amplification of the nucleic acid bound to the bead. In some
embodiments, the amplification is carried out using isothermal
amplification using a composite RNA/DNA primer, RNase H, and a
polymerase with strand displacement activity. For this embodiment,
the sequence (A') acts as the site to which the composite RNA/DNA
amplification primer hybridizes, allowing for amplification. In
some embodiments, for example where random sequences at the 3' end
of the first and/or second primer are used, a plurality of
different nucleic acids bound to a solid surface is created in
which each of the nucleic acids has a specific sequence (A') at its
3' end (and in some embodiments also a specific sequence (B) at its
5' end), and where the different nucleic acids have different
intervening sequences, wherein the intervening sequences are
identical to or substantially identical to the sequences in the
target nucleic acid. The set of bound nucleic acids thus generated
can be analyzed, for example, by sequencing in order to provide
information about the sequence of the target nucleic acid.
[0377] In some embodiments, the sample comprising the target RNA is
in a sample that also comprises DNA. In such cases, it can be
advantageous to add a selective DNA dependent DNA polymerase
inhibitor such as actinomycin such that it is present during step
(a) to selectively inhibit the production of extension product
complementary to the DNA during step (a). The presence of a DNA
dependent DNA polymerase inhibitor such as actinomycin is
particularly advantageous when a first primer comprising a random
sequence is used, as the inhibitor allows for the selective
creation of first primer extension products to RNA without the need
of separating the RNA from the DNA. This is also advantageous when
the priming is carried out at specific target sequences since the
sequence may be the same on the DNA when the DNA and RNA in the
sample represent total nucleic acid from the same biological
entity, for example, human tissue, animal tissue, and the like. The
use of DNA dependent DNA polymerase inhibitors such as actinomycin
is described in co-pending application.
[0378] One aspect of the invention comprises amplification of the
nucleic acid bound to the bead. In some embodiments, the
amplification is carried out using isothermal amplification using a
composite RNA/DNA primer, RNase H, and a polymerase with strand
displacement activity. For this embodiment, the sequence (A') acts
as the site to which the composite RNA/DNA amplification primer
hybridizes, allowing for amplification.
[0379] In some embodiments, the amplification comprises the steps
of: (i) cleaving the RNA region from the first polynucleotide
product hybridized to the third primer extension product using
RNase H; (j) annealing an amplification primer to sequence (A') on
the single-stranded portion of the third primer extension product,
wherein the amplification primer has a DNA portion and a 5' RNA
portion; (k) extending the amplification primer with an enzyme
having strand displacement activity to produce an amplified product
hybridized to the third primer extension product on the solid
support; (l) repeating steps (i) to (k) to produce multiple copies
of an amplified product wherein the amplified product comprises
sequence (B') at its 3' end; and (m) capturing the amplified
product on the solid support wherein the solid support comprises
sequence (B).
[0380] In some embodiments, for example where random sequences at
the 3' end of the first and/or second primer are used, a plurality
of different nucleic acids bound to a solid surface is created in
which each of the bound nucleic acids has a specific sequence (A')
at its 3' end and also a specific sequence (B) at its 5' end, and
where the different nucleic acids have different intervening
sequences, wherein the intervening sequences are identical to or
substantially identical to the sequences in the target RNA. The set
of bound nucleic acids thus generated can be analyzed, for example,
by sequencing in order to provide information about the sequence of
the target RNA.
[0381] The solid surface can be any of a variety of surfaces, some
described in more detail below. The solid surface can be, for
example a planar surface, for example, a planar array. In some
embodiments the solid surface comprises a plurality of beads. In
some embodiments the beads are magnetic.
[0382] The step of attaching or binding the polynucleotides to the
solid surface through the sequence (B), step (g), can be carried
out such that only one nucleic acid is bound to an isolated area of
a surface or only one nucleic acid is bound to a single bead. This
isolated binding of nucleic acids can be used for clonal
amplification of the specific bound nucleic acid in that area or on
that bead. Such bound, isolated nucleic acids can also be stored
and archived for later analysis, for example by sequencing. The
bound, isolated nucleic acids can be amplified, stored, and
analyzed multiple times.
[0383] In some embodiments, the method further comprises treating
the solid surface with reagents to produce multiple copies of an
amplification product that are substantially complementary to the
third primer extension product. This step comprises carrying out an
amplification reaction wherein the bound nucleic acid acts as a
template for the amplification. Generally, the amplification is
carried out using the sequence (A') on the third primer extension
product for the hybridization of primer. In some embodiments the
amplification is an isothermal amplification reaction comprising a
composite RNA/DNA primer, RNase H, and a DNA polymerase with strand
displacement activity. In some embodiments, the amplification is
carried out using polymerase chain reaction (PCR). For example
where the third primer extension product comprises both as sequence
(B) at or near its 5' end and a sequence (A') at or near its 3'
end, a set of primers, one designed to hybridize to all or a
portion of the sequence (A') and the other designed to hybridize to
sequence (B'), the complement of sequence (B), can be used to carry
out a PCR reaction to exponentially produce double stranded
amplified product.
[0384] In some embodiments the amplification is carried out such
that the amplified product is not attached to the substrate, but is
freely dissolved in the solution. In other embodiments, the
amplification is carried out such that the amplified product
remains bound to the substrate, for example by performing solid
phase PCR such as bridge PCR. In yet other embodiments, an
amplified product is generated that may float freely in solution,
but which comprises a sequence, for example sequence (A) or
sequence (B'), that allows it to be captured to another solid
surface or other portion of the solid surface by hybridization to a
complementary sequence bound to such surface, e.g. sequence (A') or
sequence (B). In some embodiments, the amplified product is a
single-stranded product and, because it is generated at the solid
surface, the amplified product readily captured by complementary
sequences, e.g. sequence (B), bound to the surface.
[0385] In one aspect of the invention, a plurality of beads is
used, and the methods described above are carried out such that on
average, one or fewer first primer extension product molecules are
bound per bead. The beads are dispersed into an aqueous solution,
and a plurality of microreactors, e.g. droplets, are produced such
that on average one or fewer beads is contained within each of the
plurality of microreactors. The amplification of the first primer
extension products bound to the beads is then carried out such that
the clonal amplification of each of the plurality of second primer
extension products in the separate microreactors is achieved. This
clonal amplification in microreactors can be performed on a sample
of target RNA, such as whole transcriptome or total RNA, wherein
the plurality of first primer extension products comprise sequences
that correspond to most, to substantially all, or to all of the
sequences in the target RNA. In some embodiments, the amplified
products are captured by bead having attached thereto a plurality
of oligonucleotides comprising complementary sequences bound to
such surface, e.g. sequence (A') or sequence (B), which are
complementary to sequence (A) or sequence (B') on the amplified
product.
[0386] In some embodiments, the plurality of beads, produced as
described above, with each bead comprising a single first primer
extension product can comprise a library. These libraries can be
stored, then later clonally amplified. In some embodiments, a
library of beads can comprise a plurality of beads wherein each
bead had multiple copies of a single amplification product
generated from a second primer extension product. These libraries
can be analyzed, for example by sequencing. The libraries can be
stored, and later analyzed. In some embodiments the libraries can
be stored, then analyzed multiple times.
[0387] A schematic exemplary of an embodiment of the invention
relating to this method for generating a polynucleotide for binding
to a solid surface from an RNA target is shown in FIG. 14. The
figure shows a target RNA and a chimeric RNA/DNA first primer. The
primer is first annealed to the target RNA. Step I illustrates
extension of the first primer comprising a DNA segment and a 5' RNA
segment, wherein a 3' portion of the primer is complementary to a
target RNA and a 5' portion, sequence (A), of the of the primer is
not complementary to the target RNA, to form a first primer
extension product hybridized to the target RNA, forming an RNA/DNA
hybrid. The sequence complementary to a target RNA can be a
specific sequence, a sequence that will hybridize to Poly-A, a
sequence common to a plurality of regions (consensus sequence), or
a random sequence. Step II represents separation of the target RNA
from the RNA/DNA hybrid. The separation can be accomplished
thermally, chemically, or enzymatically, e.g. with RNase H. The
second primer comprising a 5' sequence (B) is then annealed to the
first primer extension product. Step III illustrate extending a
second primer, comprising a 5' sequence (B) and a 3' segment
complementary to a portion of the first primer extension product,
to produce a double stranded product with a DNA/RNA heteroduplex at
one end; wherein the double stranded product comprises a second
primer extension product hybridized to the first primer extension
product, and whereby a portion of the 3' end of the second primer
extension product comprises a sequence (A') that is complementary
to the sequence (A) of the of the first primer. This embodiment
provides for attachment of the first primer extension product to
the solid surface by creating a sequence (B'), allowing attachment
to a solid surface comprising sequences (B) attached thereto. Step
IV shows the removal of 3' nucleotides from the 3' region of the
first primer extension product that is not hybridized to the second
primer extension product. This step may be done using exonucleases
or using DNA polymerase comprising exonuclease activity. Step V
shows extension of the first primer extension product by DNA
polymerase to generate a sequence (B'), complementary to the
sequence (B) of the second primer extension product. Step VI shows
the denaturation of the first and second primer extension product
by methods described previously. The first primer extension product
comprising a sequence (B') and a defined sequence (A) at its 3' end
is useful for storage, archiving and analysis as it has a sequence
(B') capable of binding to a solid surface. Such first primer
extension product also comprises a sequence that is representative
of (identical to or substantially identical to) a sequence in the
target RNA, so analysis of this product provides information about
the target RNA. Step VII shows the binding of sequence (B') of the
first primer extension product to a sequence (B) on a solid
surface, whereby the first primer extension product becomes bound
to the solid surface. Step VIII shows extension of the immobilized
sequence (B) oligonucleotide on the solid support using DNA and
RNA-dependent DNA polymerase, resulting in another DNA/RNA
heteroduplex.
[0388] In FIG. 15, Step I illustrates amplification using single
primer isothermal linear amplification (SPIA) wherein RNaseH
cleaves the RNA from the DNA/RNA heteroduplex, a chimeric RNA/DNA
primer binds to sequence (A'), and DNA polymerase with strand
displacement activity is used to extend the chimeric primer to
produce amplified product of the first primer extension product.
Step II illustrates amplified products that are produced from
repeated rounds of SPIA from Step I of the figure. Since the
amplification products are generated in close proximity to access
sequence (B) on the solid support, the amplified products are
captured on the solid support through hybridization of the 3'
sequence (B') to sequence (B) for further manipulations as
described herein such as clonal amplification.
Alternative Method for Generating a Polynucleotide for Binding to a
Solid Surface from a DNA Target
[0389] The methods of the present invention can be used to analyze
the DNA (e.g. genomic DNA) samples that are important for many
studies. The methods can be used for high-throughput genomic
analysis, and can be used for forensic and paleoarcheology work
which can be severely limited by nucleic acid sample size. The
methods can be used, for example, for the genotyping of multiple
loci in the study of complex diseases. The methods can also be used
for the determination of genomic instability in various
pathological conditions such as cancer, which is most precisely
carried out in well defined cell populations, such as that obtained
by laser capture micro-dissection or cell sorting. The DNA
amplification technologies described herein provide global
amplification of very small polynucleotide samples, for example,
from one or a very few cells.
[0390] One aspect of the invention is a method for generating from
a DNA target a polynucleotide comprising a sequence for binding to
a solid surface comprising the steps of: (a) denaturing a
double-stranded target DNA. Double stranded DNA can be denatured,
for example by heating, or by the addition of denaturing agents, or
using a combination of heating the sample and adding denaturing
agents.
[0391] The method further comprises step: (b) annealing to the
target DNA and extending with a DNA polymerase comprising strand
displacement activity, a first primer comprising a DNA segment and
a 5' RNA segment, wherein a 3' portion of the primer comprises a
random sequence, and a 5' portion of the primer comprises sequence
(A), which is not complementary to the target DNA; to form a
plurality of first primer extension product hybridized to the
target DNA and comprising sequence (A) at its 5' end. The enzyme
that carries out step (b) is generally a DNA polymerase. In some
cases a mixture of DNA polymerases can be used. This extension is
generally performed with an enzyme comprising DNA-dependent DNA
polymerase activity. The sequence that is complementary to the
target DNA comprises a random sequence, such that the extension of
the first primer results in a plurality of first primer extension
products complementary to the sequences adjacent to the sequence
where each random species hybridizes. The use of a random sequence
at the 3' end of the primer can be useful for performing a global
amplification of a DNA target, generating a plurality of sequences
which together can represent, for example substantially the whole
sequence of the target DNA. In some embodiments, the relative
amounts of the various sequences can be used to quantitate the
relative amount of a given sequence in a sample, for example to
determine the number of gene copies in a DNA sample, or obtaining
sequence information. In some embodiments, the extension of one
first primer, will result in the release of a downstream first
primer extension product. This can occur throughout the target DNA
resulting in the release of multiple first primer extension
products from the target DNA. This process can occur simultaneously
on both of the strands of the double-stranded DNA target, thus
creating first primer extension products complementary to sequences
in both strands.
[0392] In some embodiments, the first primer extension step is
carried out with a DNA polymerase capable of extension at elevated
temperature that is not compatible with subsequent hybridization of
the random sequence to the displaced primer-extension product. For
example, Bst DNA polymerase can be used which is active at elevated
temperature. The reaction can be carried out stepwise, first with
incubation at a lower temperature such as about 25.degree. C.,
followed by incubation at higher temperature such as about
50.degree. C. In some embodiments, the first incubation is carried
out below about 30.degree. C., and the second incubation is carried
out above about 40.degree. C. In some embodiments, a DNA polymerase
which is active at temperatures above about 45.degree. C. is used
to extend the first primer. Mixtures of DNA polymerases can also be
useful.
[0393] The method further comprises step: (c) separating the first
primer extension product from the target DNA. In some embodiments,
the separation can be affected by denaturing the complex comprising
the first primer extension product and the nucleic acid.
Denaturation can be performed, for example by heating the sample,
or by adding a denaturing agent, or using a combination of heating
the sample and adding denaturing agents. The amount of cleaving
required is that amount which will allow the extension of the
second primer.
[0394] The method further comprises step: (d) annealing to the
first primer extension product and extending a second primer
comprising a 3' complementary DNA region that comprises a random
sequence, wherein the second primer is a tailed primer comprising a
5' sequence (B), to form a double stranded product comprising a
first primer extension product and a second primer extension
product, whereby a double-stranded product with a DNA/RNA
heteroduplex at one end is generated. In some embodiments, a DNA
polymerase comprising both DNA and RNA dependent DNA polymerase
activities is used here. In other embodiments, both a RNA dependent
DNA polymerase and a DNA dependent DNA polymerase are used. This
step may be carried out with or without prior denaturation. If
carried out without denaturation, generally, only the single
stranded displaced first primer extension product will hybridize to
the second primer. Generally the second primer does not comprise
RNA. The extension of the second primer is carried out with a DNA
polymerase as described herein. The second primer comprises a
random primer sequence that randomly binds to the first primer
extension product. Extension of the second primer comprising a
random sequence produces a plurality of second primer extension
products. The use of a random sequence at the 3' end of the primer
is useful, for example, in performing global amplification of a
target DNA, whereby a plurality of second primer extension products
are produced which is representative of the sequence of the target
DNA. In some embodiments, for example where the first primer is
designed to hybridize to a specific sequence on a target DNA, or a
sequence common to a family of DNA targets, random priming by the
second primer ensures amplification of the entire selected target
or family of selected targets. In this embodiment, the second
primer extension products comprise sequences which are the same or
substantially the same as the sequences in the target DNA.
[0395] The second primer extension product is extended such that
the 3' portion of the second primer extension product comprises a
sequence (A') which is complementary to sequence (A) of the first
composite primer. Since sequence (A) on the first primer extension
product comprises RNA, both DNA dependent DNA polymerase activity
and RNA dependent DNA polymerase activity are used in step (d). The
primer extension results in a product that is at least partially
double stranded since sequence (B) not does hybridize to the first
primer extension product. The method produces a nucleic acid that
comprises a sequence (B), allowing it to be bound to a solid
surface by hybridization to its complement, which is immobilized on
the solid surface and that has a specific sequence (A') at its 3'
end. The specific, or universal, sequence (A') can be a site for
primer hybridization and further analysis or amplification of the
nucleic acid bound to the bead.
[0396] The method further comprises step: (e) adding an exonuclease
to the double-stranded DNA product, whereby single stranded 3'
nucleotides are removed from the 3' region of the first primer
extension product that is not hybridized to the second primer
extension product. Non-limiting examples of an exonuclease include
single-strand specific 3'-exonucleases such as exonuclease 1. The
exonuclease should remove all of the single-stranded 3' nucleotides
which are not hybridized to sequence (B). In some embodiments, the
exonuclease may remove additional 3' nucleotides which are
hybridized to the second primer extension product. In other
embodiments, a polymerase comprising exonuclease activity may be
used. Non-limiting examples include a T4 polymerase comprising 3'
exonuclease activity.
[0397] The method further comprises step: (f) extending the first
primer extension product to produce a sequence (B'), complementary
to sequence (B) on the second primer extension product. The
extension of the first primer extension product is carried out with
a DNA polymerase as described herein and is generally carried out
with a DNA-dependent DNA polymerase if the second primer extension
product contains only DNA or with RNA-dependent DNA polymerase if
the second primer extension product contains a RNA sequence. The
primer extension results in a product that is double stranded and
comprises sequences (A) and (B') on the first primer extension
product and sequences (B) and (A') on the second primer extension
product.
[0398] The method further comprises step: (g) denaturing the
double-stranded DNA product. The first primer extension product can
be separated from the second primer extension product by
denaturation. Denaturation can be performed, for example by heating
the sample, or by adding a denaturing agent, or using a combination
of heating the sample and adding denaturing agents.
[0399] The method further comprises step: (h) attaching the
single-stranded first primer extension product to a solid support
by annealing sequence (B') to the solid support comprising an
oligonucleotide attached thereto, comprising a sequence (B),
whereby a plurality of first primer extension products become bound
to the solid surface. The oligonucleotide or third primer comprises
a sequence or oligo (B) that is complementary to the sequence (B')
of the first primer extension product and results in attaching the
single stranded first primer extension product to the solid
surface. The method produces a nucleic acid that is hybridize to a
sequence on the solid surface and has a specific sequence (A) at
its 5' end.
[0400] Step (h) of binding the polynucleotides to the solid surface
sequence (B) can be carried out such that only one nucleic acid is
bound to an isolated area of a surface or only one nucleic acid is
bound to a single bead. This isolated binding of nucleic acids can
be used for clonal amplification of the specific bound nucleic acid
in that area or on that bead. Such bound, isolated nucleic acids
can also be stored and archived for later analysis, for example by
sequencing. The bound, isolated nucleic acids can be amplified,
stored, and analyzed multiple times.
[0401] The method further comprises step: (i) extending sequence
(B) on the solid support to produce a third primer extension
product, hybridized to the first primer extension product, wherein
the third primer extension product comprises a 3' sequence (A'),
whereby a DNA/RNA heteroduplex at one end is generated.
[0402] The extension of the third primer is carried out with a DNA
polymerase as described herein. In some embodiments, a DNA
polymerase comprising both DNA and RNA dependent DNA polymerase
activities is used here. In other embodiments, both a RNA dependent
DNA polymerase and a DNA dependent DNA polymerase are used. The
primer extension results in a product that is double stranded and
comprises sequences (A) and (B') on the first primer extension
product and sequences (B) and (A') on the third primer extension
product.
[0403] The method produces a nucleic acid that is bound to a solid
surface that has a specific sequence (A') at its 3' end. The
specific, or universal, sequence (A') can be a site for primer
hybridization and further analysis or amplification of the nucleic
acid bound to the bead. One aspect of the invention comprises
amplification of the nucleic acid bound to the bead. In some
embodiments, the amplification is carried out using isothermal
amplification using a composite RNA/DNA primer, RNase H, and a
polymerase with strand displacement activity. For this embodiment,
the sequence (A') acts as the site to which the composite RNA/DNA
amplification primer hybridizes, allowing for amplification. In
some embodiments, for example where random sequences at the 3' end
of the first and/or second primer are used, a plurality of
different nucleic acids bound to a solid surface is created in
which each of the nucleic acids has a specific sequence (A') at its
3' end (and in some embodiments also a specific sequence (B) at its
5' end), and where the different nucleic acids have different
intervening sequences, wherein the intervening sequences are
identical to or substantially identical to the sequences in the
target nucleic acid. The set of bound nucleic acids thus generated
can be analyzed, for example, by sequencing in order to provide
information about the sequence of the target nucleic acid.
[0404] One aspect of the invention comprises amplification of the
nucleic acid bound to the bead. In some embodiments, the
amplification is carried out using isothermal amplification using a
composite RNA/DNA primer, RNase H, and a polymerase with strand
displacement activity. For this embodiment, the sequence (A') acts
as the site to which the composite RNA/DNA amplification primer
hybridizes, allowing for amplification.
[0405] In some embodiments, the amplification comprises the steps
of: (j) cleaving the RNA from the first polynucleotide product
hybridized to the amplified product using RNase H; (k) annealing an
amplification primer to the single-stranded portion of the
amplified product complementary to sequence (A'), wherein the
amplification primer has a DNA portion and a 5' RNA portion; (l)
extending the amplification primer with an enzyme having strand
displacement activity to produce an amplified product hybridized to
the third primer extension product on the bead or isolated area;
(m) repeating steps (j) to (l) to produce multiple copies of an
amplified product wherein the amplified product comprises sequence
(B') at its 3' end; and (n) capturing the amplified product on the
solid support comprising sequence (B).
[0406] In some embodiments, a plurality of different nucleic acids
bound to a solid surface is created in which each of the nucleic
acids has a specific sequence (A') at its 3' end and also a
specific sequence (B) at its 5' end, and where the different
nucleic acids have different intervening sequences, wherein the
intervening sequences are identical to or substantially identical
to the sequences in the target DNA. The set of bound nucleic acids
thus generated can be analyzed, for example, by sequencing in order
to provide information about the sequence of the target DNA.
[0407] The solid surface can be any of a variety of surfaces, some
described in more detail below. The solid surface can be, for
example a planar surface, for example, a planar array. In some
embodiments the solid surface comprises a plurality of beads. In
some embodiments the beads are magnetic.
