U.S. patent application number 12/099670 was filed with the patent office on 2009-03-12 for global amplification using a randomly primed composite primer.
Invention is credited to Nurith Kurn, Shenglong Wang.
Application Number | 20090068709 12/099670 |
Document ID | / |
Family ID | 33303107 |
Filed Date | 2009-03-12 |
United States Patent
Application |
20090068709 |
Kind Code |
A1 |
Kurn; Nurith ; et
al. |
March 12, 2009 |
Global Amplification Using A Randomly Primed Composite Primer
Abstract
The invention relates to the field of polynucleotide
amplification. More particularly, the invention provides methods,
compositions and kits for amplification of (i.e., making multiple
copies of) a multiplicity of different polynucleotide template
sequences using a randomly primed RNA/DNA composite primer.
Inventors: |
Kurn; Nurith; (Palo Alto,
CA) ; Wang; Shenglong; (San Mateo, CA) |
Correspondence
Address: |
WILSON SONSINI GOODRICH & ROSATI
650 PAGE MILL ROAD
PALO ALTO
CA
94304-1050
US
|
Family ID: |
33303107 |
Appl. No.: |
12/099670 |
Filed: |
April 8, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10824829 |
Apr 14, 2004 |
7402386 |
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12099670 |
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60462962 |
Apr 14, 2003 |
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60462965 |
Apr 14, 2003 |
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Current U.S.
Class: |
435/91.5 |
Current CPC
Class: |
C12P 19/34 20130101;
C12Q 1/6844 20130101; C12Q 1/6844 20130101; C12Q 2525/179 20130101;
C12Q 2525/203 20130101; C12Q 1/6853 20130101; C12Q 2537/143
20130101 |
Class at
Publication: |
435/91.5 |
International
Class: |
C12P 19/34 20060101
C12P019/34 |
Claims
1. A method for amplification of a template polynucleotide,
comprising: (a) incubating a reaction mixture, said reaction
mixture comprising: (i) a template polynucleotide; (ii) a first
primer, wherein the first primer is a composite primer that is
hybridizable to a multiplicity of template polynucleotide sites,
wherein the composite primer comprises an RNA portion and a 3' DNA
portion; (iii) a DNA-dependent DNA polymerase; and (iv) an
RNA-dependent DNA polymerase; wherein the incubation is under
conditions that permit first primer random hybridization to the
template polynucleotide, and primer extension, whereby a complex
comprising a RNA/DNA heteroduplex is generated; and (b) incubating
a reaction mixture, said reaction mixture comprising (i) at least a
portion of the reaction products generated according to step (a);
(ii) an amplification primer, wherein said amplification primer is
a composite primer comprising an RNA portion and a 3' DNA portion;
(iii) an DNA-dependent DNA polymerase; and (iv) an agent that
cleaves RNA from an RNA/DNA hybrid; wherein the incubation is under
conditions that permit RNA cleavage, primer hybridization, primer
extension, and displacement of the primer extension product when
its RNA is cleaved and another amplification primer binds to the
template and is extended by strand displacement, whereby multiple
copies of a polynucleotide amplification product are generated.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S. Ser.
No. 10/824,829, filed Apr. 14, 2004, which claims the benefit of
U.S. Provisional Patent Application Ser. Nos. 60/462,962, filed
Apr. 14, 2003 and 60/462,965, filed Apr. 14, 2003, which
applications are incorporated herein by reference in their
entirety.
TECHNICAL FIELD
[0002] The invention relates to the field of polynucleotide
amplification. More particularly, the invention provides methods,
compositions and kits for amplifying (i.e., making multiple copies
of) a multiplicity of different polynucleotide template sequences
using a randomly primed RNA/DNA composite primer.
BACKGROUND ART
[0003] The quality and quantity of nucleic acid (e.g. genomic DNA)
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.
[0004] 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, including RNAs that are not polyadenylated, are
of great importance.
[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.