[0408] The step of binding the polynucleotides to the solid surface
through the sequence (B), step (h), can be carried out such that
only one nucleic acid is bound to an isolated area of a surface or
only one nucleic acid is bound to a single bead. This isolated
binding of nucleic acids can be used for clonal amplification of
the specific bound nucleic acid in that area or on that bead. Such
bound, isolated nucleic acids can also be stored and archived for
later analysis, for example by sequencing. The bound, isolated
nucleic acids can be amplified, stored, and analyzed multiple
times.
[0409] In some embodiments, the method further comprises treating
the solid surface with reagents to produce multiple copies of an
amplification product that are substantially complementary to the
third primer extension product. This step comprises carrying out an
amplification reaction wherein the bound nucleic acid acts as a
template for the amplification. Generally, the amplification is
carried out using the sequence (A') on the third primer extension
product for the hybridization of primer. In some embodiments the
amplification is an isothermal amplification reaction comprising a
composite RNA/DNA primer, RNase H, and a DNA polymerase with strand
displacement activity. In some embodiments, the amplification is
carried out using polymerase chain reaction (PCR). For example
where the third primer extension product comprises both as sequence
(B) at or near its 5' end and a sequence (A') at or near its 3'
end, a set of primers, one designed to hybridize to all or a
portion of the sequence (A') and the other designed to hybridize to
sequence (B'), the complement of sequence (B), can be used to carry
out a PCR reaction to exponentially produce double stranded
amplified product.
[0410] In some embodiments the amplification is carried out such
that the amplified product is not attached to the substrate, but is
freely dissolved in the solution. In other embodiments, the
amplification is carried out such that the amplified product
remains bound to the substrate, for example by performing solid
phase PCR such as bridge PCR. In yet other embodiments, an
amplified product is generated that may float freely in solution,
but which comprises a sequence, for example sequence (A) or
sequence (B'), that allows it to be captured to another solid
surface or other portion of the solid surface by hybridization to a
complementary sequence bound to such surface, e.g. sequence (A') or
sequence (B). In some embodiments, the amplified product is a
single-stranded product and, because it is generated at the solid
surface, the amplified product readily captured by complementary
sequences, e.g. sequence (B), bound to the surface.
[0411] In one aspect of the invention, a plurality of beads is
used, and the methods described above are carried out such that on
average, one or fewer first primer extension product molecules are
bound per bead. The beads are dispersed into an aqueous solution,
and a plurality of microreactors, e.g. droplets, are produced such
that on average one or fewer beads is contained within each of the
plurality of microreactors. The amplification of the second primer
extension products bound to the beads is then carried out such that
the clonal amplification of each of the plurality of second primer
extension products in the separate microreactors is achieved. This
clonal amplification in microreactors can be performed on a sample
of target DNA, such as genomic DNA, wherein the plurality of second
primer extension products comprise sequences that correspond to
most, to substantially all, or to all of the sequences in the
target DNA. In some embodiments, the amplified products are
captured by bead having attached thereto a plurality of
oligonucleotides comprising complementary sequences bound to such
surface (e.g. sequence (A') or sequence (B)), which are
complementary to sequence (A) or sequence (B') on the amplified
product.
[0412] In some embodiments, the plurality of beads, produced as
described above, with each bead comprising a single first primer
extension product can comprise a library. These libraries can be
stored, then later clonally amplified. In some embodiments, a
library of beads can comprise a plurality of beads wherein each
bead had multiple copies of a single amplification product
generated from a second primer extension product. These libraries
can be analyzed, for example by sequencing. The libraries can be
stored, and later analyzed. In some embodiments the libraries can
be stored, then analyzed multiple times.
[0413] A schematic exemplary of an embodiment of the invention
relating to method for generating a polynucleotide comprising a
sequence (B) for binding to a solid surface from a DNA target is
shown in FIG. 16. Step I represents denaturing a double-stranded
target DNA, for example by raising the temperature. Steps II
illustrate annealing to the target DNA and extending with a DNA
polymerase comprising strand displacement activity, a first primer
comprising a DNA segment and a 5' RNA segment, wherein a 3' portion
of the primer comprises a random sequence, and a 5' portion of the
of the primer comprises sequence (A), which is not complementary to
the target DNA; to form a plurality of first primer extension
products, each with sequence (A) at its 5' end. The enzyme that
carries out Step II is generally a DNA polymerase. In some cases a
mixture of DNA polymerases can be used. In some embodiments, a DNA
polymerase with strand displacement activity is used such that a
growing first primer extension product can displace a downstream
first primer extension product, producing a plurality of first
primer extension products, representing different regions of the
sequence of the target DNA are produced. Step III illustrates
annealing a second primer to the first primer extension product
that has been denatured from the target DNA. Step IV illustrates
extending a second primer comprising a 3' DNA region that comprises
a random sequence, wherein the primer is a tailed primer comprising
a nucleic acid sequence (B) that is 5' of the random sequence, to
form a plurality of double-stranded products each comprising a
first primer extension product and a second primer extension
product. Step V shows the removal of single stranded 3' nucleotides
from the 3' region of the first primer extension product that is
not hybridized to the second primer extension product. This step
may be done using exonucleases. Step VI shows extension of the
first primer extension product by DNA polymerase to generate a
sequence (B'), complementary to sequence (B) of the second primer
extension product. Step VII shows the denaturation of the first and
second primer extension product by methods described previously.
Step VIII shows the binding of sequence (B') of the first primer
extension product to a sequence (B) on a solid surface, whereby the
first primer extension product becomes hybridized to the solid
surface. The immobilized sequence (B) oligonucleotide on the solid
support is extended using DNA and RNA-dependent DNA polymerase,
resulting in another DNA/RNA heteroduplex, as illustrated in step
IX. Amplification of the first primer extension product takes place
using repeated cycles of single primer isothermal linear
amplification (SPIA), which comprises the following steps not shown
in the figure: RNaseH cleaves the RNA from the DNA/RNA
heteroduplex, a chimeric RNA/DNA primer binds to sequence (A'), and
DNA polymerase with strand displacement activity is used to extend
the chimeric primer to produce amplified product of the first
primer extension product. As shown in step X, since the
amplification products are generated in close proximity to access
sequence (B) on the solid support, the amplified products are
captured on the solid support through hybridization of the 3'
sequence (B') to sequence (B) for further manipulations as
described herein such as clonal amplification.
Alternative Method for Generating a Polynucleotide Having a Defined
3' and 5' Sequences from an RNA Target
[0414] One aspect of the invention is a method for generating a
polynucleotide having defined 3' and 5' sequences from a RNA
target. The method utilizes a composite RNA/DNA oligonucleotide to
generate an oligonucleotide extension product comprising sequences
(A) and (C), which will allow extension of the third primer on the
solid surface such that the third primer extension product
comprises a sequence (C') at its 3' end than can be used as a site
for isothermal amplification in a manner such that the sequence (A)
is present at or near the 5' end of the amplified product produced
in this amplification.
[0415] The method comprises the steps: (a) extending a first primer
comprising a DNA segment and a 5' RNA segment, wherein a 3' portion
of the primer is complementary to a target RNA and a 5' portion,
sequence (A), of the of the primer is not complementary to the
target RNA; to form a first primer extension product hybridized to
the target RNA, forming an RNA/DNA hybrid. This extension is
generally performed with an enzyme comprising RNA-dependent DNA
polymerase activity. In some embodiments, the 3' portion of the
primer that is complementary to the target RNA is a specific
sequence. For example, where a specific region of interest of a
target RNA that is known or suspected to be upstream of a specific
sequence on the target RNA, the sequence that is complementary to
the target RNA of the first primer can be designed to hybridize to
this specific sequence on the target RNA such that extension of the
primer results in producing a first primer extension product that
is complementary to such upstream region. The specific sequence may
be common to a family of target RNA. A combination of primers with
various specific sequences at the 3' end can also be useful. In
some embodiments, such as where the target RNA comprises mRNA, and
the mRNA comprises a plurality of sequences, each having a 3'
poly-A segment; the specific sequence that is complementary to the
target RNA can comprise a sequence that will hybridize to the
poly-A region of the mRNA, thus allowing the extension of the first
primer to produce a plurality of first primer extension products,
each of which is complementary to the region of an mRNA molecule
adjacent to the poly-A region. In some embodiments, the sequence
that is complementary to the target RNA comprises a random
sequence, such that the extension of the first primer results in a
plurality of first primer extension products complementary to the
sequences adjacent to the sequence where each random species
hybridizes. The use of a random sequence at the 3' end of the
primer can be useful for performing a global amplification of a RNA
target, generating a plurality of sequences which together can
represent, for example substantially the whole sequence of the
target RNA. In some embodiments, the relative amounts of the
various sequences can be used to quantitate the relative amount of
a given sequence in a sample, for example to determine the level of
expression in an mRNA sample. In some embodiments more than one
type of sequence that is complementary to the target RNA can be
used, for instance both a primer with a random sequence and a
primer, or combination of primers with a specific sequence
complementary to RNA can be used. In some embodiments, multiple
primers comprising different specific sequences can be used.
[0416] The method further comprises step: (b) removing the target
RNA from the RNA/DNA hybrid. In some embodiments, the cleaving of
the target RNA from the RNA/DNA hybrid involves selectively
cleaving or degrading the target RNA. In some cases the complex
comprising the first primer extension product and the nucleic acid
can be heated in reaction conditions comprising Mg, which leads to
cleaving the RNA including the RNA in the RNA/DNA hybrid. The RNA
can also be removed from the RNA/DNA hybrid by denaturation,
performed, for example by heating the sample (thermal methods), or
by adding a denaturing agent, or using a combination of heating the
sample and adding denaturing agents. The cleaving can be
accomplished with an enzyme that cleaves RNA from an RNA/DNA hybrid
such as RNase H, or a combination of RNase enzymes, or cleaving can
be accomplished chemically or by heating. In some embodiments, the
target RNA is completely cleaved. In other embodiments, the target
RNA is only partly cleaved or degraded. The amount of cleaving
required is that amount which will allow the extension of the
second primer.
[0417] The method further comprises step: (c) extending a second
primer, comprising a 3' segment complementary to a portion of the
first primer extension product and a 5' segment non-complementary
to the first primer extension product comprising sequence (B), to
produce a double-stranded product with a DNA/RNA heteroduplex at
one end; wherein the double-stranded product comprises a second
primer extension product hybridized to the first primer extension
product, and whereby a portion of the 3' end of the second primer
extension product comprises a sequence (A') that is complementary
to the sequence (A) of the of the first primer.
[0418] The extension of the second primer is carried out with a DNA
polymerase as described herein. In some embodiments, a DNA
polymerase comprising both DNA and RNA dependent DNA polymerase
activities is used here. In other embodiments, both a RNA dependent
DNA polymerase and a DNA dependent DNA polymerase are used.
[0419] The second primer can comprise RNA, DNA, or can be a
composite primer comprising both RNA and DNA. In some embodiments,
the second primer can comprise a specific primer sequence that is
designed to hybridize to a specific sequence in the first primer
extension product. In some embodiments the second primer comprises
a random primer sequence that randomly binds to the first primer
extension product. Extension of the second primer comprising a
random sequence produces a plurality of second primer extension
products. The use of a random sequence at the 3' end of the primer
is useful, for example, in performing global amplification of a
target RNA, whereby a plurality of second primer extension products
are produced which is representative of the sequence of the target
RNA. In some embodiments, for example where the first primer is
designed to hybridize to a specific sequence on a target RNA, or a
sequence common to a family of RNA targets, random priming by the
second primer ensures amplification of the entire selected target
or family of selected targets. In this embodiment, the second
primer extension products comprise sequences which are the same or
substantially the same as the sequences in the target RNA (sense
copies).
[0420] The second primer extension product is extended such that
the 3' portion of the second primer extension product comprises a
sequence (A') which is complementary to sequence (A) of the first
primer. Since sequence (A) on the first primer extension product
comprises RNA, both DNA dependent DNA polymerase activity and RNA
dependent DNA polymerase activity are used in step (c). The primer
extension results in a product that is at least partially double
stranded since sequence (B) does not hybridize to the first primer
extension product. The product further comprises a DNA-RNA
heteroduplex region. The specific, or universal, sequence (A') can
be a site for primer hybridization and further analysis or
amplification of the nucleic acid bound to the bead.
[0421] In some embodiments, the sample comprising the target RNA is
in a sample that also comprises DNA. In such cases, it can be
advantageous to add a selective DNA dependent DNA polymerase
inhibitor such as actinomycin such that it is present during step
(a) to selectively inhibit the production of extension product
complementary to the DNA during step (a). The presence of a DNA
dependent DNA polymerase inhibitor such as actinomycin is
particularly advantageous when a first primer comprising a random
sequence is used, as the inhibitor allows for the selective
creation of first primer extension products to RNA without the need
of separating the RNA from the DNA. This is also advantageous when
the priming is carried out at specific target sequences since the
sequence may be the same on the DNA when the DNA and RNA in the
sample represent total nucleic acid from the same biological
entity, for example, human tissue, animal tissue, and the like. The
use of DNA dependent DNA polymerase inhibitors such as actinomycin
is described in copending application.
[0422] The method further comprises step: (d) cleaving the RNA in
the heteroduplex from the first primer extension product such that
a portion of the second primer extension product that is
complementary to sequence (A) is single-stranded. The cleaving of
RNA can be performed, for example by treatment with RNase H, which
will selectively cleave the RNA portion of the DNA/RNA partial
heteroduplex formed in step (c).
[0423] The method further comprises step: (e) annealing to the
second primer extension product an oligonucleotide comprising a
3'-DNA sequence (A) that is complementary to sequence (A') and a
5'-RNA segment comprising sequence (C) that is non-complementary to
the second primer extension product. The oligonucleotide comprises
at least one DNA and at least one RNA portion. In some embodiments
the 5' DNA segment is complementary to all of sequence (A'), in
other embodiments, the 5' DNA segment is complementary to portion
of sequence (A'). In some embodiments, 5' RNA segment comprising
sequence (C) is partly complementary to sequence (A').
[0424] The method further comprises step: (f) extending the
oligonucleotide at the 3' segment to form an oligonucleotide
extension product hybridized to the second primer extension
product. In some embodiments, the oligonucleotide is extended from
its 3' end to produce an oligonucleotide extension product
hybridized to the second primer extension product and displaces the
first primer extension product. The second primer comprises a
sequence (B), such that the oligonucleotide extension product will
comprise a sequence (B') at or near its 3' end that is
complementary to sequence (B). An optional step that may be added
using a DNA polymerase that has RNA dependent DNA polymerase
activity is extension of the second primer extension product to
create a heteroduplex such that the second primer comprises a DNA
sequence (C') that is complementary to sequence (C). This step
creates an RNA/DNA heteroduplex region.
[0425] The method further comprises step: (g) denaturing the
double-stranded DNA product. The first primer extension product can
be separated from the second primer extension product by
denaturation. Denaturation can be performed, for example by heating
the sample, or by adding a denaturing agent, or using a combination
of heating the sample and adding denaturing agents.
[0426] The method further comprises step: (h) attaching the
single-stranded first primer extension product to a solid support
by annealing sequence (B') to the solid support comprising a
sequence (B). The oligonucleotide bound to the solid surface
comprises sequence (B) such that binding of sequence (B') of the
oligonucleotide primer extension product results in attaching the
oligonucleotide primer extension product to the solid surface. The
method produces a nucleic acid that is hybridized to sequence (B)
on a solid surface that has a specific sequence (A) and (C) at its
5' end.
[0427] The method further comprises step: (i) extending sequence
(B) on the solid support to produce a third primer extension
product, comprising a 3' sequence (A') and (C'), whereby a DNA/RNA
heteroduplex at one end is generated.
[0428] The extension of the third primer is carried out with a DNA
polymerase as described herein. In some embodiments, a DNA
polymerase comprising both DNA and RNA dependent DNA polymerase
activities is used here. In other embodiments, both a RNA dependent
DNA polymerase and a DNA dependent DNA polymerase are used.
[0429] The primer extension results in a product that is double
stranded and comprises sequences (B'), (A), (C) on the
oligonucleotide primer extension product and sequences (B), (A')
and (C') on the third primer extension product.
[0430] In some embodiments, the sample comprising the target RNA is
in a sample that also comprises DNA. In such cases, it can be
advantageous to add a selective DNA dependent DNA polymerase
inhibitor such as actinomycin such that it is present during step
(a) to selectively inhibit the production of extension product
complementary to the DNA during step (a). The presence of a DNA
dependent DNA polymerase inhibitor such as actinomycin is
particularly advantageous when a first primer comprising a random
sequence is used, as the inhibitor allows for the selective
creation of first primer extension products to RNA without the need
of separating the RNA from the DNA. This is also advantageous when
the priming is carried out at specific target sequences since the
sequence may be the same on the DNA when the DNA and RNA in the
sample represent total nucleic acid from the same biological
entity, for example, human tissue, animal tissue, and the like. The
use of DNA dependent DNA polymerase inhibitors such as actinomycin
is described in co-pending application.
[0431] The method produces a nucleic acid that is bound to a solid
surface that has a specific sequence (A') and (C') at its 3' end
and a sequence (B) at or near its 5' end. The specific, or
universal, sequence (A') or (C') can be a site for primer
hybridization and further analysis or amplification of the nucleic
acid bound to the bead. The specific sequence (C') can be a site
for primer hybridization and further analysis or amplification of
the nucleic acid bound to the bead. One aspect of the invention
comprises amplification of the nucleic acid bound to the bead. In
some embodiments, the amplification is carried out using isothermal
amplification using a composite RNA/DNA primer, RNase H, and a
polymerase with strand displacement activity. For this embodiment,
the sequence (C') acts as the site to which the composite RNA/DNA
amplification primer hybridizes, allowing for amplification. When
the sequence (C') acts as a site to which a composite amplification
primer binds, the amplified product that is produced has the
sequence (A) and a portion of sequence (C) at its 5' end. The third
primer comprises the sequence (B) and the amplified product also
has the sequence (B'), complementary to (B) at or near its 3' end.
Thus the method produced amplified product with defined sequences
at or near both its 3' and 5' ends.
[0432] In some embodiments, for example where random sequences at
the 3' end of the first and/or second primer are used, a plurality
of different nucleic acids hybridized to a solid surface is created
in which each of the nucleic acids has a specific sequence (A) and
(C) at its 5' end and also a specific sequence (B') at its 3' end,
and where the different nucleic acids have different intervening
sequences, wherein the intervening sequences are identical to or
substantially identical to the sequences in the target RNA. The set
of bound nucleic acids thus generated can be analyzed, for example,
by sequencing in order to provide information about the sequence of
the target RNA.
[0433] The solid surface can be any of a variety of surfaces, some
described in more detail below. The solid surface can be, for
example a planar surface, for example, a planar array. In some
embodiments the solid surface comprises a plurality of beads. In
some embodiments the beads are magnetic.
[0434] The step of binding the polynucleotides to the solid surface
through sequence (B'), step (i), can be carried out such that only
one nucleic acid is bound to an isolated area of a surface or only
one nucleic acid is bound to a single bead. This isolated binding
of nucleic acids can be used for clonal amplification of the
specific bound nucleic acid in that area or on that bead. Such
bound, isolated nucleic acids can also be stored and archived for
later analysis, for example by sequencing. The bound, isolated
nucleic acids can be amplified, stored, and analyzed multiple
times.
[0435] In some embodiments, the method further comprises treating
the solid surface with reagents to produce multiple copies of an
amplification product that are substantially complementary to the
third primer extension product. This step comprises carrying out an
amplification reaction wherein the bound nucleic acid acts as a
template for the amplification.
[0436] Generally, the amplification is carried out using the
sequence (C') on the third primer extension product for the
hybridization of a primer such as a composite RNA/DNA amplification
primer hybridizes, allowing for amplification. In some embodiments
the amplification is an isothermal amplification reaction
comprising a composite RNA/DNA primer, RNase H, and a DNA
polymerase with strand displacement activity. In some embodiments,
the amplification is carried out using polymerase chain reaction,
(PCR). For example where the third primer extension product
comprises both a sequence (B) at or near its 5' end and a sequence
(C') at or near its 3' end, a set of primers, one designed to
hybridize to all or a portion of the sequence (C') and the other
designed to hybridize to sequence (B), can be used to carry out a
PCR reaction to exponentially produce double stranded amplified
product.
[0437] In some embodiments, the amplification is performed by a
method comprising the following steps: (j) cleaving the RNA from
the heteroduplex polynucleotide product hybridized to the amplified
product using RNase H to produce a single-stranded portion of the
third primer extension product corresponding to sequence (C'); (k)
annealing an amplification primer to the single-stranded portion of
the third primer extension product complementary to sequence (C'),
wherein the amplification primer has a DNA portion and a 5' RNA
portion; (l) extending the amplification primer with an enzyme
having strand displacement activity to produce an amplified product
hybridized to the third primer extension product on the solid
support; (m) repeating steps (j) to (l) to produce multiple copies
of the amplified product comprising sequences (A) and (B'); and (n)
capturing the amplified product on the solid support wherein the
solid support comprises sequence (B).
[0438] This amplification method, utilizing a sequence (B) and
(C'), allows for the production of an amplified product comprising
a sequence (B') at or near its 3' end that is substantially
complementary to sequence (B), and a sequence (A) near its 5' end
that is complementary to sequence (A'), thus producing an amplified
polynucleotide product with defined 3' and 5' ends.
[0439] In some embodiments the amplification is carried out such
that the amplified product is not attached to the substrate, but is
freely dissolved in the solution. In other embodiments, the
amplification is carried out such that the amplified product
remains bound to the substrate, for example by performing solid
phase PCR such as bridge PCR. In yet other embodiments, an
amplified product is generated that may float freely in solution,
but which comprises a sequence, for example sequence (A) or
sequence (B'), that allows it to be captured to another solid
surface or other portion of the solid surface by hybridization to a
complementary sequence bound to such surface, e.g. sequence (A') or
sequence (B). In some embodiments, the amplified product is a
single-stranded product and, because it is generated at the solid
surface, the amplified product readily captured by complementary
sequences, e.g. sequence (B), bound to the surface.
[0440] In one aspect of the invention, a plurality of beads is
used, and the methods described above are carried out such that on
average, one or fewer oligonucleotide primer extension product
molecules are bound per bead. The beads are dispersed into an
aqueous solution, and a plurality of microreactors, e.g. droplets,
are produced such that on average one or fewer beads is contained
within each of the plurality of microreactors. The amplification of
the third primer extension products bound to the beads is then
carried out such that the clonal amplification of each of the
plurality of second primer extension products in the separate
microreactors is achieved. This clonal amplification in
microreactors can be performed on a sample of target RNA, such as
whole transcriptome or total RNA, wherein the plurality of third
primer extension products comprise sequences that correspond to
most, to substantially all, or to all of the sequences in the
target RNA. In some embodiments, the amplified products are
captured by bead having attached thereto a plurality of
oligonucleotides comprising complementary sequences bound to such
surface (e.g. sequence (A') or sequence (B)), which are
complementary to sequence (A) or sequence (B') on the amplified
product.
[0441] In some embodiments, the plurality of beads, produced as
described above, with each bead comprising a single third primer
extension product can comprise a library. These libraries can be
stored, then later clonally amplified. In some embodiments, a
library of beads can comprise a plurality of beads wherein each
bead had multiple copies of a single amplification product
generated from a second primer extension product. These libraries
can be analyzed, for example by sequencing. The libraries can be
stored, and later analyzed. In some embodiments the libraries can
be stored, then analyzed multiple times.
[0442] In some embodiments, a bead or isolated area of the solid
surface comprises covalently attached thereto multiple
oligonucleotides comprising the sequence (B) at their 5' ends,
whereby upon the amplification of step (n) multiple copies of
amplified product comprising sequence (B') at their 3' end are
hybridized to the bead or isolated area. For example, where beads
are used, a plurality of beads in a plurality of microreactors
wherein, the plurality of beads has, on average one or fewer third
primer extension products bound to it and there are, on average,
one or fewer beads in each microreactor, a clonal amplification of
the plurality of third primer extension products can be carried
out, and the amplified products in each of the microreactors will
bind to the bead through the sequence (B') on the amplified product
to the sequence (B) on the beads. This approach produces a
plurality of beads, each with multiple copies of a different
sequence bound to it. Where these sequences are representative of
the target RNA, the plurality of beads can constitute a library
representative of such RNA.
[0443] Since the amplification products are generated in close
proximity to access sequence (B) on the solid support, the
amplified products are captured on the solid support through
hybridization of the 3' sequence (B') to sequence (B) for further
manipulations as described herein. The (B) sequences on the beads
can be extended along the amplified product by a DNA polymerase or
mixture of polymerases to produce a multiple polynucleotides
covalently attached to the bead or isolated area that are
substantially complementary to the amplified product and also
comprise sequence (A') near their 3' ends. This method provides for
the production of beads with polynucleotides complementary to
amplified product covalently attached to the beads. Covalently
attached polynucleotides such as those produce here are more robust
than nucleotides that are attached only by hybridization to the
beads. Thus, the covalently attached polynucleotides can be more
stable and can be used with analysis methods and sequencing methods
that have harsher conditions which would result in the displacement
of polynucleotides bound only by hybridization.
[0444] In some embodiments, the amplified product is removed from
the covalently bound polynucleotide to render the polynucleotide
single stranded. Such single stranded covalently bound
polynucleotides comprise a specific sequence at their 3' ends
comprising sequence (A') and a portion of sequence (C'). Here, the
portion of sequence (C') is the DNA portion of the chimeric
amplification primer (C) that does is generally not cleaved by
RNase H and therefore becomes incorporated into the amplified
product. This specific sequence at the 3' end of the covalently
bound polynucleotide can act as a hybridization site for a primer
complementary to sequence (A') that can act as a primer to carry
out sequencing by any of a variety of sequencing methods, for
example, those described herein.
[0445] The sequencing methods can comprise the use of cleavable
labeled terminators. The sequencing method can comprise
pyrophosphate detection. The sequencing method can comprise an
isothermal sequencing method, for example using chimeric primers,
RNase H, and a polymerase with strand displacement activity. The
sequencing method can also comprise cycle sequencing.
[0446] In some embodiments the methods of the invention provide for
performing bridge PCR comprising making amplified product as
described above with defined 3' and 5' ends, and further comprising
the steps of exposing the amplified product to a solid substrate
comprising oligonucleotide sequences attached thereto complementary
to the defined 3' and 5' sequences, for example, A and B'
sequences, on the amplified product in the presence of components
necessary for polymerase chain reaction, and thermal cycling the
system to perform bridge PCR amplification.
[0447] In some embodiments the methods of the invention provide for
making amplified product as described above with defined 3' and 5'
ends and further performing rolling circle amplification comprising
performing the steps of: (o) hybridizing the amplified product to a
nucleic acid sequence comprising regions complementary to A and B'
sequences in close proximity; (p) optionally extending the gap with
a DNA polymerase enzyme; (q) ligating to form a circular nucleic
acid comprising the amplified product, and performing rolling
circle amplification by extending a primer that is complementary to
a sequence in the circular nucleic acid.
[0448] In some embodiments, the rolling circle amplification uses
primers complementary to sequence (A), sequence (B'), or a sequence
that was between sequences (A) and (B') in the amplified product.
In some cases, such a primer can be an oligonucleotide attached to
a solid surface, thus resulting in amplified product bound to the
surface
[0449] In some embodiments the methods of the invention provide for
performing PCR comprising making amplified product as described
above with defined 3' and 5' ends, further comprising the steps of
amplifying the amplified product using primers complementary to
sequences (A) and (B), or using primers complementary to sequences
(A') and (B').
[0450] In some embodiments the methods of the invention provide for
performing strand displacement amplification (SDA) comprising
making amplified product as described above with defined 3' and 5'
ends, wherein the defined 3' and 5' ends, for example, sequences
(A) and (B'), in the amplified product are designed to be cleaved
by a restriction enzyme, and performing strand displacement
amplification on the amplified product.
[0451] A schematic exemplary of an embodiment of the invention
relating to generating a polynucleotide having a defined 3' and 5'
sequences is shown in FIG. 17. The figure shows a target RNA and a
chimeric RNA/DNA first primer. The primer is first annealed to the
target RNA. Step I illustrates extension of the first primer
comprising a DNA segment and a 5' RNA segment, wherein a 3' portion
of the primer is complementary to a target RNA and a 5' portion,
sequence (A), of the of the primer is not complementary to the
target RNA, to form a first primer extension product hybridized to
the target RNA, forming an RNA/DNA hybrid. The sequence
complementary to a target RNA can be a specific sequence, a
sequence that will hybridize to Poly-A, a sequence common to a
plurality of regions (consensus sequence), or a random sequence.
Step II represents separation of the target RNA from the RNA/DNA
hybrid. The separation can be accomplished thermally, chemically,
or enzymatically, e.g. with RNase H. The second primer comprising a
5' sequence (B) is then annealed to the first primer extension
product. Step III illustrate extending a second primer, comprising
a 5' sequence (B) and a 3' segment complementary to a portion of
the first primer extension product, to produce a double stranded
product with a DNA/RNA heteroduplex at one end; wherein the double
stranded product comprises a second primer extension product
hybridized to the first primer extension product, and whereby a
portion of the 3' end of the second primer extension product
comprises a sequence (A') that is complementary to the sequence (A)
of the of the first primer. In step IV, cleavage of the RNA from
the first primer extension product in the DNA-RNA heteroduplex
occurs such that a portion of the second primer extension product
that is complementary to sequence (A) is single stranded. As shown,
the cleavage is performed using RNase H. Chemical and thermal means
can alternatively be employed. Step V illustrates annealing to the
second primer extension product a chimeric oligonucleotide
comprising a 3'-DNA segment that is complementary to sequence (A')
and a 5' RNA segment comprising sequence (C). Step VI is an
optional step and may occur when there is DNA polymerase comprising
RNA-dependent DNA polymerase activity. In this step the second
primer extension product is extended along sequence C. Step VII
illustrates extension of the oligonucleotide at the 3' end,
generating an oligonucleotide extension product which is hybridized
to the second primer extension product and comprises a sequence
(B'), complementary to sequence (B) on the second primer extension
product. The first primer extension product is displaced during
3'-extension of the oligonucleotide. Step VIII illustrates the
denaturation of the chimeric oligonucleotide extension product from
the second primer extension product. Step IX illustrates binding of
the chimeric oligonucleotide extension product to a third primer
comprising sequence (B) on the solid surface. Step X illustrates
extension of the third primer to create a strand complementary to
the chimeric oligonucleotide extension product comprising sequence
(C') and (A').
[0452] In FIG. 18, Step I illustrates the steps of: cleaving the
RNA from the DNA-RNA heteroduplex created in step X above to
produce a single-stranded portion of the third primer extension
product corresponding to sequence (C'); annealing an amplification
primer to sequence (C'), wherein the amplification primer has a DNA
portion and a 5' RNA portion, to the single stranded portion of the
second primer extension product complementary to sequence (C');
extending the amplification primer with a DNA polymerase having
strand displacement activity to produce an amplified product. These
steps can be repeated to produce multiple copies of amplified
product wherein the 5' portion of the amplified product has a
sequence complementary to sequence (A'). In the embodiment
illustrated, the amplified product shown comprises a defined
sequences on both the 5' and 3' ends. Step II illustrates that the
amplification products are generated in close proximity to the
sequence (B) immobilized on the bead and will thus allow the
amplification product to be captured on the bead via hybridization
of its sequence (B'). DNA polymerase extends immobilized sequence
(B) along the hybridized amplification product to generate bound
nucleic acid comprising specific sequences (B) and (A'). Also shown
on the amplified product is a portion of sequence (C') that is the
DNA portion of the chimeric amplification primer (C). This portion
does not generally become cleaved by RNase H and therefore becomes
incorporated into the amplified product.
Alternative Method for Generating a Polynucleotide Having a Defined
3' and 5' Sequences from a DNA Target
[0453] The method utilizes a composite RNA/DNA oligonucleotide to
generate an oligonucleotide extension product comprising sequences
(A) and (C), which will allow extension of the third primer on the
solid surface such that the third primer extension product
comprises a sequence (C') at its 3' end than can be used as a site
for isothermal amplification in a manner such that the sequence (A)
is present at or near the 5' end of the amplified product produced
in this amplification, and where a second primer comprising
sequence (B) is used, amplified products with defined sequences at
both the 3' and 5' ends can be produced.
[0454] The method comprises the step: (a) denaturing a
double-stranded target DNA. Double stranded DNA can be denatured,
for example by heating, or by the addition of denaturing
agents.
[0455] The method further comprises step: (b) annealing to the
target DNA and extending with a DNA polymerase comprising strand
displacement activity, a first primer comprising a DNA segment and
a 5' RNA segment, wherein a 3' portion of the primer comprises a
random sequence, and a 5' portion of the of the primer comprises
sequence (A), which is not complementary to the target DNA; to form
a first primer extension product hybridized to the target DNA and
comprising sequence (A) at its 5' end. This extension is generally
performed with an enzyme comprising DNA-dependent DNA polymerase
activity. The sequence that is complementary to the target DNA
comprises a random sequence, such that the extension of the first
primer results in a plurality of first primer extension products
complementary to the sequences adjacent to the sequence where each
random species hybridizes. The use of a random sequence at the 3'
end of the primer can be useful for performing a global
amplification of a target DNA, generating a plurality of sequences
which together can represent, for example substantially the whole
sequence of the target DNA. In some embodiments, the relative
amounts of the various sequences can be used to quantitate the
relative amount of a given sequence in a sample, for example to
determine the number of gene copies in a DNA sample, or obtaining
sequence information. In some embodiments, the extension of one
first primer, will result in the release of a downstream first
primer extension product. This can occur throughout the target DNA
resulting in the release of multiple first primer extension
products from the target DNA. This process can occur simultaneously
on both of the strands of the double-stranded DNA target, thus
creating first primer extension products complementary to sequences
in both strands.
[0456] In some embodiments, the first primer extension step is
carried out with a DNA polymerase capable of extension at elevated
temperature that is not compatible with subsequent hybridization of
the random sequence to the displaced primer-extension product. For
example, Bst DNA polymerase can be used which is active at elevated
temperature. The reaction can be carried out stepwise, first with
an incubation at a lower temperature such as about 25.degree. C.,
followed by an incubation at higher temperature such as about
50.degree. C. In some embodiments, the first incubation is carried
out below about 30.degree. C., and the second incubation is carried
out above about 40.degree. C. In some embodiments, a DNA polymerase
which is active at temperatures above about 45.degree. C. is used
to extend the first primer. Mixtures of DNA polymerases can also be
useful.
[0457] The method further comprises step: (c) separating the first
primer extension product from the target DNA. In some embodiments,
the separation can be affected by denaturing the complex comprising
the first primer extension product and the nucleic acid.
Denaturation can be performed, for example by heating the sample,
or by adding a denaturing agent, or using a combination of heating
the sample and adding denaturing agents. The amount of cleaving
required is that amount which will allow the extension of the
second primer.
[0458] The method further comprises step: (d) annealing to the
first primer extension product and extending a second primer
comprising a 3' complementary DNA region that comprises a random
sequence, wherein the second primer is a tailed primer comprising a
5' sequence (B), to form a double-stranded product comprising a
first primer extension product and a second primer extension
product, whereby a double-stranded product with a DNA/RNA
heteroduplex at one end is generated. In some embodiments, a DNA
polymerase comprising both DNA and RNA dependent DNA polymerase
activities is used here. In other embodiments, both a RNA dependent
DNA polymerase and a DNA dependent DNA polymerase are used.
[0459] This step may be carried out with or without prior
denaturation. If carried out without denaturation, generally, only
the single stranded displaced first primer extension product will
hybridize to the second primer. Generally the second primer does
not comprise RNA. The extension of the second primer is carried out
with a DNA polymerase as described herein. The second primer
comprises a random primer sequence that randomly binds to the first
primer extension product. Extension of the second primer comprising
a random sequence produces a plurality of second primer extension
products. The use of a random sequence at the 3' end of the primer
is useful, for example, in performing global amplification of a
target DNA, whereby a plurality of second primer extension products
are produced which is representative of the sequence of the target
DNA. In some embodiments, for example where the first primer is
designed to hybridize to a specific sequence on a target DNA, or a
sequence common to a family of DNA targets, random priming by the
second primer ensures amplification of the entire selected target
or family of selected targets. In this embodiment, the second
primer extension products comprise sequences which are the same or
substantially the same as the sequences in the target DNA.
[0460] The second primer extension product is extended such that
the 3' portion of the second primer extension product comprises a
sequence (A') which is complementary to sequence (A) of the first
composite primer. Since sequence (A) on the first primer extension
product comprises RNA, both DNA dependent DNA polymerase activity
and RNA dependent DNA polymerase activity are used in step (d). The
primer extension results in a product that is at least partially
double stranded since sequence (B) does not hybridize to the first
primer extension product. The method produces a nucleic acid that
comprises a sequence (B), allowing it to be bound to a solid
surface by hybridization to its complement, which is immobilized on
the solid surface and that has a specific sequence (A') at its 3'
end. The specific, or universal, sequence (A') can be a site for
primer hybridization and further analysis or amplification of the
nucleic acid bound to the bead.
[0461] The method further comprises step: (e) cleaving the RNA in
the heteroduplex from the first primer extension product such that
a portion of the second primer extension product that is
complementary to sequence (A) is single stranded. The cleaving of
RNA can be performed, for example by treatment with RNase H, which
will selectively cleave the RNA portion of the DNA/RNA partial
heteroduplex formed in step (d).
[0462] The method further comprises step: (f) annealing to the
second primer extension product an oligonucleotide comprising a
3'-DNA segment that is complementary to sequence (A') and a 5' RNA
segment comprising sequence (C).
[0463] The oligonucleotide comprises at least one DNA and at least
one RNA portion. In some embodiments the 5' DNA segment is
complementary to all of sequence (A'), in other embodiments, the 5'
DNA segment is complementary to portion of sequence (A'). In some
embodiments, 5' RNA segment comprising sequence (C) is partly
complementary to sequence (A').
[0464] The method further comprises step: (g) extending the
oligonucleotide along the second primer extension product to form
an oligonucleotide extension product comprising a sequence (B'),
complementary to sequence (B) on the second primer extension
product. In some embodiments, the oligonucleotide is extended from
its 3' end to produce an oligonucleotide extension product
hybridized to the second primer extension product and displaces the
first primer extension product. The second primer comprises a
sequence (B), such that the oligonucleotide extension product will
comprise a sequence (B') at or near its 3' end that is
complementary to sequence (B). An optional step that may be added
using a DNA polymerase that has RNA dependent DNA polymerase
activity is extension of the second primer extension product to
create a heteroduplex such that the second primer comprises a DNA
sequence (C') that is complementary to sequence (C). This step
creates an RNA/DNA heteroduplex region.
[0465] The method further comprises step: (h) denaturing the
double-stranded DNA product. The oligonucleotide primer extension
product can be separated from the second primer extension product
by denaturation. Denaturation can be performed, for example by
heating the sample, or by adding a denaturing agent, or using a
combination of heating the sample and adding denaturing agents.
[0466] The method further comprises step: (i) attaching the
single-stranded oligonucleotide extension product to solid support
by annealing sequence (B') to the bead or isolated area comprising
a sequence (B). The third primer comprises an oligonucleotide
sequence (B) that is complementary to the sequence (B') of the
oligonucleotide primer extension product and results in attaching
the single stranded oligonucleotide primer extension product to the
solid surface. The method produces a nucleic acid that is
hybridized to a solid surface that has a specific sequence (A) at
its 5' end.
[0467] Step (i) of binding the polynucleotides to the solid surface
sequence (B) can be carried out such that only one nucleic acid is
bound to an isolated area of a surface or only one nucleic acid is
bound to a single bead. This isolated binding of nucleic acids can
be used for clonal amplification of the specific bound nucleic acid
in that area or on that bead. Such bound, isolated nucleic acids
can also be stored and archived for later analysis, for example by
sequencing. The bound, isolated nucleic acids can be amplified,
stored, and analyzed multiple times.
[0468] The method further comprises the step of: (j) extending
sequence (B) on the solid support to produce a third primer
extension product, hybridized to the oligonucleotide extension
product, comprising a 3' sequence (A') and (C'), whereby a DNA/RNA
heteroduplex at one end is generated. The extension of the third
primer is carried out with a DNA polymerase as described herein. In
some embodiments, a DNA polymerase comprising both DNA and RNA
dependent DNA polymerase activities is used here. In other
embodiments, both a RNA dependent DNA polymerase and a DNA
dependent DNA polymerase are used. The primer extension results in
a product that is double stranded and comprises sequences (B'),
(A), (C) on the first primer extension product and sequences (B),
(A') and (C') on the third primer extension product.
[0469] The method produces a nucleic acid that is bound to a solid
surface that has a specific sequence (A') and (C') at its 3' end
and a sequence (B) at or near its 5' end. The specific, or
universal, sequence (A') or (C') can be a site for primer
hybridization and further analysis or amplification of the nucleic
acid bound to the bead. The specific, or universal, sequence (C')
can be a site for primer hybridization and further analysis or
amplification of the nucleic acid bound to the bead. One aspect of
the invention comprises amplification of the nucleic acid bound to
the bead. In some embodiments, the amplification is carried out
using isothermal amplification using a composite RNA/DNA primer,
RNase H, and a polymerase with strand displacement activity. For
this embodiment, the sequence (C') acts as the site to which the
composite RNA/DNA amplification primer hybridizes, allowing for
amplification. In some embodiments, for example where random
sequences at the 3' end of the first and/or second primer are used,
a plurality of different nucleic acids bound to a solid surface is
created in which each of the nucleic acids has a specific sequence
(C') at its 3' end and also a specific sequence (B) at its 5' end,
and where the different nucleic acids have different intervening
sequences, wherein the intervening sequences are identical to or
substantially identical to the sequences in the target nucleic
acid. The set of bound nucleic acids thus generated can be
analyzed, for example, by sequencing in order to provide
information about the sequence of the target nucleic acid.
[0470] In some embodiments, for example where random sequences at
the 3' end of the first and/or second primer are used, a plurality
of different nucleic acids hybridized to a solid surface is created
in which each of the nucleic acids has a specific sequence (A) and
(C) at its 5' end and also a specific sequence (B') at its 3' end,
and where the different nucleic acids have different intervening
sequences, wherein the intervening sequences are identical to or
substantially identical to the sequences in the target DNA. The set
of bound nucleic acids thus generated can be analyzed, for example,
by sequencing in order to provide information about the sequence of
the target DNA.
[0471] The solid surface can be any of a variety of surfaces, some
described in more detail below. The solid surface can be, for
example a planar surface, for example, a planar array. In some
embodiments the solid surface comprises a plurality of beads. In
some embodiments the beads are magnetic.
[0472] The step of binding the polynucleotides to the solid surface
through sequence (B'), step (i), can be carried out such that only
one nucleic acid is bound to an isolated area of a surface or only
one nucleic acid is bound to a single bead. This isolated binding
of nucleic acids can be used for clonal amplification of the
specific bound nucleic acid in that area or on that bead. Such
bound, isolated nucleic acids can also be stored and archived for
later analysis, for example by sequencing. The bound, isolated
nucleic acids can be amplified, stored, and analyzed multiple
times.
[0473] In some embodiments, the method further comprises treating
the solid surface with reagents to produce multiple copies of an
amplification product that are substantially complementary to the
third primer extension product. This step comprises carrying out an
amplification reaction wherein the bound nucleic acid acts as a
template for the amplification.
[0474] Generally, the amplification is carried out using the
sequence (C') on the third primer extension product for the
hybridization of a primer such as a composite RNA/DNA amplification
primer hybridizes, allowing for amplification. In some embodiments
the amplification is an isothermal amplification reaction
comprising a composite RNA/DNA primer, RNase H, and a DNA
polymerase with strand displacement activity. In some embodiments,
the amplification is carried out using polymerase chain reaction,
(PCR). For example where the second primer extension product
comprises both as sequence (B) at or near its 5' end and a sequence
(C') at or near its 3' end, a set of primers, one designed to
hybridize to all or a portion of the sequence (C') and the other
designed to hybridize to sequence (B), can be used to carry out a
PCR reaction to exponentially produce double stranded amplified
product.
[0475] In some embodiments, the amplification is performed by a
method comprising the following steps: (k) cleaving the RNA from
the heteroduplex polynucleotide product hybridized to the amplified
product using RNase H to produce a single-stranded portion of the
second primer extension product corresponding to sequence (C'); (l)
annealing an amplification primer to the single-stranded portion of
the amplified product complementary to sequence (C'), wherein the
amplification primer has a DNA portion and a 5' RNA portion; (m)
extending the amplification primer with an enzyme having strand
displacement activity to produce an amplified product hybridized to
the amplified product on the bead or isolated area; and (n)
repeating steps (k) to (m) to produce multiple copies of the second
polynucleotide product comprising sequences (A) and (B'). This
amplification method, utilizing a sequence (B) and (C'), allows for
the production of an amplified product comprises a sequence (B') at
or near its 3' end that is substantially complementary to sequence
(B), and a sequence (A) near its 5' end that is complementary to
sequence (A'), thus producing an amplified polynucleotide product
with defined 3' and 5' ends.