[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. Although these methods are most
commonly used, they have some significant drawbacks: a) the
reactions require thermocycling; b) the products are double
stranded, thus rendering them less accessible to binding to probes;
and c) the reactions are prone to contamination with products of
prior amplification, thus requiring strict containment of reaction
mixtures. Other current RNA amplification methods use initiation of
replication of mRNA from the poly-A tail at their 3' ends. However,
not all RNA transcripts have a mRNA tail (for example, prokaryotic
RNAs and non-coding RNAs). In addition, due to sample preparation
procedures, the RNA transcript structural integrity is compromised.
Thus, it may be desirable in certain circumstances to use RNA
amplification methods that do not require initiation of replication
at the defined poly-A tail. Although analysis of non-amplified RNA
is feasible, a significant amount of starting RNA would be
required. 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.
[0007] Therefore, there is a need for improved amplification
methods, particularly methods which can globally amplify DNA or RNA
polynucleotide targets. The invention described herein fulfills
this need.
[0008] All references cited herein, including patent applications
and publications, are incorporated by reference in their
entirety.
DISCLOSURE OF THE INVENTION
[0009] The invention provides methods, compositions, and kits for
isothermal global amplification using a randomly hybridized RNA/DNA
composite primer, as well as applications of the amplification
methods.
[0010] Accordingly, in one aspect, the invention provides methods
for amplification of a template polynucleotide, said methods
comprising: (a) incubating a reaction mixture, said reaction
mixture comprising: (i) a template polynucleotide; (ii) a first
primer, wherein the first primer is a composite primer that is
hybridizable to a multiplicity of template polynucleotide sites,
wherein the composite primer comprises an RNA portion and a 3' DNA
portion; (iii) a DNA-dependent DNA polymerase; and (iv) an
RNA-dependent DNA polymerase (which may be present as a separate
enzyme or as an enzyme comprising both DNA-dependent DNA polymerase
and RNA-dependent DNA polymerase activities); wherein the
incubation is under conditions that permit composite primer random
hybridization, primer extension and, in some embodiments,
displacement of the primer extension product from template
polynucleotide, whereby a complex comprising an RNA/DNA partial
heteroduplex is generated; and (b) incubating a reaction mixture,
said reaction mixture comprising (i) the reaction products
generated according to step (a) (or an aliquot thereof); (ii) a
composite primer (which may be the same as the first primer, or may
be a different primer), wherein the composite primer comprises an
RNA portion and a 3' DNA portion; (iii) an DNA-dependent DNA
polymerase; and (iv) an agent (such as an enzyme) that cleaves RNA
from an RNA/DNA hybrid; wherein the incubation is under conditions
that permit RNA cleavage from an RNA/DNA heteroduplex, primer
hybridization, primer extension, and displacement of the primer
extension product from the complex of (a) when its RNA is cleaved
and another composite primer binds to the template and is extended,
whereby multiple copies of a polynucleotide (generally, DNA)
amplification product are generated. In embodiments wherein the
template polynucleotide is RNA, the reaction mixture of step (a)
further comprises (v) an agent (such as an enzyme) that cleaves RNA
from an RNA/DNA hybrid, whereby template RNA is cleaved form the
complex comprising template RNA and first primer extension product.
In some embodiments, the reaction mixture of step (b) comprises the
reaction mixture according to step (a) (or an aliquot thereof). In
other embodiments, step (b) is initiated by the addition of an
agent that cleaves RNA from a partial RNA/DNA heteroduplex (such as
RNase H), and optionally, a DNA-dependent DNA polymerase, to the
reaction mixture of step (a). In some embodiments, the reaction
mixture of step (a) and/or (b) further comprises auxiliary primers.
In some embodiments (generally embodiments in which the template
polynucleotide is DNA), the RNA-dependent DNA polymerase may be
omitted from reaction mixture (a).