[0476] In some embodiments the amplification is carried out such
that the amplified product is not attached to the substrate, but is
freely dissolved in the solution. In other embodiments, the
amplification is carried out such that the amplified product
remains bound to the substrate, for example by performing solid
phase PCR such as bridge PCR. In yet other embodiments, an
amplified product is generated that may float freely in solution,
but which comprises a sequence, for example sequence (A) or
sequence (B'), that allows it to be captured to another solid
surface or other portion of the solid surface by hybridization to a
complementary sequence bound to such surface, e.g. sequence (A') or
sequence (B). In some embodiments, the amplified product is a
single-stranded product and, because it is generated at the solid
surface, the amplified product readily captured by complementary
sequences, e.g. sequence (B), bound to the surface.
[0477] In one aspect of the invention, a plurality of beads is
used, and the methods described above are carried out such that on
average, one or fewer oligonucleotide primer extension product
molecules are bound per bead. The beads are dispersed into an
aqueous solution, and a plurality of microreactors, e.g. droplets,
are produced such that on average one or fewer beads is contained
within each of the plurality of microreactors. The amplification of
the third primer extension products bound to the beads is then
carried out such that the clonal amplification of each of the
plurality of second primer extension products in the separate
microreactors is achieved. This clonal amplification in
microreactors can be performed on a sample of target DNA, such as
whole transcriptome or total DNA, wherein the plurality of third
primer extension products comprise sequences that correspond to
most, to substantially all, or to all of the sequences in the
target RNA. In some embodiments, the amplified products are
captured by bead having attached thereto a plurality of
oligonucleotides comprising complementary sequences bound to such
surface (e.g. sequence (A') or sequence (B)), which are
complementary to sequence (A) or sequence (B') on the amplified
product.
[0478] In some embodiments, the plurality of beads, produced as
described above, with each bead comprising a single oligonucleotide
primer extension product can comprise a library. These libraries
can be stored, then later clonally amplified. In some embodiments,
a library of beads can comprise a plurality of beads wherein each
bead had multiple copies of a single amplification product
generated from a third primer extension product. These libraries
can be analyzed, for example by sequencing. The libraries can be
stored, and later analyzed. In some embodiments the libraries can
be stored, then analyzed multiple times.
[0479] In some embodiments, a bead or isolated area of the solid
surface comprises covalently attached thereto multiple
oligonucleotides comprising the sequence (B) at their 5' ends,
whereby upon the amplification of step (m) multiple copies of
amplified product comprising sequence (B') at their 3' end are
hybridized to the bead or isolated area. For example, where beads
are used, a plurality of beads in a plurality of microreactors
wherein, the plurality of beads has, on average one or fewer
oligonucleotide primer extension products bound to it and there
are, on average, one or fewer beads in each microreactor, a clonal
amplification of the plurality of third primer extension products
can be carried out, and the amplified products in each of the
microreactors will bind to the bead through the sequence (B') on
the amplified product to the sequence (B) on the beads. This
approach produces a plurality of beads, each with multiple copies
of a different sequence bound to it. Where these sequences are
representative of the target DNA, the plurality of beads can
constitute a library representative of such DNA.
[0480] After the amplified products are bound to the beads by
hybridization, the (B) sequences on the beads can be extended along
the amplified product by a DNA polymerase or mixture of polymerases
to produce a multiple polynucleotides covalently attached to the
bead or isolated area that are substantially complementary to the
amplified product and also comprise sequence (A') near their 3'
ends. This method provides for the production of beads with
polynucleotides complementary to amplified product covalently
attached to the beads. Covalently attached polynucleotides such as
those produce here are more robust than nucleotides that are
attached only by hybridization to the beads. Thus, the covalently
attached polynucleotides can be more stable and can be used with
analysis methods and sequencing methods that have harsher
conditions which would result in the displacement of
polynucleotides bound only by hybridization.
[0481] In some embodiments, the amplified product is removed from
the covalently bound polynucleotide to render the polynucleotide
single stranded. Such single stranded covalently bound
polynucleotides comprise a specific sequence at their 3' ends
comprising sequence (A') and a portion of sequence (C'). Here, the
portion of sequence (C') is the DNA portion of the chimeric
amplification primer (C) that does is generally not cleaved by
RNase H and therefore becomes incorporated into the amplified
product. This specific sequence at the 3' end of the covalently
bound polynucleotide can act as a hybridization site for a primer
complementary to sequence (A') that can act as a primer to carry
out sequencing by any of a variety of sequencing methods, for
example, those described herein.
[0482] The sequencing methods can comprise the use of cleavable
labeled terminators. The sequencing method can comprise
pyrophosphate detection. The sequencing method can comprise an
isothermal sequencing method, for example using chimeric primers,
RNase H, and a polymerase with strand displacement activity. The
sequencing method can also comprise cycle sequencing.
[0483] In some embodiments the methods of the invention provide for
performing bridge PCR comprising making amplified product as
described above with defined 3' and 5' ends, and further comprising
the steps of exposing the amplified product to a solid substrate
comprising oligonucleotide sequences attached thereto complementary
to the defined 3' and 5' sequences, for example, A and B'
sequences, on the amplified product in the presence of components
necessary for polymerase chain reaction, and thermal cycling the
system to perform bridge PCR amplification.
[0484] In some embodiments the methods of the invention provide for
making amplified product as described above with defined 3' and 5'
ends and further performing rolling circle amplification comprising
performing the steps of: (o) hybridizing the amplified products to
a target nucleic acid comprising regions complementary to A and B'
sequences in close proximity; (p) optionally extending the gap with
a polymerase enzyme; and (q) ligating to form a circular nucleic
acid comprising the amplified product, and performing rolling
circle amplification by extending a primer that is complementary to
a sequence in the circular nucleic acid.
[0485] In some embodiments, the rolling circle amplification uses
primers complementary to sequence (A), sequence (B'), or a sequence
that was between sequences (A) and (B') in the amplified product.
In some cases, such a primer can be an oligonucleotide attached to
a solid surface, thus resulting in amplified product bound to the
surface
[0486] In some embodiments the methods of the invention provide for
performing PCR comprising making amplified product as described
above with defined 3' and 5' ends, further comprising the steps of
amplifying the amplified product using primers complementary to
sequences (A) and (B), or using primers complementary to sequences
(A') and (B').
[0487] In some embodiments the methods of the invention provide for
performing strand displacement amplification (SDA) comprising
making amplified product as described above with defined 3' and 5'
ends, wherein the defined 3' and 5' ends, for example, sequences
(A) and (B'), in the amplified product are designed to be cleaved
by a restriction enzyme, and performing strand displacement
amplification on the amplified product.
[0488] A schematic exemplary of an embodiment of the invention
relating to generating a polynucleotide having a defined 3' and 5'
sequences is shown in FIG. 19. The figure shows a double stranded
target DNA that is denatured in step I. In step II, a chimeric
RNA/DNA first primer is first annealed to the target DNA and is
extended to form a first primer extension product hybridized to the
target DNA, forming a RNA/DNA hybrid. The first primer comprises a
DNA segment and a 5' RNA segment, wherein a 3' portion of the
primer is complementary to a target RNA and a 5' portion, sequence
(A), of the of the primer is not complementary to the target DNA.
The sequence complementary to a target DNA can be a specific
sequence, a sequence that will hybridize to Poly-A, a sequence
common to a plurality of regions (consensus sequence), or a random
sequence. Step III represents separation of the target DNA from the
RNA/DNA hybrid. The separation can be accomplished thermally,
chemically, or enzymatically, e.g. with RNase H. A second primer
comprising a 5' sequence (B) is then annealed to the first primer
extension product. Step IV illustrate extension of the second
primer, comprising a 5' sequence (B) and a 3' segment complementary
to a portion of the first primer extension product, to produce a
double stranded product with a DNA/RNA heteroduplex at one end;
wherein the double stranded product comprises a second primer
extension product hybridized to the first primer extension product,
and whereby a portion of the 3' end of the second primer extension
product comprises a sequence (A') that is complementary to the
sequence (A) of the of the first primer. In step V, cleavage of the
RNA from the first primer extension product in the DNA-RNA
heteroduplex occurs such that a portion of the second primer
extension product that is complementary to sequence (A) is single
stranded. As shown, the cleavage is performed using RNase H.
Chemical and thermal means can alternatively be employed. Step VI
illustrates annealing to the second primer extension product a
chimeric oligonucleotide comprising a 3'-DNA segment that is
complementary to sequence (A') and a 5' RNA segment comprising
sequence (C). Step VII is an optional step and may occur when there
is DNA polymerase comprising RNA-dependent DNA polymerase activity.
In this step the second primer extension product is extended along
sequence C. Step VIII illustrates extension of the oligonucleotide
at the 3' end, generating an oligonucleotide extension product
which is hybridized to the second primer extension product and
comprises a sequence (B'), complementary to sequence (B) on the
second primer extension product. The first primer extension product
is displaced during 3'-extension of the oligonucleotide. Step IX
illustrates the denaturation of the chimeric oligonucleotide
extension product from the second primer extension product. Step X
illustrates binding of the chimeric oligonucleotide extension
product to a third primer comprising sequence (B) on the solid
surface. Step XI illustrates extension of the third primer to
create a strand complementary to the chimeric oligonucleotide
extension product comprising sequence (C') and (A'). Amplification
of the oligonucleotide extension product comprising specific
sequences (B) and (A') at the 3' and 5' ends, respectively, take
place by carrying out the steps illustrated in FIG. 18.
[0489] In one aspect of the present invention, methods are provided
for amplifying a target nucleic acid or its complement on a solid
support to form a plurality of amplified products comprising
clonally amplifying said target sequence or its complement by
linear amplification. In some embodiments, the method comprises
amplification using a single primer. In some embodiments, the
method comprises amplification from a double-stranded nucleic acid
having a single-stranded 3' overhang at one end. In some
embodiments, the method comprises amplification using a DNA-RNA
chimeric primer. In some embodiments, the method comprises a
combination of linear amplification using a single primer from a
double-stranded nucleic acid having a single-stranded 3' overhang
at one end using a DNA-RNA chimeric primer. In some embodiments,
the target nucleic acid sequence is coupled to a solid support. In
some cases, the amplification is isothermal. In some cases, the
solid support is a bead. In some cases, the amplification results
in at least 10,000; 100,000; one million or more copies of the
target sequence, its complement, or a portion thereof. In some
cases, the solid surface comprises a plurality of primers of
substantially identical sequence. In some cases, the solid surface
consists of a plurality of primers of substantially identical
sequence. In some cases, the target nucleic acid sequence is a
linear template. In some cases, the target nucleic acid sequence is
greater than about 150, 200, 250, 300, 400, 500, 1 kb, 2 kb, 5 kb,
10 kb or more in length. In some cases, the target nucleic acid is
less than about 1 megabase, 100 kb, 50 kb, 10 kb, 5 kb, or less in
length.
[0490] In one aspect of the present invention, methods are provided
for clonally amplifying a target nucleic acid sequence or its
complement by delivering the target nucleic acid sequence into an
emulsion and performing linear amplification of the target nucleic
acid sequence inside the emulsion. In some cases, the method
further comprises the step of forming the emulsion first. In some
cases, the emulsion is formed around the target nucleic acid and/or
the target nucleic acid in a reaction mixture comprising
amplification reagents including but not limited to buffers; one or
more enzymes such as a DNA polymerase with substantial strand
displacement activity, exonuclease, and RNase H; salts; primers
including chimeric primers, amplification primers, and all-DNA
primers, oligonucleotides including chimeric oligonucleotides; and
dNTPs. In some cases, the method may further comprise amplifying
the target nucleic acid in the presence of a solid surface such as
a bead, a substantially planar array, an isolated surface, or a
well in a plate. In some cases the amplification results in a
plurality of non-multimerized individual amplification products.
These are physically separated or chemically separated amplified
products. In some cases, the amplification results in a lower error
rate than PCR such as for example fewer errors than 2 in every
100,000 nucleotides incorporated into amplified product.
General Techniques
[0491] The practice of the invention will employ, unless otherwise
indicated, conventional techniques of molecular biology (including
recombinant techniques), microbiology, cell biology, biochemistry,
and immunology, which are within the skill of the art. Such
techniques are explained fully in the literature, such as,
"Molecular Cloning: A Laboratory Manual", second edition (Sambrook
et al., 1989); "Oligonucleotide Synthesis" (M. J. Gait, ed., 1984);
"Animal Cell Culture" (R. I. Freshney, ed., 1987); "Methods in
Enzymology" (Academic Press, Inc.); "Current Protocols in Molecular
Biology" (F. M. Ausubel et al., eds., 1987, and periodic updates);
"PCR: The Polymerase Chain Reaction", (Mullis et al., eds.,
1994).
[0492] The terms "Polynucleotide," or "nucleic acid," as used
interchangeably herein, refer to polymers of nucleotides of any
length, and include DNA and RNA. The nucleotides can be
deoxyribonucleotides, ribonucleotides, modified nucleotides or
bases, and/or their analogs, or any substrate that can be
incorporated into a polymer by DNA or RNA polymerase. A
polynucleotide may comprise modified nucleotides, such as
methylated nucleotides and their analogs. If present, modification
to the nucleotide structure may be imparted before or after
assembly of the polymer. The sequence of nucleotides may be
interrupted by non-nucleotide components. A polynucleotide may be
further modified after polymerization, such as by conjugation with
a labeling component. Other types of modifications include, for
example, "caps", substitution of one or more of the naturally
occurring nucleotides with an analog, internucleotide modifications
such as, for example, those with uncharged linkages (e.g., methyl
phosphonates, phosphotriesters, phosphoamidates, cabamates, etc.)
and with charged linkages (e.g., phosphorothioates,
phosphorodithioates, etc.), those containing pendant moieties, such
as, for example, proteins (e.g., nucleases, toxins, antibodies,
signal peptides, ply-L-lysine, etc.), those with intercalators
(e.g., acridine, psoralen, etc.), those containing chelators (e.g.,
metals, radioactive metals, boron, oxidative metals, etc.), those
containing alkylators, those with modified linkages (e.g., alpha
anomeric nucleic acids, etc.), as well as unmodified forms of the
polynucleotide(s). Further, any of the hydroxyl groups ordinarily
present in the sugars may be replaced, for example, by phosphonate
groups, phosphate groups, protected by standard protecting groups,
or activated to prepare additional linkages to additional
nucleotides, or may be conjugated to solid supports. The 5' and 3'
terminal OH can be phosphorylated or substituted with amines or
organic capping groups moieties of from 1 to 20 carbon atoms. Other
hydroxyls may also be derivatized to standard protecting groups.
Polynucleotides can also contain analogous forms of ribose or
deoxyribose sugars that are generally known in the art, including,
for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or
2'-azido-ribose, carbocyclic sugar analogs, alpha.-anomeric sugars,
epimeric sugars such as arabinose, xyloses or lyxoses, pyranose
sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic
nucleoside analogs such as methyl riboside. One or more
phosphodiester linkages may be replaced by alternative linking
groups. These alternative linking groups include, but are not
limited to, embodiments wherein phosphate is replaced by
P(O)S("thioate"), P(S)S ("dithioate"), "(O)NR.sub.2 ("amidate"),
P(O)R, P(O)OR', CO or CH.sub.2 ("formacetal"), in which each R or
R' is independently H or substituted or unsubstituted alkyl (1-20
C) optionally containing an ether (--O--) linkage, aryl, alkenyl,
cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a
polynucleotide need be identical. The preceding description applies
to all polynucleotides referred to herein, including RNA and
DNA.
[0493] "Oligonucleotide," as used herein, generally refers to
short, generally single stranded, generally synthetic
polynucleotides that are generally, but not necessarily, less than
about 200 nucleotides in length. The oligonucleotide(s) are
generally comprised of a sequence of at least 5 nucleotides,
generally from about 10 to about 100 nucleotides, about 20 to about
50 nucleotides, and often about 10 to about 30 nucleotides in
length. The oligonucleotides of the invention can be DNA, RNA,
DNA-RNA, or other polynucleotide. The terms oligo or sequence may
be used interchangeable herein.
[0494] Various techniques can be employed for preparing an
oligonucleotide utilized in the present invention. Such
oligonucleotide can be obtained by biological synthesis or by
chemical synthesis. For short sequences (up to about 100
nucleotides) chemical synthesis will frequently be more economical
as compared to the biological synthesis. In addition to economy,
chemical synthesis provides a convenient way of incorporating low
molecular weight compounds and/or modified bases during the
synthesis step. Furthermore, chemical synthesis is very flexible in
the choice of length and region of the target polynucleotide
binding sequence. The oligonucleotide can be synthesized by
standard methods such as those used in commercial automated nucleic
acid synthesizers. Chemical synthesis of DNA on a suitably modified
glass or resin can result in DNA covalently attached to the
surface. This may offer advantages in washing and sample handling.
For longer sequences standard replication methods employed in
molecular biology can be used such as the use of M13 for single
stranded DNA as described by J. Messing (1983) Methods Enzymol,
101, 20-78.
[0495] In the present invention, nucleoside triphosphates are
incorporated by a polymerase enzyme in the extension of the primer
to produce an extension product. Nucleoside triphosphates are
generally nucleosides having a 5'-triphosphate substituent. The
nucleosides are pentose sugar derivatives of nitrogenous bases of
either purine or pyrimidine derivation, covalently bonded to the
1'-carbon of the pentose sugar, which is usually a deoxyribose or a
ribose. The purine bases include adenine (A), guanine (G), inosine
(I), and derivatives and analogs thereof. The pyrimidine bases
include cytosine (C), thymine (T), uracil (U), and derivatives and
analogs thereof. Nucleoside triphosphates include
deoxyribonucleoside triphosphates such as the four common
triphosphates dATP, dCTP, dGTP and dTTP and ribonucleoside
triphosphates such as the four common triphosphates rATP, rCTP,
rGTP and rUTP. The term "nucleoside triphosphates" also includes
derivatives and analogs thereof, which are exemplified by those
derivatives that are recognized in a similar manner to the
underivatized nucleoside triphosphates. Examples of such
derivatives or analogs, by way of illustration and not limitation,
are those which are biotinylated, amine modified, alkylated, and
the like and also include phosphorothioate, phosphite, ring atom
modified derivatives, and the like.
[0496] As used herein, the term "nucleotide" generally refers to a
base-sugar-phosphate combination that is the monomeric unit of
nucleic acid polymers, i.e., DNA and RNA. In some aspects of the
invention modified nucleotides are used, for example, where a
nucleotide is connected to a ligand. A modified nucleotide is
generally the unit in a nucleic acid polymer that results from the
incorporation of a modified nucleoside triphosphate during an
amplification reaction and therefore becomes part of the nucleic
acid polymer.
[0497] As used herein, a nucleoside is generally a base-sugar
combination or a nucleotide lacking a phosphate moiety.
[0498] A "primer" is generally a nucleotide sequence (i.e. a
polynucleotide), generally with a free 3'-OH group, that hybridizes
with a template sequence (such as a target RNA, or a primer
extension product) and is capable of promoting polymerization of a
polynucleotide complementary to the template. A "primer" can be,
for example, an oligonucleotide. It can also be, for example, a
sequence of the template (such as a primer extension product or a
fragment of the template created following RNase cleavage of a
template-DNA complex) that is hybridized to a sequence in the
template itself (for example, as a hairpin loop), and that is
capable of promoting nucleotide polymerization. Thus, a primer can
be an exogenous (e.g., added) primer or an endogenous (e.g.,
template fragment) primer.
[0499] The primers of the invention are usually oligonucleotide
primers. A primer is generally an oligonucleotide that is employed
in an extension on a polynucleotide template. The oligonucleotide
primer is usually a synthetic nucleotide that is single stranded,
containing a sequence at its 3'-end that is capable of hybridizing
with a sequence of the target polynucleotide. Normally, the 3'
region of the primer that hybridizes with the target nucleic acid
has at least 80%, preferably 90%, more preferably 95%, most
preferably 100%, complementarity to a sequence or primer binding
site. The number of nucleotides in the hybridizable sequence of a
specific oligonucleotide primer should be such that stringency
conditions used to hybridize the oligonucleotide primer will
prevent excessive random non-specific hybridization. Usually, the
number of nucleotides in the hybridizing portion of the
oligonucleotide primer will be at least as great as the defined
sequence of the target polynucleotide, namely, at least ten
nucleotides, at least about 15 nucleotides and generally from about
10 to about 200, usually about 20 to about 50 nucleotides.
[0500] A "random primer," as used herein, is a primer that
comprises a sequence that is designed not necessarily based on a
particular or specific sequence in a sample, but rather is based on
a statistical expectation (or an empirical observation) that the
sequence of the random primer is hybridizable (under a given set of
conditions) to one or more sequences in the sample. The random
primers used herein are generally tailed random primers comprising
a 3' segment that acts as a random primer to the target
polynucleotide, and a 5' sequence that generally does not hybridize
to the target polynucleotide. The sequence of a random primer (or
its complement) may or may not be naturally-occurring, or may or
may not be present in a pool of sequences in a sample of interest.
The amplification of a plurality of polynucleotides, e.g. DNA or
RNA species in a single reaction mixture would generally, but not
necessarily, employ a multiplicity, preferably a large
multiplicity, of random primers. As is well understood in the art,
a "random primer" can also refer to a primer that is a member of a
population of primers (a plurality of random primers) which
collectively are designed to hybridize to a desired and/or a
significant number of target sequences. A random primer may
hybridize at a plurality of sites on a nucleic acid sequence. The
use of random primers provides a method for generating primer
extension products complementary to a target polynucleotide which
does not require prior knowledge of the exact sequence of the
target. In some embodiments one portion of a primer is random, and
another portion of the primer comprises a defined sequence. For
example, in some embodiments, a 3' portion of the primer will
comprise a random sequence, while the 5' portion of the primer
comprises a defined sequence. In some embodiments a 3' random
portion of the primer will comprise DNA, and a 5' portion defined
portion of the primer will comprise RNA, in other embodiments, both
the 3' and 5' portions will comprise DNA.
[0501] Composite primers are employed in certain embodiments of the
invention. Composite primers are primers that are composed of RNA
and DNA portions. In some aspects, the composite primer is a tailed
composite primer comprising, for example, a 3' DNA portion and a 5'
RNA portion. In the tailed composite primer, a 3' portion, all or a
portion of which comprises DNA is complementary to a
polynucleotide; and a 5' portion, all or a portion of which
comprises RNA, is not complementary to the polynucleotide and does
not hybridize to the polynucleotide under conditions in which the
3' portion of the tailed composite primer hybridizes to the
polynucleotide target. When the tailed composite primer is extended
with a DNA polymerase, a primer extension product with a 5' RNA
portion comprising a defined sequence can be created. This primer
extension product can then have a second primer anneal to it, which
can be extended with a DNA polymerase to create a double stranded
product with an RNA/DNA heteroduplex comprising a defined sequence
at one end. The RNA portion can be selectively cleaved from the
partial heteroduplex to create a double stranded DNA with a 3'
single stranded overhang which can be useful for a various aspects
of the present invention including allowing for isothermal
amplification using a composite amplification primer.
[0502] In other aspects, the composite primer is an amplification
composite primer. In the amplification composite primer, both the
RNA and the DNA portions are generally complementary and hybridize
to a sequence in the polynucleotide to be copied or amplified. In
some embodiments, a 3' portion of the amplification composite
primer is DNA and a 5' portion of the composite amplification
primer is RNA. The composite amplification primer is designed such
that the primer is extended from the 3' DNA portion to create a
primer extension product. The 5'RNA portion of this primer
extension product, in a DNA-RNA heteroduplex is susceptible to
cleavage by RNase H, thus freeing a portion of the polynucleotide
to the hybridization of an additional composite amplification
primer. The extension of the additional composite primer by a DNA
polymerase with strand displacement activity releases the primer
extension product from the original primer and creates another copy
of the sequence of the polynucleotide. Repeated rounds of primer
hybridization, primer extension with strand displacement DNA
synthesis, and RNA cleavage create multiple copies of the sequence
of the polynucleotide. Composite primers are described in more
detail below.
[0503] Polymerases are used in the methods of the invention, for
example to extend primers to produce extension products. A
polymerase, or nucleotide polymerase, is a catalyst, usually an
enzyme, for forming an extension of a polynucleotide along a DNA or
RNA template where the extension is complementary thereto. The
nucleotide polymerase is a template dependent polynucleotide
polymerase and utilizes nucleoside triphosphates as building blocks
for extending the 3'-end of a polynucleotide to provide a sequence
complementary with the polynucleotide template. Usually, the
catalysts are enzymes, such as DNA polymerases, for example,
prokaryotic DNA polymerase (I, II, or III), T4 DNA polymerase, T7
DNA polymerase, Klenow fragment, Bst DNA polymerase, reverse
transcriptase, Bca polymerase, Vent DNA polymerase, Pfu DNA
polymerase, Taq DNA polymerase, and the like, derived from any
source such as cells, bacteria, such as E. coli, plants, animals,
virus, thermophilic bacteria, and so forth. RNA polymerases include
T7 RNA polymerase, AMV polymerase, Q-beta-replicase, and so forth.
In some cases, Bst DNA Polymerase Large Fragment can be used. Bst
DNA polymerase Large Fragment is the portion of the Bacillus
stearothermophilus DNA Polymerase protein that contains the
5'.fwdarw.3' polymerase activity, but lacks the 5'.fwdarw.3'
exonuclease domain. Where the polymerase forms an extension product
on a DNA template, it is referred to herein as a DNA dependent
polymerase. Where the polymerase forms an extension product on a
RNA template, it is referred to herein as a RNA dependent
polymerase.
[0504] A "labeled dNTP," or "labeled rNTP," as used herein, refers,
respectively, to a dNTP or rNTP, or analogs thereof, that is
directly or indirectly attached with a label. For example, a
"labeled" dNTP or rNTP, may be directly labeled with, for example,
a dye and/or a detectable moiety, such as a member of a specific
binding pair (such as biotin-avidin). A "labeled" dNTP or rNTP, may
also be indirectly labeled by its attachment to, for example, a
moiety to which a label is/can be attached. A dNTP or rNTP, may
comprise a moiety (for example, an amine group) to which a label
may be attached following incorporation of the dNTP or rNTP into an
extension product. Useful labels in the present invention include
fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,
rhodamine, green fluorescent protein and the like), radioisotopes
(e.g., .sup.3H, .sup.35S, .sup.32P, .sup.33P, .sup.125I, or
.sup.14C), enzymes (e.g., LacZ, horseradish peroxidase, alkaline
phosphatase), digoxigenin, and colorimetric labels such as
colloidal gold or colored glass or plastic (e.g., polystyrene,
polypropylene, latex, etc.) beads.
[0505] The term "double stranded product" is used herein to refer
to products that are produced by the extension of a primer. It is
understood that the products are at least partially double
stranded, for example, in the region comprising the primer
extension product and its complement. The double stranded product
need not be completely double-stranded, and may have single
stranded regions. It is also understood that the double stranded
product can have heteroduplex regions in which one strand comprises
RNA and the complementary strand comprised DNA in that region.
Amplification
[0506] Some aspects of the invention comprise the amplification of
polynucleotide molecules or sequences within the polynucleotide
molecules. Amplification generally refers to a method that results
in the formation of one or more copies of a nucleic acid or
polynucleotide molecule or in the formation of one or more copies
of the complement of a nucleic acid or polynucleotide molecule.
Amplifications can be used in the invention, for example, to
amplify or analyze a polynucleotide bound to a solid surface. The
amplifications can be performed, for example, after archiving the
samples in order to analyze the archived polynucleotide.
[0507] In some aspects of the invention, exponential amplification
of nucleic acids or polynucleotides is used. These methods often
depend on the product catalyzed formation of multiple copies of a
nucleic acid or polynucleotide molecule or its complement. The
amplification products are sometimes referred to as "amplicons."
One such method for the enzymatic amplification of specific double
stranded sequences of DNA is known as the polymerase chain reaction
(PCR). This in vitro amplification procedure is based on repeated
cycles of denaturation, oligonucleotide primer annealing, and
primer extension by thermophilic template dependent polynucleotide
polymerase, resulting in the exponential increase in copies of the
desired sequence of the polynucleotide analyte flanked by the
primers. The two different PCR primers, which anneal to opposite
strands of the DNA, are positioned so that the polymerase catalyzed
extension product of one primer can serve as a template strand for
the other, leading to the accumulation of a discrete double
stranded fragment whose length is defined by the distance between
the 5' ends of the oligonucleotide primers.
[0508] Another method for amplification involves amplification of a
single stranded polynucleotide using a single oligonucleotide
primer. The single stranded polynucleotide that is to be amplified
contains two non-contiguous sequences that are complementary to one
another and, thus, are capable of hybridizing together to form a
stem-loop structure. This single stranded polynucleotide already
may be part of a polynucleotide analyte or may be created as the
result of the presence of a polynucleotide analyte.
[0509] Another method for achieving the result of an amplification
of nucleic acids is known as the ligase chain reaction (LCR). This
method uses a ligase enzyme to join pairs of preformed nucleic acid
probes. The probes hybridize with each complementary strand of the
nucleic acid analyte, if present, and ligase is employed to bind
each pair of probes together resulting in two templates that can
serve in the next cycle to reiterate the particular nucleic acid
sequence.
[0510] Another method for achieving a nucleic acid amplification is
the nucleic acid sequence based amplification (NASBA). This method
is a promoter-directed, enzymatic process that induces in vitro
continuous, homogeneous and isothermal amplification of a specific
nucleic acid to provide RNA copies of the nucleic acid. The
reagents for conducting NASBA include a first DNA primer with a 5'
tail comprising a promoter, a second DNA primer, reverse
transcriptase, RNAse-H, T7 RNA polymerase, NTPs and dNTPs.
[0511] Another method for amplifying a specific group of nucleic
acids is the Q-beta-replicase method, which relies on the ability
of Q-beta-replicase to amplify its RNA substrate exponentially. The
reagents for conducting such an amplification include "midi-variant
RNA" (amplifiable hybridization probe), NTPs, and
Q-beta-replicase.
[0512] Another method for amplifying nucleic acids is known as 3 SR
and is similar to NASBA except that the RNAse-H activity is present
in the reverse transcriptase. Amplification by 3 SR is an RNA
specific target method whereby RNA is amplified in an isothermal
process combining promoter directed RNA polymerase, reverse
transcriptase and RNase H with target RNA.
[0513] Another method for amplifying nucleic acids is the
Transcription Mediated Amplification (TMA) used by Gen-Probe. The
method is similar to NASBA in utilizing two enzymes in a
self-sustained sequence replication. See U.S. Pat. No.
6,946,254.
Another method for amplification of nucleic acids is Strand
Displacement Amplification (SDA) (Westin et al 2000, Nature
Biotechnology, 18, 199-202; Walker et al 1992, Nucleic Acids
Research, 20, 7, 1691-1696), which is an isothermal amplification
technique based upon the ability of a restriction endonuclease such
as HinchII or BsoBI to nick the unmodified strand of a
hemiphosphorothioate form of its recognition site, and the ability
of an exonuclease deficient DNA polymerase such as Klenow exo minus
polymerase, or Bst polymerase, to extend the 3'-end at the nick and
displace the downstream DNA strand. Exponential amplification
results from coupling sense and antisense reactions in which
strands displaced from a sense reaction serve as targets for an
antisense reaction and vice versa.
[0514] Another method for amplification of nucleic acids is Rolling
Circle Amplification (RCA) (Lizardi et al. 1998, Nature Genetics,
19:225-232). RCA can be used to amplify single stranded molecules
in the form of circles of nucleic acids. In its simplest form, RCA
involves the hybridization of a single primer to a circular nucleic
acid. Extension of the primer by a DNA polymerase with strand
displacement activity results in the production of multiple copies
of the circular nucleic acid concatenated into a single DNA
strand.
[0515] In some embodiments of the invention, RCA is coupled with
ligation. For example, a single oligonucleotide can be used both
for ligation and as the circular template for RCA. This type of
polynucleotide can be referred to as a "padlock probe" or a "RCA
probe". For a padlock probe, both termini of the oligonucleotide
contains sequences complementary to a domain within a nucleic acid
sequence of interest. The first end of the padlock probe is
substantially complementary to a first domain on the nucleic acid
sequence of interest, and the second end of the padlock probe is
substantially complementary to a second domain, adjacent to the
first domain near the first domain. Hybridization of the
oligonucleotide to the target nucleic acid results in the formation
of a hybridization complex. Ligation of the ends of the padlock
probe results in the formation of a modified hybridization complex
containing a circular polynucleotide. In some cases, prior to
ligation, a polymerase can fill in the gap by extending one end of
the padlock probe. The circular polynucleotide thus formed can
serve as a template for RCA that with the addition of a polymerase
results in the formation of an amplified product nucleic acid. The
methods of the invention described herein, can produce amplified
products with defined sequences on both the 5' and 3' ends. Such
amplified products can be used as padlock probes.
[0516] Some aspects of the invention utilize the linear
amplification of nucleic acids or polynucleotides. Linear
amplification generally refers to a method that involve the
formation of one or more copies of the complement of only one
strand of a nucleic acid or polynucleotide molecule, usually a
nucleic acid or polynucleotide analyte. Thus, the primary
difference between linear amplification and exponential
amplification is that the latter is auto catalyzed, that is, the
product serves to catalyze the formation of more product, whereas
in the former process the starting sequence catalyzes the formation
of product but is not itself replicated. In linear amplification
the amount of product formed increases as a linear function of time
as opposed to exponential amplification where the amount of product
formed is an exponential function of time.
[0517] In some embodiments, amplification methods can be
solid-phase amplification, polony amplification, colony
amplification, emulsion PCR, bead RCA, surface RCA, surface SDA,
etc., as will be recognized by one of skill in the art. In some
embodiments, amplification methods that results in amplification of
free DNA molecules in solution or tethered to a suitable matrix by
only one end of the DNA molecule can be used. Methods that rely on
bridge PCR, where both PCR primers are attached to a surface (see,
e.g., Adessi et al., Nucleic Acids Research (2000): 28(20): E87)
can be used. In some cases the methods of the invention can create
a "polymerase colony technology", or "polony", referring to a
multiplex amplification that maintains spatial clustering of
identical amplicons (see Harvard Molecular Technology Group and
Lipper Center for Computational Genetics website). These include,
for example, in situ polonies (Mitra and Church, Nucleic Acid
Research 27, e34, Dec. 15, 1999), in situ rolling circle
amplification (RCA) (Lizardi et al., Nature Genetics 19, 225, July
1998), bridge PCR (U.S. Pat. No. 5,641,658), picotiter PCR (Leamon
et al., Electrophoresis 24, 3769, November 2003), and emulsion PCR
(Dressman et al., PNAS 100, 8817, Jul. 22, 2003). The methods of
the invention provide new methods for generating and using
polonies.
[0518] In some embodiments, amplification methods such as
amplification that maintains spatial clustering of identical
amplicons, amplification that produces in situ polonies, picotiter
amplification, amplification inside microdroplets, amplification
inside the aqueous phase of microdroplets of an oil and water
emulsion or the like, may provide clonal amplification. "Clonal
amplification" as used herein is the amplification of a target
sequence or a set of target sequences in a spatially or physically
separated manner such that the amplified products are spatially or
physically separated from the amplified products of other target
sequences. For example, some of the methods of the present
invention provide for amplification of a set of target sequences
attached to, bound to, or hybridized to a solid surface such as a
set of beads, isolated surfaces, or wells in a plate such that each
surface on average contains one or fewer target sequences. In some
cases, the amplification is carried out under conditions that the
amplified products of the target sequence of a given surface are
attached to, bound to, or hybridized to that surface. Further, in
some cases, the given surface comprises few, substantially no, or
no other amplified products from other target sequences. Similarly,
clonal amplification may be carried out by separating the target
sequences and their products from other target sequences and their
products in other ways such as in a set of microreactors such as
microdroplets such as in an emulsion, in a set of wells, or by
dilution. Additionally clonal amplification may be performed using
a combination of compositions and techniques such as by amplifying
a set of target sequences on a solid surface or set of solid
surfaces such as a bead or set of beads in a set of microdroplets
in an emulsion.
Single Primer Isothermal Amplification Using a Complex Comprising
an RNA/DNA Partial Heteroduplex as a Template
[0519] In some aspects of the invention, the amplification method
that is used is a single primer isothermal amplification using a
complex comprising an RNA/DNA partial heteroduplex as a template.
In this method, termed single primer isothermal amplification, a
complex comprising an RNA/DNA partial heteroduplex is a substrate
for further amplification as follows: an enzyme which cleaves RNA
sequence from an RNA/DNA hybrid (such as RNase H) cleaves RNA from
the partial heteroduplex, leaving a partially double stranded
polynucleotide complex comprising a 3' single stranded DNA
sequence. The 3' single stranded sequence (formed by cleavage of
RNA in the complex comprising an RNA/DNA partial heteroduplex) is
generally the complement of the amplification composite primer, and
thus forms a specific binding site for a composite primer.
Extension of a bound composite primer by a DNA-dependent DNA
polymerase with strand displacement activity produces a primer
extension product, which displaces the previously bound cleaved
primer extension product, whereby polynucleotide (generally, DNA)
product accumulates. See, for example, U.S. Pat. Nos. 6,251,639 and
6,692,918.
[0520] Amplification using a complex comprising an RNA/DNA partial
heteroduplex as a template for further amplification (also termed
single primer isothermal amplification) generally occurs under
conditions permitting composite primer hybridization, primer
extension by a DNA polymerase with strand displacement activity,
cleavage of RNA from an RNA/DNA hybrid and strand displacement. In
so far as the composite primer hybridizes to the 3' single stranded
portion (of the partially double stranded polynucleotide which is
formed by cleaving RNA in the complex comprising an RNA/DNA partial
heteroduplex) comprising, generally, the complement of at least a
portion of the composite primer sequence, composite primer
hybridization may be under conditions permitting specific
hybridization.
[0521] In some embodiments, the methods of the invention result in
amplification of a multiplicity, a large multiplicity, or a very
large multiplicity of template polynucleotide sequences. In some
embodiments, essentially all of the template polynucleotide present
in the initial sample (e.g., all of the mRNA or all of the genomic
DNA) is amplified. In other embodiments, at least 1, at least 5, at
least 10, at least 20. at least 50, at least 100, at least 200, at
least 300, or more distinct sequences (such as a gene or other
subsegment of a polynucleotide, transcripts of a nucleic acid
target, a marker (such as a SNP or other polymorphism) are
amplified, as assessed, e.g., by analysis of marker sequences known
to be present in the template sample under analysis, using methods
known in the art. Template polynucleotide sequences that are
amplified may be present on the same polynucleotide (e.g., a
chromosome or portion of a chromosome for genomic DNA template or
on the same RNA for RNA template) or on different template
polynucleotides (e.g., different chromosome or portions of
chromosomes for DNA template, or different RNAs for RNA template).
In some case, amplification of genomic DNA is exemplified herein,
it will be understood by those of skill in the art, however, that
the global amplification methods of the invention are suitable for
amplification of any pool or subset of polynucleotides.
[0522] In some embodiments, the methods of the invention are used
to globally amplify double stranded DNA target. It is understood
that in these cases, the amplified product generally is a mixture
of sense and antisense copies of the template DNA. In some
embodiments, the methods of the invention are used to globally
amplify a single stranded DNA or RNA target. In these cases, the
amplification product will generally be a copy of either the target
polynucleotide (sense copy) or of the complement to the target
nucleotide (antisense copy). Whether the sense or antisense copy is
produced will depend on the method, as will be understood by one of
ordinary skill in the art. In some embodiments, the amplification
product of different senses can be annealed to form a double
stranded (or partially double stranded) complex. In other
embodiments, they can be prevented from annealing (or subsequently
denatured) to produce a mixture of single stranded amplification
products. The amplified products may be of differing lengths.
[0523] As illustrated in these embodiments, all steps are
isothermal (in the sense that thermal cycling is not required),
although the temperatures for each of the steps may or may not be
the same. It is understood that various other embodiments may be
practiced, given the general description provided above. For
example, as described and exemplified herein, certain steps may be
performed as temperature is changed (e.g., raised, or lowered).
[0524] For simplicity, the isothermal amplification methods of the
invention are described as two distinct steps or phases, above. It
is understood that the two phases may occur simultaneously in some
embodiments (for example, if the enzyme that cleaves RNA from
RNA/DNA hybrid is included in the first reaction mixture).
[0525] Although generally only one composite primer is described
above, it is further understood that the amplification methods may
be performed in the presence of two or more different composite
primers that randomly prime template polynucleotide. In addition,
the amplification polynucleotide products of two or more separate
amplification reactions conducted using two or more different
composite primers that randomly prime template polynucleotide can
be combined.
Extension of Primers
[0526] The methods of the present invention involve the extension
of primers. In general, primers hybridize to, and are extended
along (chain extended), a sequence within the target polynucleotide
and, thus, the target polynucleotide acts as a template. The
extended primers are chain "extension products." The sequence over
which the primer is extended may lie between two defined sequences
but need not be. In general, the primers hybridize with a sequence
within the target polynucleotide. The target sequence usually
contains from about 30 to 5,000 or more nucleotides, often 50 to
1,000 nucleotides. The target polynucleotide may be a fraction of a
larger molecule or it may be substantially the entire molecule
(polynucleotide analyte).
Composite Primers
[0527] Generation of primers suitable for extension by
polymerization is well known in the art, such as described in PCT
Pub. No. WO99/42618 (and references cited therein). The composite
primer comprises a combination of RNA and DNA, with the 3'-end
nucleotide being a nucleotide suitable for nucleic acid extension.
The 3'-end nucleotide can be any nucleotide or analog that when
present in a primer, is extendable by a DNA polymerase when
hybridized to a polynucleotide template. Generally, the 3'-end
nucleotide has a 3'-OH. Suitable primers include those that
comprise at least one portion of RNA and at least one portion of
DNA. For example, composite primers can comprise a 5'-RNA portion
and a 3'-DNA portion (in which the RNA portion is adjacent to the
3'-DNA portion); or 5'- and 3'-DNA portions with an intervening RNA
portion. Accordingly, in one embodiment, the composite primer
comprises a 5' RNA portion and a 3'-DNA portion, preferably wherein
the RNA portion is adjacent to the 3'-DNA portion. In another
embodiment, the composite primer comprises 5'- and 3'-DNA portions
with at least one intervening RNA portion (i.e., an RNA portion
between the two DNA portions). In yet another embodiment, the
composite primer of the invention comprises a 3'-DNA portion and at
least one intervening RNA portion (i.e., an RNA portion between DNA
portions).
Composite Amplification Primers
[0528] Composite amplification primers are RNA/DNA composite
primers that can be used to create multiple copies of (amplify) a
polynucleotide sequence isothermally using RNA cleavage, and DNA
polymerase activity with strand displacement. Amplification with
such primers is described, for example in U.S. Pat. Nos. 6,251,639,
6,692,918, and 6,946,251. The composite amplification primer
comprises sequences capable of hybridizing to a portion of a DNA
template, and most often comprises sequences hybridizable to a
defined 3'-portion of the DNA.
[0529] A composite amplification primer comprises at least one RNA
portion that is capable of (a) binding (hybridizing) to a sequence
on a DNA template independent of hybridization of the DNA
portion(s) to a sequence on the same extension product; and being
cleaved with a ribonuclease when hybridized to the DNA template.
The composite amplification primers bind to the DNA template to
form a partial heteroduplex in which only the RNA portion of the
primer is cleaved upon contact with a ribonuclease such as RNase H,
while the DNA template remains intact, thus enabling annealing of
another composite primer.
[0530] The composite amplification primers also comprise a 3' DNA
portion that is capable of hybridization to a sequence on the DNA
template such that its hybridization to the DNA is favored over
that of the nucleic acid strand that is displaced from the DNA
template by the DNA polymerase. Such primers can be rationally
designed based on well known factors that influence nucleic acid
binding affinity, such as sequence length and/or identity, as well
as hybridization conditions. In a preferred embodiment,
hybridization of the 3' DNA portion of the composite primer to its
complementary sequence in the second strand cDNA is favored over
the hybridization of the homologous sequence in the 5' end of the
displaced strand to the second strand cDNA.
Ligands and Receptors
[0531] The present invention utilizes ligands to bind
polynucleotides to a solid surface. A ligand is a compound which
can bind to a receptor. The ligand and receptor (antiligand) can be
members of a specific binding pair of two different molecules. In
some cases, the receptor and or ligand have one or more areas on
the surface or in a cavity which gives rise to specific binding.
The ligand can be complementary with a particular spatial and polar
organization of the receptor. The specific binding pair may be
members of an immunological pair such as antigen-antibody, or may
be operator-repressor, nuclease-nucleotide, biotin-avidin,
hormones-hormone receptors, nucleic acid duplexes, IgG-protein A,
DNA-DNA, DNA-RNA, and the like. Examples of ligands and/or
receptors include, but are not limited to, agonists and antagonists
for cell membrane receptors, toxins and venoms, viral epitopes,
hormones such as steroids, hormone receptors, peptides, enzymes and
other catalytic polypeptides, enzyme substrates, cofactors, drugs
including small organic molecule drugs, opiates, opiate receptors,
lectins, sugars, saccharides including polysaccharides, proteins,
and antibodies including monoclonal antibodies and synthetic
antibody fragments, cells, cell membranes and moieties therein
including cell membrane receptors, and organelles. Examples of
ligand-receptor pairs include antibody-antigen;
lectin-carbohydrate; peptide-cell membrane receptor; protein
A-antibody; hapten-antihapten; digoxigenin-anti-digoxigenin; avidin
and biotin, enzyme-cofactor and enzyme-substrate.
[0532] In one embodiment, the receptor may comprise an antibody. As
used herein, the term "antibody" refers to an immunoglobulin
molecule or a fragment of an immunoglobulin molecule having the
ability to specifically bind to a particular antigen. The antibody
may be an anti-receptor antibody specific for the receptor used in
the assay. Thus, the antibody may be capable of specifically
binding the receptor as the antigen. Antibodies and methods for
their manufacture are well known in the art of immunology. The
antibody may be produced, for example, by hybridoma cell lines, by
immunization to elicit a polyclonal antibody response, or by
recombinant host cells that have been transformed with a
recombinant DNA expression vector that encodes the antibody.
Illustrative receptors include naturally occurring receptors, e.g.,
thyroxine binding globulin, antibodies, enzymes, Fab fragments,
lectins, nucleic acids, repressors, protection enzymes, protein A,
complement component Clq, DNA binding proteins or ligands and the
like. In some embodiments, the receptor is an antibody, and the
ligand is a molecule or portion of a molecule that is recognized by
the antibody such as an epitope, or a hapten.
[0533] In some embodiments it is desirable that the ligand be a
small organic molecule, for example, a compound of molecular weight
less than about 1500 g/mol, generally between about 100 to about
1000 g/mol, often between about 300 to about 600 g/mol such as
biotin or other haptens. The small organic molecule can provide a
means for attachment of a nucleotide sequence to a label or to a
support.
[0534] Various anti-ligands and ligands can be used (as labels
themselves or as a means for attaching a label). In the case of a
ligand that has a natural anti-ligand, such as biotin, thyroxine
and cortisol, the ligand can be used in conjunction with labeled
anti-ligands.
[0535] Attaching the receptors that are specific binders to the
ligands to a support or surface may be accomplished by well-known
techniques, commonly available in the literature. See, for example,
"Immobilized Enzymes," Ichiro Chibata, Halsted Press, New York
(1978) and Cuatrecasas, J. Biol. Chem., 245:3059 (1970).
[0536] The ligand and receptor can be members of capture pairs. For
example, capture pairs can employ reversible (e.g., cleavable) or
irreversible linkages. Non-limiting examples of reversible linkages
include thiol-thiol, digoxigenin/anti-digoxigenin, and linkages
using VECTREX (E Avidin DLA (Vector Laboratories, Burlingame,
Calif.), CaptAvidin.TM., NeutrAvidinN, and D-desthiobiotin
(Molecular Probes, Inc., Eugene, Oreg.).
[0537] In some embodiments, the ligand-receptor pair can be pairs
of reactive molecules that can react to form a covalent bond, thus
binding the polynucleotide to the surface. For example, the ligand
can comprise an amine group bound to the polynucleotide that can be
reacted with a functional group attached to the solid surface to
form a covalent bond. The amine on the polynucleotide can react,
for example with an activated carbonyl group attached to the
surface, e.g. an N-hydroxy succinimide (NHS) ester. Functional
groups such as N-acylimidazole, 2- or 3-bromoacrylate, cyanuric
chloride, disulfide, N-hydroxysuccinimide ester, hydrazide,
iodoacetyl, imidoester, isocyanate, isothiocyanate, maleimide,
succinimidyl carbonate, acyl chloride, and sulfonyl chloride can be
used in order to form a covalent linkage to the solid surface.
DNA Polymerase, and an Agent Capable of Cleaving an RNA-DNA
Hybrid
[0538] The isothermal amplification methods of the invention employ
the following enzymes: an RNA-dependent DNA polymerase, a
DNA-dependent DNA polymerase, and an agent capable of cleaving an
RNA strand of an RNA-DNA hybrid (for example, a ribonuclease such
as RNase H). One or more of these activities may be found and used
in a single enzyme. For example, RNase H activity may be supplied
by an RNA-dependent DNA polymerase (such as reverse transcriptase)
or may be provided in a separate enzyme. Reverse transcriptases
useful for this method may or may not have RNase H activity. Many
reverse transcriptases, such as those from avian myeloblastosis
virus (AMV-RT), and Moloney murine leukemia virus (MMLV-RT)
comprise more than one activity (for example, polymerase activity
and ribonuclease activity) and can function in the formation of the
double stranded cDNA molecules. However, in some instances, it is
preferable to employ a reverse transcriptase which lacks or has
reduced levels RNase H activity. Reverse transcriptase devoid of
RNase H or with reduced levels of RNase H activity are known in the
art, including those comprising a mutation of the wild type reverse
transcriptase where the mutation eliminates the RNase H activity.
In these cases, the addition of an RNase H from other sources, such
as that isolated from E. coli, can be employed for the formation of
the double stranded cDNA. The RNA-dependent DNA polymerase activity
and DNA-dependent DNA polymerase activity may be provided by the
same enzyme (for example, Bst polymerase), or these activities may
be provided in separate enzymes. DNA polymerases with strand
displacement activity are also useful.
[0539] One aspect of the invention is the formation of a complex
comprising an RNA/DNA partial heteroduplex. This process generally
utilizes the enzymatic activities of an RNA-dependent DNA
polymerase, a DNA-dependent DNA polymerase. Generally, RNA in the
RNA/DNA partial heteroduplex is cleaved by an agent (such as an
enzyme, such as a ribonuclease) capable of cleaving RNA from an
RNA/DNA hybrid, generating a 3' single stranded portion with
sequences that are complementary to RNA in a composite primer (and
thus forming a binding site for a composite primer).
[0540] RNA-dependent DNA polymerases for use in the methods and
compositions of the invention are capable of effecting extension of
a primer according to the methods of the invention. Accordingly, a
preferred RNA-dependent DNA polymerase is one that is capable of
extending a nucleic acid primer along a nucleic acid template that
is comprised at least predominantly of ribonucleotides. Suitable
RNA-dependent DNA polymerases for use in the methods and
compositions of the invention include reverse transcriptase and,
for example, a DNA polymerase that possesses both DNA-dependent and
RNA-dependent DNA polymerase activity, such as Bst DNA
polymerase.
[0541] DNA-dependent DNA polymerases for use in the methods and
compositions of the invention are capable of effecting extension of
the composite primer according to the methods of the invention.
Accordingly, a preferred polymerase is one that is capable of
extending a nucleic acid primer along a nucleic acid template that
is comprised at least predominantly of deoxynucleotides. The
formation of the complex comprising the RNA/DNA partial
heteroduplex can be carried out by a DNA polymerase which comprises
both RNA-dependent DNA polymerase and DNA-dependent DNA polymerase
activities (such as Bst DNA polymerase, or a reverse
transcriptase). Amplification of an RNA sequence according to
methods of the invention involves the use of a DNA polymerase that
is able to displace a nucleic acid strand from the polynucleotide
to which the displaced strand is bound, and, generally, the more
strand displacement capability the polymerase exhibits (i.e.,
compared to other polymerases which do not have as much strand
displacement capability) is preferable. Preferably, the DNA
polymerase has high affinity for binding at the 3'-end of an
oligonucleotide hybridized to a nucleic acid strand. Preferably,
the DNA polymerase does not possess substantial nicking activity.
Generally, the DNA polymerase preferably has little or no 5' to 3'
exonuclease activity so as to minimize degradation of primer, or
primer extension products. Generally, this exonuclease activity is
dependent on factors such as pH, salt concentration, whether the
template is double stranded or single stranded, and so forth, all
of which are familiar to one skilled in the art. Mutant DNA
polymerases in which the 5' to 3' exonuclease activity has been
deleted, are known in the art and are suitable for the
amplification methods described herein. Mutant DNA polymerases
which lack both 5' to 3' nuclease and 3' to 5' nuclease activities
have also been described, for example, exo.sup.-/--Klenow DNA
polymerase. It is preferred that the DNA polymerase displaces
primer extension products from the template nucleic acid in at
least about 25%, more preferably at least about 50%, even more
preferably at least about 75%, and most preferably at least about
90%, of the incidence of contact between the polymerase and the 5'
end of the primer extension product. In some embodiments, the use
of thermostable DNA polymerases with strand displacement activity
is preferred. Such polymerases are known in the art, such as
described in U.S. Pat. No. 5,744,312 (and references cited
therein). Generally, the DNA polymerase has little to no
proofreading activity
[0542] Suitable DNA polymerases for use in the methods and
compositions of the invention include those disclosed in U.S. Pat.
Nos. 5,648,211 and 5,744,312, which include exo.sup.- Vent (New
England Biolabs), exo.sup.- Deep Vent (New England Biolabs), Bst
(BioRad), exo.sup.- Pfu (Stratagene), Bca (Panvera), sequencing
grade Taq (Promega), exo.sup.-/- Klenow DNA polymerase, and
thermostable DNA polymerases from Thermoanaerobacter
thermohydrosulfuricus.
[0543] One of the ribonuclease for use in the methods and
compositions of the invention is capable of cleaving
ribonucleotides in an RNA/DNA hybrid or heteroduplex. Preferably,
the ribonuclease cleaves ribonucleotides in an RNA/DNA hybrid
regardless of the identity and type of nucleotides adjacent to the
ribonucleotide to be cleaved. It is preferred that the ribonuclease
cleaves independent of sequence identity. Examples of suitable
ribonucleases for the methods and compositions of the invention are
well known in the art, including ribonuclease H(RNase H), e.g.,
Hybridase.
[0544] As is well known in the art, DNA-dependent DNA polymerase
activity, RNA-dependent DNA polymerase activity, and the ability to
cleave RNA from a RNA/DNA hybrid may be present in different
enzymes, or two or more activities may be present in the same
enzyme. Accordingly, in some embodiments, the same enzyme comprises
RNA-dependent DNA polymerase activity and cleaves RNA from an
RNA/DNA hybrid. In some embodiments, the same enzyme comprises
DNA-dependent DNA polymerase activity and cleaves RNA from an
RNA/DNA hybrid. In some embodiments, the same enzyme comprises
DNA-dependent DNA polymerase activity, RNA-dependent DNA polymerase
activity and cleaves RNA from an RNA/DNA hybrid. In some
embodiments, different enzymes comprise RNA-dependent DNA
polymerase activity and DNA-dependent DNA polymerase activity. In
some embodiments, different enzymes comprise RNA-dependent DNA
polymerase activity and cleave RNA from an RNA/DNA hybrid. In some
embodiments, different enzymes comprise DNA-dependent DNA
polymerase activity and cleave RNA from an RNA/DNA hybrid. In other
embodiments the RNA targets are degraded by the use of other RNases
such as RNase 1, for example, for removing the target RNA following
extension of all DNA first primer along the RNA targets by
transcription, or a combination of RNase H and other RNases.
Nucleic Acid Target
[0545] The DNA, RNA, or polynucleotide target is generally a
polymeric nucleotide, which in the intact natural state can have
about 30 to 5,000,000 or more nucleotides and in an isolated state
can have about 20 to 50,000 or more nucleotides, usually about 100
to 20,000 nucleotides, more frequently 500 to 10,000 nucleotides.
The polynucleotide target to be amplified includes nucleic acids
from any source in purified or unpurified form, which can be DNA
(dsDNA and ssDNA) or RNA, mitochondrial DNA and RNA, chloroplast
DNA and RNA, DNA-RNA hybrids, or mixtures thereof, genes,
chromosomes, plasmids, the genomes of biological material such as
microorganisms, e.g., bacteria, yeasts, viruses, viroids, molds,
fungi, plants, animals, humans, and fragments thereof. Exemplary
RNAs include, but are not limited to, mRNAs, tRNAs, snRNAs, rRNAs,
retroviruses, small non-coding RNAs, microRNAs, polysomal RNAs,
pre-mRNAs, intronic RNA, viral RNA and fragments thereof. Exemplary
DNAs include, but are not limited to, genomic DNA, plasmid DNA,
phage DNA, nucleolar DNA, mitochondrial DNA, chloroplast DNA, cDNA,
synthetic DNA, yeast artificial chromosomal DNA ("YAC"), bacterial
artificial chromosome DNA ("BAC"), other extrachromosomal DNA,
primer extension products and fragments thereof. Target
polynucleotide includes DNA (e.g., genomic DNA, including human
genomic DNA, and mammalian genomic DNA (such as mouse, rat,)) and
RNA (e.g., mRNA, ribosomal RNA, and total RNA). It should be
understood that template RNA includes coding and non-coding RNA.
The sequences can be naturally occurring or recombinant nucleic
acid targets, including cloned nucleic fragments of interest. The
terms "polynucleotide target", "nucleic acid target", "target
polynucleotide" and "polynucleotide target" are used
interchangeably. The term "DNA target" is interchangeable with the
term "target DNA", and the term "RNA target" is interchangeable
with the term "target RNA". The target nucleic acid may be a
mixture of DNA and RNA targets.
[0546] The target polynucleotide can be only a minor fraction of a
complex mixture such as a biological sample and can be obtained
from various biological material by procedures well known in the
art. Polynucleotides can be obtained from sources containing very
small quantities of nucleic acid, such a single cells, small
numbers of cells, patient samples, forensic samples, and
archeological samples. Obtaining and purifying nucleic acids use
standard techniques in the art, including methods designed to
isolate one or a very small number of cells, such a cell sorting or
laser capture micro-dissection. The methods of the invention are
particularly suited for use with genomic DNA (e.g., human and other
mammalian genomic DNA), as well as RNA (e.g., total RNA or mRNA
samples) or fragments thereof.
[0547] The target polynucleotide(s) can be known or unknown and may
contain more than one desired specific nucleic acid sequence of
interest, each of which may be the same or different from each
other. If the target polynucleotide is double stranded (e.g.,
double stranded DNA or a double stranded DNA/RNA hybrid, such as is
produced by first strand cDNA synthesis), the target may first be
treated to render it single stranded (e.g., by denaturation or by
cleavage of the RNA portion of a DNA/RNA hybrid). Denaturation may
also be carried out to remove secondary structure present in a
single stranded target molecule (e.g., RNA). In some cases, double
stranded DNA target polynucleotide may be first cleaved by one or
more restriction endonuclease enzymes.
[0548] When the target polynucleotide is DNA, the initial step of
the amplification of a target nucleic acid sequence can be
rendering the target single stranded. If the target nucleic acid is
a double stranded (ds) DNA, the initial step can be target
denaturation. The denaturation step may be thermal denaturation or
any other method known in the art, such as alkali treatment. If the
target nucleic acid is present in a DNA-RNA hybrid, the initial
step can be denaturation of the hybrid to obtain a DNA, or removal
of the RNA strand using other means known in the art, such as
thermal treatment, digestion with an enzyme that cleaves RNA from
an RNA/DNA hybrid (such as RNase H) or alkali treatment, to
generate single stranded DNA. When the target is RNA, the initial
step may be the synthesis of a single stranded cDNA. Techniques for
the synthesis of cDNA from RNA are known in the art, and include
reverse transcription of RNA strand using a primer that binds to a
specific target, such as the poly-A tail of eukaryotic mRNAs or
other specific or consensus sequences. In addition, reverse
transcription can be primed by a population of degenerate or
partially degenerate primers. First strand cDNA can be separated
from the complex of RNA and first strand cDNA as described
herein.
[0549] RNAs can be from any source in purified or unpurified form,
which can be RNA such as total RNA, tRNA, mRNA, rRNA, mitochondrial
RNA, chloroplast RNA, DNA-RNA hybrids, or mixtures thereof, from
any source and/or species, including human, animals, plants, and
microorganisms such as bacteria, yeasts, viruses, viroids, molds,
fungi, plants, and fragments thereof. It is understood that the RNA
can be coding or noncoding RNA (such as untranslated small RNAs).
RNAs can be obtained and purified using standard techniques in the
art. Use of a DNA target (including genomic DNA target) can involve
initial transcription of the DNA target into RNA form, which can be
achieved using methods disclosed in Kurn, U.S. Pat. No. 6,251,639
B1, and by other techniques (such as expression systems) known in
the art. Thus, RNA template can be itself generated from a DNA
source (such as genomic DNA), using methods known in the art,
including Kurn, U.S. Pat. No. 6,251,639. RNA copies of genomic DNA
would generally include untranscribed sequences generally not found
in mRNA, such as introns, regulatory and control elements, etc. RNA
targets may also be generated from cloned genomic DNA sequences
that can be subjected to in vitro transcription. Use of a DNA-RNA
hybrid can involve denaturation of the hybrid to obtain a single
stranded RNA, denaturation followed by transcription of the DNA
strand to obtain an RNA, or other methods known in the art such as
digestion with an RNase H to generate single stranded DNA.
Reaction Conditions and Detection
[0550] Appropriate reaction media and conditions for carrying out
the methods of the invention include those that permit nucleic acid
extension, copying, and amplification according to the methods of
the invention. Such media and conditions are known to persons of
skill in the art, and are described in various publications, such
as U.S. Pat. Nos. 5,554,516; 5,716,785; 5,130,238; 5,194,370;
6,090,591; 5,409,818; 5,554,517; 5,169,766; 5,480,784; 5,399,491;
5,679,512; and PCT Pub. No. WO99/42618. For example, a buffer may
be Tris buffer, although other buffers can also be used as long as
the buffer components are non-inhibitory to enzyme components of
the methods of the invention. The pH is preferably from about 5 to
about 11, more preferably from about 6 to about 10, even more
preferably from about 7 to about 9, and most preferably from about
7.5 to about 8.5. The reaction medium can also include bivalent
metal ions such as Mg.sup.2+, or Mn.sup.2+, at a final
concentration of free ions that is within the range of from about
0.01 to about 15 mM, and most preferably from about 1 to 10 mM. The
reaction medium can also include other salts, such as KCl or NaCl,
that contribute to the total ionic strength of the medium. For
example, the range of a salt such as KCl is preferably from about 0
to about 125 mM, more preferably from about 0 to about 100 mM, and
most preferably from about 0 to about 75 mM. The reaction medium
can further include additives that could affect performance of the
amplification reactions, but that are not integral to the activity
of the enzyme components of the methods. Such additives include
proteins such as BSA, single stranded binding protein (for example,
T4 gene 32 protein), and non-ionic detergents such as NP40 or
Triton. Reagents, such as DTT, that are capable of maintaining
enzyme activities can also be included. Such reagents are known in
the art. Where appropriate, an RNase inhibitor (such as RNasin)
that does not inhibit the activity of the RNase employed in the
method can also be included. Any aspect of the methods of the
invention can occur at the same or varying temperatures. In some
embodiments, the amplification reactions (particularly, primer
extension and transcription; and generally not the step of
denaturing) are performed isothermally, which substantially avoids
the thermocycling process. The isothermal amplification reaction is
carried out at a temperature that permits hybridization of the
oligonucleotides (primer) of the invention to the template
polynucleotide and that does not substantially inhibit the activity
of the enzymes employed. The temperature can be in the range of
preferably about 25.degree. C. to about 85.degree. C., more
preferably about 30.degree. C. to about 80.degree. C., and most
preferably about 37.degree. C. to about 75.degree. C. The
temperature for the transcription steps can be lower than the
temperature(s) for the preceding steps. The temperature of the
transcription steps can be in the range of preferably about
25.degree. C. to about 85.degree. C., more preferably about
30.degree. C. to about 75.degree. C., and most preferably about
37.degree. C. to about 70.degree. C.
[0551] Nucleotide and/or nucleotide analogs, such as
deoxyribonucleoside triphosphates, that can be employed for
synthesis of the primer extension products in the methods of the
invention are provided in the amount of from preferably about 50 to
about 2500 .mu.M, more preferably about 100 to about 2000 .mu.M,
even more preferably about 200 to about 1700 .mu.M, and most
preferably about 250 to about 1500 .mu.M. Nucleotides and/or
analogs, such as ribonucleoside triphosphates, that can be employed
for synthesis of the RNA transcripts in the methods of the
invention are provided in the amount of from preferably about 0.25
to about 6 mM, more preferably about 0.5 to about 5 mM, even more
preferably about 0.75 to about 4 mM, and most preferably about 1 to
about 3 mM.
[0552] The oligonucleotide components of the reactions of the
invention are generally in excess of the number of target nucleic
acid sequence to be amplified. They can be provided at about or at
least about any of the following: 10, 10.sup.2, 10.sup.4, 10.sup.6,
10.sup.8, 10.sup.10 times the amount of target nucleic acid.
Primers can be provided at about or at least about any of the
following concentrations: 50 nM, 100 nM, 500 nM, 1000 nM, 2500 nM,
5000 nM.
[0553] In the methods of the invention, the steps may be carried
out in the order listed or, in some cases, may be carried out in a
different order. In some methods a later step depends on the
formation of a product from an earlier step, in which case such
steps must be carried out in the order listed. One of ordinary
skill in the art will understand which steps should be carried out
in the order listed, and which steps can be carried out in a
different order.
[0554] In some embodiments, the foregoing components are added
simultaneously at the initiation of the isothermal amplification
process. In another embodiment, components are added in any order
prior to or after appropriate timepoints during the amplification
process, as required and/or permitted by the amplification
reaction. Such timepoints, some of which are noted below, can be
readily identified by a person of skill in the art. The enzymes
used for nucleic acid amplification according to the methods of the
invention can be added to the reaction mixture either prior to a
denaturation step, following the denaturation step, or following
hybridization of a primer to a polynucleotide template, as
determined by their thermal stability and/or other considerations
known to the person of skill in the art. The first primer extension
product and the second primer extension product synthesis reactions
can be performed consecutively, followed by an amplification steps.
In these embodiments, the reaction conditions and components may be
varied between the different reactions.
[0555] In some embodiments, the amplification reactions can be
stopped at various timepoints, and resumed at a later time. Said
timepoints can be readily identified by a person of skill in the
art. One timepoint is at the end of a first primer extension
product synthesis. Another timepoint is at the end of a second
primer extension product synthesis. Methods for stopping the
reactions are known in the art, including, for example, cooling the
reaction mixture to a temperature that inhibits enzyme activity or
heating the reaction mixture to a temperature that destroys an
enzyme. Methods for resuming the reactions are also known in the
art, including, for example, raising the temperature of the
reaction mixture to a temperature that permits enzyme activity or
replenishing a destroyed (or depleted) enzyme. In some embodiments,
one or more of the components of the reactions is replenished prior
to, at, or following the resumption of the reactions.
Alternatively, the reaction can be allowed to proceed (i.e., from
start to finish) without interruption.
[0556] In some embodiments the reaction can be allowed to proceed
without purification of intermediate complexes, for example, to
remove primer. Products can be purified at various timepoints,
which can be readily identified by a person of skill in the art.
One timepoint is at the end of first primer extension product
synthesis. Another timepoint is at the end of second primer
extension synthesis. In some embodiments, the removal of primers
and/or target at the end of a defined step by enzymes with
appropriate nuclease activities are also useful, for example,
cleavage of the RNA portion of free composite tailed primer prior
to the isothermal amplification step by treatment with RNase 1.
[0557] The detection of the amplification product can be indicative
of the presence of a target sequence. Quantitative analysis is also
an aspect of the instant invention. Direct and indirect detection
methods (including quantitation) are well known in the art. For
example, by comparing the amount of product amplified from a test
sample containing an unknown amount of a polynucleotide containing
a target sequence to the product of amplification of a reference
sample that has a known quantity of a polynucleotide that contains
the target sequence, the amount of target sequence in the test
sample can be determined. The amplification methods of the
invention can also be extended to analysis of sequence alterations
and sequencing of the target nucleic acid. Further, detection could
be effected by, for example, examination of translation products
from RNA amplification products. The global amplification by the
methods of the invention and/or the amplification of selected
targets, when present in the sample, are useful for various methods
which enable highly parallel nucleic acid interrogations.
Characterization of Nucleic Acids
[0558] The methods of the invention are amenable to quantitative
analysis, as in some embodiments, amplification can yield
sufficient single stranded polynucleotide (generally, DNA and RNA)
products which accurately reflect the representation of the various
DNA or RNA sequences (e.g. genomic DNA or mRNA) in the starting
material. The amplified products can be analyzed using, for
example, probe hybridization techniques known in the art, such as
Northern blotting, and hybridizing to probe arrays. In addition,
the single stranded polynucleotide products may serve as starting
material for other starting material for other analytical and/or
quantification methods known in the art, such as real time PCR,
quantitative TaqMan, quantitative PCR using molecular beacons,
methods described in Kurn, U.S. Pat. No. 6,251,639, etc. Thus, the
invention includes those further analytical and/or quantification
methods as applied to any of the products of the methods
herein.
[0559] In another embodiment, the amplification methods of the
invention are utilized to generate multiple copies of single
stranded polynucleotide products from RNA or DNA targets that are
labeled by the incorporation of labeled nucleotides during DNA
polymerization. For example, amplification according to the methods
of the invention can be carried out with suitable labeled dNTPs or
rNTPs. These labeled nucleotides can be directly attached to a
label, or can comprise a moiety which could be attached to a label.
The label may be attached covalently or non-covalently to the
amplification products. Suitable labels are known in the art, and
include, for example, a ligand which is a member of a specific
binding pair which can be detected/quantified using a detectable
second member of the binding pair. Thus, amplification of total RNA
or mRNA according to the methods of the invention in the presence
of, for example, Cy3-dUTP or Cy5-dUTP results in the incorporation
of these nucleotides into the amplification products.
[0560] The labeled amplified products are suitable for analysis
(for example, detection and/or quantification) by contacting them
with, for example, microarrays (of any suitable surface, which
includes glass, chips, plastic), beads, or particles, that comprise
suitable probes such as cDNA and/or oligonucleotide probes. Thus,
the invention provides methods to characterize (for example, detect
and/or quantify) a DNA or RNA sequence of interest by generating
labeled polynucleotide (generally, DNA) products using
amplification methods of the invention, and analyzing the labeled
products. Analysis of labeled products can be performed by, for
example, hybridization of the labeled amplification products to,
for example, probes immobilized at, for example, specific locations
on a solid or semi-solid substrate, probes immobilized on defined
particles, or probes immobilized on blots (such as a membrane), for
example arrays. Other methods of analyzing labeled products are
known in the art, such as, for example, by contacting them with a
solution comprising probes, followed by extraction of complexes
comprising the labeled amplification products and probes from
solution. The identity of the probes provides characterization of
the sequence identity of the amplified products, and thus by
extrapolation the identity of the target DNA or target RNA present
in a sample. Hybridization of the labeled products is detectable,
and the amount of specific labels that are detected is proportional
to the amount of the labeled amplification products of a specific
DNA or RNA sequence of interest. This measurement is useful for,
for example, measuring the relative amounts of the various RNA
species in a sample, which are related to the relative levels of
gene expression, as described herein or to detect the presence or
absence of defined target DNA or RNA in a sample. The measurement
is also useful for measuring the relative amounts of various DNA
sequences corresponding, for example, to genetic regions in the
sample. The amount of labeled products (as indicated by, for
example, detectable signal associated with the label) hybridized at
defined locations on an array can be indicative of the detection
and/or quantification of the corresponding target DNA or target RNA
species in the sample.
Sequencing of the Polynucleotide Products of the Invention
[0561] As described above, the methods can be used to obtain
sequence information about a target RNA or target DNA of interest.
The sequencing can be carried out on the primer extension products
or amplification products produced by the methods herein. In some
embodiments the sequencing is performed on the polynucleotides
attached to solid surfaces as described herein. In one embodiment
sequencing is performed on polynucleotides that are attached to the
beads through oligonucleotides attached to the beads which capture
amplified product, and are extended to produce a polynucleotide
attached to the surface comprising a defined sequence at its 3'
end.
[0562] The methods of the invention are useful, for example, for
sequencing of a polynucleotide sequence of interest. The sequencing
process is carried out as described for the methods described
herein.
[0563] Known methods for sequencing include, for example, those
described in: Sanger, F. et al., Proc. Natl. Acad. Sci. U.S.A. 75,
5463-5467 (1977); Maxam, A. M. & Gilbert, W. Proc Natl Acad Sci
USA 74, 560-564 (1977); Ronaghi, M. et al., Science 281, 363,365
(1998); Lysov, 1. et al., Dokl Akad Nauk SSSR 303, 1508-1511
(1988); Bains W. & Smith G. C. J. Theor Biol 135, 303-307
(1988); Drnanac, R. et al., Genomics 4, 114-128 (1989); Khrapko, K.
R. et al., FEBS Lett 256.118-122 (1989); Pevzner P. A. J Biomol
Struct Dyn 7, 63-73 (1989); and Southern, E. M. et al., Genomics
13, 1008-1017 (1992). Pyrophosphate-based sequencing reaction as
described, e.g., in U.S. Pat. Nos. 6,274,320, 6,258,568 and
6,210,891), may also be used. In some cases, the methods above
require that the nucleic acid attached to the solid surface be
single stranded. In such cases, the unbound strand may be melted
away using any number of commonly known methods such as addition of
NaOH, application of low ionic (e.g., salt) strength, enzymatic
degradation or displacement of the second strand, or heat
processing. Where the solid surface comprises a plurality of beads,
following this strand removal step, the beads can be pelleted and
the supernatant discarded. The beads can then be resuspended in a
buffer, and a sequencing primer or other non-amplification primer
can be added. The primer is annealed to the single stranded
amplification product. This can be accomplished by using an
appropriate annealing buffer and temperature conditions, e.g., as
according to standard procedures in the art.
[0564] The methods of the invention are useful, for example, for
sequencing of an RNA sequence of interest. The sequencing process
can be carried out by processing and amplifying a target RNA
containing the sequence of interest by any of the methods described
herein. Addition of nucleotides during primer extension can be
analyzed using methods known in the art, for example, incorporation
of a terminator nucleotide or sequencing by synthesis (e.g.
pyrosequencing).
[0565] In embodiments wherein the end product is in the form of DNA
primer extension products, in addition to the nucleotides, such as
natural deoxyribonucleotide triphosphates (dNTPs), that are used in
the amplification methods, appropriate nucleotide triphosphate
analogs, which may be labeled or unlabeled, that upon incorporation
into a primer extension product effect termination of primer
extension, may be added to the reaction mixture. Preferably, the
dNTP analogs are added after a sufficient amount of reaction time
has elapsed since the initiation of the amplification reaction such
that a desired amount of second primer extension product or
fragment extension product has been generated. Said amount of the
time can be determined empirically by one skilled in the art.
[0566] Suitable dNTP analogs include those commonly used in other
sequencing methods and are well known in the art. Examples of dNTP
analogs include dideoxyribonucleotides. Examples of rNTP analogs
(such as RNA polymerase terminators) include 3'-dNTP. Sasaki et
al., Biochemistry (1998) 95:3455-3460. These analogs may be
labeled, for example, with fluorochromes or radioisotopes. The
labels may also be labels which are suitable for mass spectroscopy.
The label may also be a small molecule which is a member of a
specific binding pair, and can be detected following binding of the
other member of the specific binding pair, such as biotin and
streptavidin, respectively, with the last member of the binding
pair conjugated to an enzyme that catalyzes the generation of a
detectable signal that could be detected by methods such as
colorimetry, fluorometry or chemiluminescence. All of the above
examples are well known in the art. These are incorporated into the
primer extension product or RNA transcripts by the polymerase and
serve to stop further extension along a template sequence. The
resulting truncated polymerization products are labeled. The
accumulated truncated products vary in length, according to the
site of incorporation of each of the analogs, which represent the
various sequence locations of a complementary nucleotide on the
template sequence.
[0567] Analysis of the reaction products for elucidation of
sequence information can be carried out using any of various
methods known in the art. Such methods include gel electrophoresis
and detection of the labeled bands using appropriate scanner,
sequencing gel electrophoresis and detection of the radiolabeled
band directly by phosphorescence, capillary electrophoresis adapted
with a detector specific for the labels used in the reaction, and
the like. The label can also be a ligand for a binding protein
which is used for detection of the label in combination with an
enzyme conjugated to the binding protein, such as biotin-labeled
chain terminator and streptavidin conjugated to an enzyme. The
label is detected by the enzymatic activity of the enzyme, which
generates a detectable signal. As with other sequencing methods
known in the art, the sequencing reactions for the various
nucleotide types (A, C, G, T or U) are carried out either in a
single reaction vessel, or in separate reaction vessels (each
representing one of the various nucleotide types). The choice of
method to be used is dependent on practical considerations readily
apparent to one skilled in the art, such as the nucleotide tri
phosphate analogs and/or label used. Thus, for example, when each
of the analogs is differentially labeled, the sequencing reaction
can be carried out in a single vessel. The considerations for
choice of reagent and reaction conditions for optimal performance
of sequencing analysis according to the methods of the invention
are similar to those for other previously described sequencing
methods. The reagent and reaction conditions should be as described
above for the nucleic acid amplification methods of the
invention.
The Solid Surface
[0568] In various exemplary embodiments, a solid surface may have a
wide variety of forms, including membranes, slides, plates,
micromachined chips, microparticles, beads and the like. Solid
surfaces may comprise a wide variety of compositions including, but
not limited to, glass, plastic, silicon, alkanethiolate derivatized
gold, cellulose, low cross linked and high cross linked
polystyrene, silica gel, polyamide, and the like, and can have
various shapes and features (e.g., wells, indentations, channels,
etc.). As used herein, the terms "solid surface" and "solid
substrate" are used interchangeably. In some cases these will be
referred to as the surface or the support. The surface can be
hydrophilic or capable of being rendered hydrophilic and may
comprise inorganic powders such as silica, magnesium sulfate, and
alumina; natural polymeric materials, particularly cellulosic
materials and materials derived from cellulose, such as fiber
containing papers, e.g., filter paper, chromatographic paper, etc.;
synthetic or modified naturally occurring polymers, such as
nitrocellulose, cellulose acetate, poly (vinyl chloride),
polyacrylamide, cross linked dextran, agarose, polyacrylate,
polyethylene, polypropylene, poly(4-methylbutene), polystyrene,
polymethacrylate, poly(ethylene terephthalate), nylon, poly(vinyl
butyrate), etc.; either used by themselves or in conjunction with
other materials; glass available as Bioglass, ceramics, metals, and
the like. Natural or synthetic assemblies such as liposomes,
phospholipid vesicles, and cells can also be employed. The surface
can have any one of a number of shapes, such as strip, rod,
particle, including bead, and the like.
[0569] In some embodiments, the solid surface comprises a bead or
plurality of beads. The beads may be of any convenient size and
fabricated from any number of known materials. Example of such
materials include: inorganics, natural polymers, and synthetic
polymers. Specific examples of these materials include: cellulose,
cellulose derivatives, acrylic resins, glass, silica gels,
polystyrene, gelatin, polyvinyl pyrrolidone, co-polymers of vinyl
and acrylamide, polystyrene cross-linked with divinylbenzene or the
like (as described, e.g, in Merrifield, Biochemistry 1964, 3,
1385-1390), polyacrylamides, latex gels, polystyrene, dextran,
rubber, silicon, plastics, nitrocellulose, natural sponges, silica
gels, control pore glass, metals, cross-linked dextrans (e.g.,
Sephadex) agarose gel (Sepharose.TM.), and other solid phase
supports known to those of skill in the art. The beads are
generally about 2 to about 100 um in diameter, or about 5 to about
80 pm in diameter, in some cases, about 10 to about 40, um in
diameter. In some embodiments the beads can be magnetic. Having
magnetic beads can be useful for isolation and purification of the
beads comprising nucleic acids described herein. Other methods to
separate beads can also be used. For example, the capture beads may
be labeled with a fluorescent moiety which would make the nucleic
acid-bead complex fluorescent. The target capture bead complex may
be separated, for example, by flow cytometry or fluorescence cell
sorter.
Attachment of Oligonucleotides to the Solid Surfaces
[0570] One aspect of the invention involves attaching
oligonucleotides to solid surfaces such that the oligonucleotides
can hybridize with polynucleotides produced by the methods of the
invention. Many methods of attaching oligonucleotides to surfaces
are known. In some embodiments, the oligonucleotide is attached
covalently to the solid surface. In some embodiments such attached
oligonucleotides act to capture nucleic acids such as amplification
products, and in some cases such attached oligonucleotides also act
as primers. Methods of attaching oligonucleotides and primers to a
surface are known in the art (see, e.g., Beier et al., 1999,
Nucleic Acids Res. 27(9):1970-1977; Brison et al., 1982, Molecular
and Cellular Biology 2:578 587; Cheung et al., 1999, Nat. Genet. 21
(1 Suppl): 15-19; Chrisey et al., 1996, Nucleic Acids Res.
24(15):3031-3039; Cohen et. al., 1997, Nucleic Acids Res. 1997 Feb.
15; 25(4):911-912; Devivar et al., 1999, Bioorg. Med. Chem. Lett.
9(9):1239-1242; Heme et al., 1997. J. Am. Chem. Soc. 119:8916-8920;
Kumar et al., 2000, Nucleic Acis Res. 28(14):e71; Lipshutz et al.,
1999, Nat. Genet. 21(1 Suppl):20-24; Milner et al., 1997, Nat.
Biotechnol. June; 15(6):537-541; Morozov et al., 1999, Anal. Chem.
71(15):3110-3117; Proudnikov et al., 1998, Anal Biochem.
259(1):34-41; Rasmussen et al., 1991, Anal Biochem. 198(1):138-142;
Rogers et al., 1999, Anal. Biochem. 266(1):23-30; Salo et al.,
1999, Bioconjug Chem. 10(5):815-823; Singh-Gasson et al., 1999,
Nat. Biotechnol. 17(10):974-978, and Pierce Chemical Company
Catalog 1994, pp. 155-200), incorporated herein by reference).
Solid Surfaces in Microreactors or Emulsions
[0571] One aspect of the invention comprises methods and libraries
relating to the attachment of nucleic acids on beads in an
microreactors such as in an emulsion. As used herein, a
microreactor is a small volume of fluid, generally in the volume
range of microliters to nanoliters. The small volumes of the
microreactors are isolated from one another allowing reactions to
occur within each microreactor without significant contamination
from or mixing with other microreactors unless desired. Emulsions
or microemulsions comprise one approach to microreactors, in which
the individual drops each can represent a microreactor.
Microreactors such as emulsions can allow for the clonal
amplification from a single molecule of a nucleic acid to produce a
population of identical nucleic acids within the microreactor.
Generally water-in-oil emulsions, also referred to as inverse
emulsions are used. These emulsions have small water droplets
dispersed in a hydrophobic medium. As used herein, the term oil is
used broadly to refer to a hydrophobic fluid in which aqueous
droplets can be dispersed. various exemplary embodiments a
hydrophobic medium can be can include an oil (e.g., mineral oil,
light mineral oil, silicon oil) or a hydrocarbon (e.g., hexane,
heptane, octane, nonane, decane, etc.) and the like. Fluorinated
hydrocarbons can also be used.
[0572] In some embodiments, solid surfaces can be included within
each microreactor. For example, where a bead comprises the solid
surface, an emulsion can be produced such that the solid surfaces
are included within the emulsion. In some embodiments, an emulsion
is produced such that a plurality of microreactors within the
emulsion have one or fewer beads. Related methods are described,
for example in WO/2004/069849, and WO/05010145A2. In some aspects
of the invention, the nucleic acids are attached to the beads by
binding of a ligand attached to the nucleic acid to a receptor
bound to the solid surface.
[0573] The emulsion can be formed either in bulk, for example, by
mixing, or can be formed by injection, for example using a
microfluidic device. For use with the present invention, beads with
or without attached nucleic acid template are suspended in a
water-in-oil emulsion. Where the amplification reaction requires
thermal cycling, such as a PCR reaction, a heat stable emulsion
must be used. Where isothermal amplification is employed, the need
for thermal stability may be reduced, expanding the types of
emulsions that can be used. In some embodiments, a plurality of the
microreactors include only one template nucleic acid species and
one bead. There may be many droplets that do not contain a template
nucleic acid or which do not contain a bead. Likewise there may be
droplets that contain more than one copy of a template. The
emulsion may be formed according to any suitable method known in
the art. One method of creating emulsion is described below but any
method for making an emulsion may be used. These methods are known
in the art and include adjuvant methods, counter-flow methods,
cross-current methods, rotating drum methods, and membrane methods.
Furthermore, the size of the microcapsules may be adjusted by
varying the flow rate and speed of the components. For example, in
dropwise addition, the size of the drops and the total time of
delivery may be varied. In some embodiments, the emulsion contains
a density of about 3,000 beads encapsulated per microliter.
[0574] Various emulsions that are suitable for biologic reactions
are referred to in Griffiths and Tawfik, EMBO, 22, pp. 24-35
(2003); Ghadessy et al., Proc. Natl. Acad. Sci. USA 98, pp.
4552-4557 (2001); U.S. Pat. No. 6,489,103 and WO 02/22869, each
fully incorporated herein by reference.
[0575] The microreactors should be sufficiently large to encompass
sufficient amplification reagents for the degree of amplification
required. However, the microreactors should be sufficiently small
so that a population of microreactors, each containing a member of
a DNA library, can be amplified by conventional laboratory
equipment. The use of microreactors as described herein allows
amplification of complex mixtures of templates (e.g., genomic DNA
samples or whole cell RNA) without intermixing of sequences, or
domination by one or more templates (e.g., PCR selection bias; see,
Wagner et al., 1994, Suzuki and Giovannoni, 1996; Chandler et al.,
1997, Polz and Cavanaugh, 1998).
[0576] In some embodiments the microreactors are produced such that
a plurality of microreactors have one bead and one copy of a
nucleic acid template. In some embodiments, limiting dilution can
be used to isolate polynucleotides in a manner that is suitable for
clonal amplification. In some embodiments the beads have the one
copy of a nucleic acid bound to the bead. Producing a plurality of
microreactors having only one bead and one nucleic acid template
can be performed by diluting the bead and the nucleic acid template
or the bead with the bound nucleic acid template to a dilution
level at which, on average, each microreactor will contain one or
fewer beads and/or one or fewer nucleic acid templates per
microreactor. Determining the level of dilution at which this
condition occurs can be done by calculation, and by experiment.
Dilution to single bead and/or single molecule is described, for
example in WO/2004/069849. For example, a sample comprising a
plurality of beads, each comprising, on average one or fewer
nucleic acids can be diluted to a concentration such that aliquots
of the diluted sample that can be divided into individual
microreactors (e.g., wells of a multi-well plate) can be predicted
to comprise on average >0 and <1 nucleic acid molecule.
Therefore, a percentage of reaction vessels can be predicted on a
statistical basis (e.g., Poisson distribution) to comprise an
isolated polynucleotide suitable for clonal amplification. Once
isolated within the reaction vessels, polynucleotides can be
amplified by various methods as described herein to yield clonal
amplification.
[0577] Subsequent to amplification, the microreactors can be
accessed in order to remove the contents, for example, the beads,
for later analysis such as by sequencing. One method of accessing
the microreactors comprises breaking the emulsion.
[0578] In one embodiment, following amplification of the nucleic
acid template and the attachment of amplification copies to the
bead, the emulsion is "broken" (also referred to as
"demulsification" in the art). There are many methods of breaking
an emulsion (see, e.g., U.S. Pat. No. 5,989,892 and references
cited therein) and one of skill in the art would be able to select
an appropriate method. In the present invention, one preferred
method of breaking the emulsion uses additional oil to cause the
emulsion to separate into two phases. The oil phase can then be
removed, and a suitable organic solvent (e.g., hexanes) is added.
After mixing, the oil/organic solvent phase is removed.
Subsequently, the aqueous layers above the beads are removed. The
beads are then washed, for example, with a mixture of an organic
solvent and buffer. Suitable organic solvents include alcohols such
as methanol, ethanol, and the like. The beads bound to
amplification products may then be resuspended in aqueous solution
for use, for example, in a sequencing reaction according to known
technologies.
[0579] In some embodiments, because the clonal amplicons that are
produced are isolated as discrete populations, clonal amplification
products can be carried analyzed, for example sequenced, in a
parallel manner. Therefore, in some embodiments, at least at least
100, 500, 1000, 10000, 50000, 100000, 300000, 500000, or 1000000
populations of clonal amplification products can be analyzed in
parallel. The skilled artisan will appreciate that various methods
can be suitable for parallel analysis of clonal amplicons.
Generally, such methods can produce a discrete detectable signal
that can be associated or linked to individual populations of
clonal amplicons.
Determination of Gene Expression Profile
[0580] The amplification methods of the invention can be used for
use in determining the levels of expression of multiple genes in a
sample since the methods described herein are capable of amplifying
multiple target RNAs in the same sample. As described above,
amplification products can be detected and quantified by various
methods, as described herein and/or known in the art. Since RNA is
a product of gene expression, the levels of the various RNA
species, such as whole transcriptome or total RNAs, in a sample is
indicative of the relative expression levels of the various genes
(gene expression profile). Thus, determination of the amount of RNA
sequences of interest present in a sample, as determined by
quantifying amplification products of the sequences, provides for
determination of the gene expression profile of the sample
source.
[0581] The methods of the present invention allow for the storage
and subsequent analysis of samples, allowing for a sample to be
bound to a solid substrate for archiving, then later to be analyzed
by the methods described herein to determine a gene expression
profile. In some embodiments, the sample can be analyzed multiple
times, and stored between analyses.
[0582] Accordingly, the invention provides methods of determining
gene expression profile in a sample, said method comprising:
amplifying single stranded product from at least one RNA sequence
of interest in the sample, using any of the methods described
herein; and determining amount of amplification products of each
RNA sequence of interest, wherein each said amount is indicative of
amount of each RNA sequence of interest in the sample, whereby the
expression profile in the sample is determined. Generally, labeled
products are generated. In one embodiment, the target RNA is mRNA.
It is understood that amount of amplification product may be
determined using quantitative and/or qualitative methods.
Determining the amount of amplification product includes
determining whether amplification product is present or absent.
Thus, an expression profile can includes information about presence
or absence of one or more RNA sequence of interest. "Absent" or
"absence" of product, and "lack of detection of product" as used
herein includes insignificant, or de minimus levels. In some cases,
the methods further provide for clonal amplification of the target
RNA or a subset of the target RNA.
[0583] The methods of expression profiling are useful in a wide
variety of molecular diagnostic, and especially in the study of
gene expression in essentially any mammalian cell (including a
single cell) or cell population. A cell or cell population (e.g. a
tissue) may be from, for example, blood, brain, spleen, bone,
heart, vascular, lung, kidney, pituitary, endocrine gland,
embryonic cells, tumors, or the like. Expression profiling is also
useful for comparing a control (normal) sample to a test sample,
including test samples collected at different times, including
before, after, and/or during development, a treatment, and the
like.
Libraries
[0584] In another embodiment, the invention encompasses a library
comprising a plurality of nucleic acid molecules, wherein each
nucleic acid molecule is separately immobilized to a different
bead. In another embodiment, the invention encompasses a library
comprising a plurality of nucleic acid molecules, wherein each
nucleic acid molecule is separately immobilized to a different bead
and wherein each bead comprises over 100,000 clonal amplification
copies of each nucleic acid molecule, wherein the library is
contained in a single vessel. As examples, the nucleic acid
molecules may be genomic DNA, cDNA, episomal DNA, BAC DNA, or YAC
DNA. The genomic DNA may be animal, plant, viral, bacterial, or
fungal genomic DNA. Preferably, the genomic DNA is human genomic
DNA or human cDNA.
Kits
[0585] One aspect of the invention comprises kits useful for
carrying out the methods of the invention.
[0586] In one aspect, the kit comprises (a) a first primer
comprising a 3'-DNA portion and a 5'-RNA portion, wherein the
3'-DNA portion comprises a random sequence or a specific sequence,
and the 5' RNA portion further comprises sequence (A), (b) a second
primer comprising a 5'-ligand. In some embodiments, the kit may
further comprise (c), an RNA dependent DNA polymerase, (d) a DNA
dependent DNA polymerase with strand displacement activity, (e)
RNase H, (f) an amplification chimeric primer comprising a 3'-DNA
portion and a 5'-RNA portion wherein the sequence of the
amplification primer is the substantial the same sequence as the
(A) sequence, or a combination thereof. In some cases, the kit may
further comprise instructions for the use of said kit.
[0587] In one aspect, the kit comprises (a) a first primer
comprising a 3'-DNA portion and a 5'-RNA portion, wherein the
3'-DNA portion comprises a random sequence and the 5' RNA portion
further comprises sequence (A), (b) a second primer comprising a
5'-ligand, and (c) a chimeric oligonucleotide comprising a 3'-DNA
portion substantially comprising sequence (A) and a 5'-RNA sequence
(C). In some cases, the kit may further comprise (d) RNase H, (e)
an RNA dependent DNA polymerase, (f) a DNA dependent DNA polymerase
with strand displacement activity, (g) a chimeric amplification
primer comprising a 3'-DNA portion and a 5'-RNA portion, wherein
the chimeric amplification primer comprises a sequence which is
substantially the same as sequence (C), or a combination thereof.
In some cases, the kit may further comprise instructions for the
use of said kit.
[0588] In some embodiments the second primer further comprises a
sequence (B) at or near the 5'-end. In some embodiments the kit
further comprises solid support with immobilized ligand binding
component on it surface.
[0589] In some embodiments the kit further comprises solid surface
with an oligonucleotide attached to the surface by the 5'-end and
comprising a sequence (B). In other embodiment the oligonucleotide
attached to the solid surface comprises a sequence hybridizable to
sequence (A) and the oligonucleotide is attached by the 5'-end.
[0590] In some embodiments the kit comprises (a) a first primer
that is a tailed DNA primer comprising a 5'-tail sequence (D), (b)
a second primer that is a chimeric primer comprising a 3'-DNA
portion and a 5'-RNA portion wherein the 5'-end comprises a tail
sequence (E), (c) a third primer which is a tailed primer
comprising a 3'-sequence that comprises a sequence substantially
the same as sequence (D), optionally a 5'-tail sequence (F), and
5'-ligand. In some cases, the kit may further comprise (d) an RNA
dependent DNA polymerase, (e) a DNA dependent DNA polymerase with
strand displacement activity, (f) RNase H, (g) a chimeric
amplification primer comprising a 3'-DNA portion and a 5'-.RNA
portion wherein the chimeric amplification primer comprises a
sequence which is substantially the same a sequence (E), or a
combination thereof. In some cases, the kit may further comprise
instructions for the use of said kit.
[0591] In some embodiments, the kit comprises (a) reagents for
forming an emulsion and (b) a DNA polymerase with substantial
strand displacement activity. Reagents for forming a suitable water
in oil emulsion are known and commercially available for example in
emPCR kits II and III (454/Roche LifeSciences). Said emulsion
forming reagents may include for example
decamethylcyclopentasiloxane, polyphenylmethylsiloxane, water
and/or buffer. In some cases, the kit may further comprise (c) one
or more RNA-DNA chimeric primers, (d) an all DNA primer, (e) a
solid surface such as a bead or set of beads, a substantially
planar array, a well or wells in a plate, or an isolated surface or
set of isolated surfaces, (f) RNase H, (g) a chimeric
oligonucleotide, or a combination thereof. In some cases, the kit
may further comprise instructions for the use of said kit.
[0592] In some embodiments the kit comprises (a) a first primer
that is a tailed DNA primer comprising a 5'-tail sequence (D), (b)
a second primer that is a chimeric primer comprising a 3'-DNA
portion and a 5'-RNA portion wherein the 5'-end comprises a tail
sequence (E), (c) a third primer which is a tailed primer
comprising a 3'-sequence that comprises a sequence substantially
the same as sequence (D), optionally a 5'-tail sequence (F), and
5'-ligand. In some cases, the kit may further comprise (d) an RNA
dependent DNA polymerase, (e) a DNA dependent DNA polymerase with
strand displacement activity, (f) RNase H, (g) a chimeric
oligonucleotide comprising a 3'-DNA sequence (E) and a 5'-RNA
sequence (G), (h) a chimeric amplification primer comprising a
3'-DNA portion and a 5'-RNA portion wherein the chimeric
amplification primer comprises a sequence which is substantially
the same as sequence (G), or a combination thereof. In some cases,
the kit may further comprise instructions for the use of said
kit.
[0593] The 3'-end sequence of the first and second primer may
comprise a specific sequence or a random sequence. In some
embodiments the kits useful for carrying out the methods of the
invention may further comprise an inhibitor of the DNA dependent
DNA polymerase, such as Actinomycin.
[0594] The components of the kits may comprise the same aspects and
embodiments as described above for the components in the
description of methods. For example, the ligands and receptors, the
primers, the enzymes and the oligonucleotides can be those
described herein to carry out the methods of the invention.
Example 1
Clonal Expansion of RNA
Step 1: Synthesis of First Primer Extension Product
[0595] 100 ng of an RNA template is provided. The provided RNA
template is produced from a biological specimen using a
commercially available kit (i.e. Qiagen RNeasy) according to the
manufacturer's instructions. A first primer extension reaction
mixture is assembled comprising a first primer consisting of a 3'
annealing sequence, a portion of which is DNA, and a 5' tail
sequence (A), a portion of which is RNA and the following reagents
in a total volume of 10 .mu.l:
100 ng of RNA template 20 pmol of primer 0.5 .mu.l dNTPs (25
mM)
0.1 .mu.l RNasin
0.1 .mu.l DTT
[0596] 2 .mu.l 5.times.AMV reverse transcriptase reaction buffer
DEPC treated water to 10 .mu.l total volume
[0597] The reaction mixture is incubated for 2 min at 75.degree.
C., and then cooled to 37.degree. C. 1 .mu.l AMV reverse
transcriptase (USB 70041Y, 15U/.mu.l) is added to each reaction and
the reaction mixture is further incubated at this temperature for
60 min. The resulting product is a first primer extension product
of DNA with a 5' RNA tail sequence (A) annealed to an RNA template.
The reaction mixture is heated to 90.degree. C. for 3 min. for
enzyme inactivation.
Step 2: Synthesis of Second Primer Extension Product (Formation of
(A)/(A') RNA/DNA Heteroduplex)
[0598] The first primer extension product is mixed with 10 .mu.l of
the second primer extension mixture containing the following:
1 .mu.l 10.times.Klenow reaction buffer 0.1 .mu.l dNTPs (25 mM) 0.5
.mu.l Klenow Exonuclease free (USB 70057Y 10U/.mu.l)) DNA
polymerase 8.4 .mu.l water
[0599] The reaction mixture is incubated for 30 min at 37.degree.
C., followed by heating to 75.degree. C. for 5 min to stop the
reaction by inactivating the enzymes. The resulting primer
extension products comprise a double stranded DNA product with a
DNA/RNA heteroduplex at one end of sequence (A')/(A) and a partial
duplex with a tailed B sequence at the other end.
Step 3: Cleavage of DNA/RNA Hybrid
[0600] To the second primer extension product reaction mixture is
added 0.02 U Hybridase (RNase H) and the reaction mixture is
incubated at 50.degree. C. for 60 min and cooled to 4.degree. C.
The resulting RNase H digested primer extension product reaction
mixture comprises a first primer extension product of DNA without a
5' RNA tail sequence (A) annealed to the second primer extension
product of DNA with a 5' tail sequence (B) and a 3' annealing
sequence (A').
Step 4: Annealing of Chimeric Oligonucleotide
[0601] Next, a chimeric oligonucleotide comprising a 3' annealing
sequence (A), a portion of which is DNA, and a 5' tail sequence
(C), a portion of which is RNA is added to the reaction mixture and
annealed to the complementary sequence (A'). The annealing step is
carried out at 50.degree. C. for 10 min. and the reaction is cooled
to 4.degree. C.
Step 5: Extension of the Second Primer Extension Product along the
Chimeric Oligonucleotide
[0602] To the reaction mixture is added 2.5 units of Exonuclease
Free Klenow polymerase and the reaction mixture is incubated for 30
min at 37.degree. C., followed by heating to 75.degree. C. for 5
min to stop the reaction by inactivating the enzymes. The resulting
first primer extension product comprises a sequence (A), a 5' tail
sequence (C), a portion of which is RNA, and a 3' tail sequence
(B') of DNA. The product is a double stranded DNA with a DNA-RNA
heteroduplex at one end (C-C').
Step 6: Attachment to a Solid Support
[0603] To the reaction mixture is added a solid support in the form
of Polystyrene beads which are derivatized with a short DNA
oligonucleotide comprised of sequence B. The reaction mixture is
heated to 98.degree. C. for 2 minutes and cooled to room
temperature to allow for annealing of the first primer extension
product to the solid support. The beads are washed in 1.times.SSC
to remove the second primer extension product. To the beads is
added 1.times. Thermopole buffer (NEB), dNTPs, 1.times.NEB BSA, Bst
DNA polymerase, and water in amounts known in the art to promote
DNA polymerase activity. The reaction mixture is incubated in a
thermocycler at 25.degree. C. for 5 minutes, 50.degree. C. for 30
min, 95.degree. C. for 5 min, and then cooled to 4.degree. C. The
resulting double stranded product, illustrated as the final product
in FIG. 17, is a SPIA substrate suitable for clonal expansion via
SPIA amplification. Enzymes, buffers and salts are removed by
washing the solid-support-bound SPIA substrate and resuspending in
a suitable volume of water.
Step 7: Clonal Expansion
[0604] SPIA amplification is carried out using buffer and enzyme
mixtures from NuGEN's WT-Ovation Pico RNA amplification system
(NuGen Technologies Inc, San Carlos Calif.). In addition, the SPIA
amplification uses a chimeric amplification primer that has a 5'
RNA sequence and a short 3' DNA sequence (approximately 7 base
pairs) and is complementary to the sequence (C') of the SPIA
substrate produced in step 6. The SPIA amplification is set up as
follows:
3 .mu.l Amplification primer (50 .mu.M stock) 10 .mu.l
Solid-support-bound SPIA substrate 17 .mu.l water 40 .mu.l
amplification buffer (WT-Ovation Pico System)
20 .mu.l Amplification Enzyme Mix (WT-Ovation Pico System)
[0605] The reactions are incubated in a thermocycler at 50.degree.
C. for 60 min, followed by 95.degree. C. for 5 min, and cooled
down. The amplification products are generated in close proximity
to the remaining unhybridized B-sequence oligonucleotides on the
solid support on which the parent SPIA substrate is bound and are
therefore also immobilized on the same solid support as they are
generated. This process ultimately provides a plurality of solid
supports (e.g. beads), each bead with a clonally expanded sequence
hybridized therein, such that the hybridized sequence has a known
sequence (B') and (C) at each end. The product of this example is
useful for such methods as archiving of nucleic acid sequences,
reducing the complexity of nucleic acid samples, and providing a
set of clonally expanded sequences on beads suitable for use in
next generation sequencing platforms such as the SOLiD system by
Applied Biosystems.
Example 2
Diagnosis and Prognosis of Cancer
[0606] A suggested course of treatment can be determined by RNA
expression analysis of a tumor biopsy. A needle biopsy is performed
on a subject to obtain tissue from the suspicious mass for further
analysis. The biopsied tissue recovered from the subject is
processed to extract and purify total RNA using a commercially
available Qiagen RNeasy kit according to the manufacturer's
instructions.
[0607] 500 pg of total RNA representing at least a portion of the
transcriptome of the biopsied material is amplified by the methods
of the present invention as described briefly herein. To the RNA in
a reaction mixture is added: 100 pmol of a first primer comprising
random first primer and a poly dT first primer, a 5' segment and a
3' segment, a portion of the 5' segment comprising RNA, and a
portion of the 3' segment comprising DNA. The 3' DNA segment of the
random first primer further comprises an annealing sequence that
comprises random hexamers. The 5' RNA segment of the random first
primer comprises a tag sequence (A).
[0608] To the reaction mixture 10 pmol of a poly-T first primer is
also added comprising a 5' segment and a 3' segment, a portion of
the 5' segment comprising RNA, and a portion of the 3' segment
comprising DNA in the reaction mixture. The 3' DNA segment of this
poly-T first primer further comprises an annealing sequence that
hybridizes to and is complementary to a portion of the poly A tail
of mRNA transcripts present in the total RNA of the reaction
mixture, and the 5' RNA segment comprises a tag sequence (A).
[0609] The volume of the reaction mixture is adjusted to 10 .mu.l
with DEPC treated water, and then the reaction mixture is heated to
75.degree. C. for 2 minutes and cooled. First strand synthesis is
carried out using the buffer and enzyme reagents provided with the
WT-Ovation Pico RNA Amplification kit, and incubation conditions
are as described in the User Guide and Quick Protocol:
(http://www.nugeninc.com/tasks/sites/nugen/assets/File/user_guides/usergu-
ide_wt_ov_pico.pdf and
http://www.nugeninc.com/tasks/sites/nugen/assets/File/quick_protocols/qp_-
wt_ov_pico.pdf.).
[0610] The first primer extension product is mixed with 10 .mu.l of
the second primer extension mixture containing the following:
[0611] second strand cDNA synthesis enzyme mixture and second
strand buffer mixture from the NuGEN's WT-Ovation Pico
amplification system (as above) 20 pmol of a second primer
comprising a 5' segment and a 3' segment, a portion of the 5'
segment comprising a tag sequence (B), and a portion of the 5'
segment comprising an annealing sequence.
[0612] The reaction mixture is incubated under the conditions
described in the use Guide for the WT-Ovation Pico RNA
Amplification system, followed by heating to 75.degree. C. for 5
minutes to stop the reaction by inactivating the enzymes. The
resulting primer extension products comprise a partial double
stranded DNA product with a DNA/RNA heteroduplex at one end of a
sequence (A')/(A). The first primer extension product in the
reaction mixture is then digested using exonuclease 1 (0.5 .mu.l at
37.degree. C. for 30 min.) followed by inactivation of the enzymes
(80.degree. C. for 20 minutes). The first and second primer
extension products are then purified using Agencourt magnetic beads
as per the manufacturer's instructions (User Guide as above).
[0613] Isothermal linear amplification (SPIA) is then carried out
in a reaction mixture containing the above purified reaction
products (10 .mu.l), 2 .mu.l chimeric amplification primer (100
.mu.M stock solution), 18 .mu.l water, 40 .mu.l amplification
buffer and 20 .mu.l amplification enzyme mixture as provided in the
WT-Ovation Pico RNA Amplification System (NuGen Technologies). The
amplification is carried out according to the instructions provided
for the WT-Ovation Pico RNA Amplification System.
[0614] The amplified product is then analyzed and quantitated by
Real Time qPCR with SYBR Green, using an MJ Opticon thermocycler.
Amplification reactions are diluted 1:100 in Tris-EDTA and 2 .mu.l
of the diluted DNA are analyzed using primer pairs specific for
abl, ras, and her2 in three separate reactions.
[0615] The results of the qPCR are analyzed to determine that abl
is overexpressed in the cells of the suspicious mass. The results
are combined with immunohistochemical and cytological analysis to
determine a suggested course of therapy.
Alternatively, a gene expression profile of the sample can be
obtained using microarrays such as GeneChip (Affymetrix). The
amplified reaction products are subjected to fragmentation and
labeling with Biotin using NuGEN's Ovation-F/L reagents and
protocol. The fragmented and labeled products are used for
hybridization to GeneChip according to the manufacturer protocol.
The resulting hybridization data provides a gene expression profile
of the sample.
Example 3
Personal Genomics
[0616] An individual is tested by a personal genomics business
using the methods of the present invention for single nucleotide
polymorphisms (SNPs) within the BRCA1, BRCA2, p53, MPO, NAT1, NAT2,
and ras coding regions that are related to increased risks for
specific types of cancer.
[0617] The individual supplies a small sample of tissue (i.e. a
cheek swab) to the personal genomics business. Genomic DNA from the
sample of tissue is isolated using a commercially available kit
(i.e. Promega's Wizard.RTM. Genomic DNA Purification Kit),
according to the manufacturer's protocol.
[0618] 1 to 10 ng of purified genomic DNA is used to clonally
amplify the sequences corresponding to the genomic regions with
known, cancer related, SNPs of the BRCA1, BRCA2, p53, MPO, NAT1,
NAT2, and ras genes on a solid support (i.e. a bead). The target
sequences are clonally amplified by isothermal linear amplification
using the steps shown in FIG. 13 steps I(b), II(b), and III(b);
FIG. 17 steps IV to X; and FIG. 18 of the present application.
[0619] The set of beads comprising the clonally amplified sequences
are loaded onto a SOLiD.TM. Analyzer and the sequences of the
regions of interest are determined using the manufacturer's
protocol using a primer complementary to the sequence (A) on the 5'
end of the amplified product.
[0620] The resulting sequences are used to determine the presence
or absence of SNPs related to cancer in the genes of interest. A
report is generated that includes the SNPs identified, the impact
of the SNPs on lifetime risk of developing specific diseases or
conditions, and suggestions for prophylactic or therapeutic
interventions.
Example 4
Analysis of Fetal DNA
[0621] A fetal sample is obtained by amniocentesis. DNA is
extracted and purified from the sample using a commercially
available kit (i.e. Promega's Wizard.RTM. Genomic DNA Purification
Kit). The DNA is attached to a solid support according to the
method outlined in FIG. 19 and clonally amplified according to the
method outlined in FIG. 18. The set of beads comprising the
clonally amplified sequences are loaded onto a Genome Sequencer FLX
Titanium Series from Roche/454 Life Sciences and the sequences of
the regions of interest are determined using the manufacturer's
protocol using a primer complementary to the sequence (A) on the 5'
end of the amplified product.
[0622] The resulting sequences are used to determine the presence
or absence of SNPs related to prenatal diseases or conditions. A
report is generated that includes the SNPs identified, the impact
of the SNPs on lifetime risk of developing specific diseases or
conditions, and suggestions for prophylactic or therapeutic
interventions.
[0623] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
* * * * *
References