[0011] In another aspect, the invention provides methods for
amplification of a template polynucleotide by incubating a reaction
mixture, said reaction mixture comprising: (a) a complex comprising
a RNA/DNA partial heteroduplex, wherein the complex is generated by
incubating a first reaction mixture, said first reaction mixture
comprising: (i) a polynucleotide template; (ii) a first primer;
wherein the first primer is a composite primer, the composite
primer comprising a RNA portion and a 3' DNA portion; and wherein
the composite primer is capable of hybridizing to a multiplicity of
template polynucleotide sites; (iii) a DNA-dependent DNA
polymerase; and (iv) an RNA-dependent DNA polymerase; wherein the
incubation is under conditions that permit composite primer random
hybridization, primer extension and displacement of the primer
extension product from template polynucleotide, whereby a complex
comprising an RNA/DNA partial heteroduplex is generated; (b) a
composite primer, wherein the composite primer comprises an RNA
portion and a 3' DNA portion; (c) a DNA-dependent DNA polymerase;
and (d) an enzyme that cleaves RNA from an RNA/DNA hybrid; wherein
the incubation is under conditions that permit primer
hybridization, primer extension, RNA cleavage from an RNA/DNA
heteroduplex, and displacement of composite primer from the complex
of step (a) when its RNA is cleaved and another composite primer
binds and is extended, whereby multiple copies of a polynucleotide
amplification product are generated. In some embodiments, the
reaction mixture and/or first reaction mixture further comprises
auxiliary primers. In some embodiments wherein the template
polynucleotide is RNA, template RNA in step (a) is cleaved
following primer extension via conditions or agents promoting
cleavage. In some embodiments, the first reaction mixture further
comprises: (v) an agent (such as an enzyme) that cleaves RNA from
an RNA/DNA hybrid, whereby template RNA is cleaved from the complex
comprising template RNA and composite primer extension product. In
some embodiments (generally those in which the template
polynucleotide is DNA), the complex comprising a RNA/DNA partial
heteroduplex may be generated without the use of RNA-dependent DNA
polymerase.
[0012] In another aspect, the invention provides methods for
amplification of a template polynucleotide by incubating a reaction
mixture, said reaction mixture comprising: (a) a complex of a first
primer extension product and a second primer extension product,
wherein the first primer extension product is generated by
extension of a randomly primed first primer hybridized to target
polynucleotide with a DNA polymerase, wherein the first primer is a
composite primer comprising an RNA portion and a 3' DNA portion,
wherein the first primer is capable of hybridizing to a
multiplicity of template polynucleotide sites, and wherein the
second primer extension product is generated by extension of a
second primer hybridized to the first primer extension product; (b)
a composite primer that is hybridizable to the second primer
extension product, wherein the composite primer comprises an RNA
portion and a 3' DNA portion; (c) a DNA-dependent DNA polymerase;
and (d) an enzyme that cleaves RNA from an RNA/DNA hybrid; wherein
the incubation is under conditions that permit primer
hybridization, primer extension, RNA cleavage from an RNA/DNA
heteroduplex, and displacement of composite primer from the complex
of step (a) when its RNA is cleaved and another composite primer
binds and is extended, whereby multiple copies of a polynucleotide
amplification product are generated. In some embodiments wherein
the template polynucleotide is RNA, the first primer extension
product is generated by extension of a randomly primed first primer
hybridized to target RNA with a RNA-dependent DNA polymerase. In
some embodiments, the first primer extension product and/or the
second primer extension product are generated in the presence of
auxiliary primers.
[0013] In another aspect, the invention provides methods for
amplification of a polynucleotide template comprising: (a) random
priming of polynucleotide template strand with a composite primer;
wherein the composite primer comprises an RNA portion and a 3' DNA
portion, and wherein the composite primer is capable of hybridizing
to a multiplicity of template polynucleotide sites; and (b)
incubating template strand in the presence of a DNA-dependent DNA
polymerase, an RNA-dependent DNA polymerase, and an agent that
cleaves RNA from an RNA/DNA, whereby multiple copies of
polynucleotide amplification product are generated via primer
extension and strand displacement. In some embodiments, random
priming occurs in the presence of a DNA polymerase. In some
embodiments, auxiliary primers are included in step (a) and/or step
(b). In some embodiments (generally those in which the
polynucleotide template is DNA), the RNA-dependent DNA polymerase
is omitted from the incubation.
[0014] In another aspect, the invention provides methods for
amplification of RNA template polynucleotides which operate as
follows: a multiplicity of template polynucleotide sequences are
amplified by incubating a reaction mixture, the reaction mixture
comprising: (a) a complex of a first primer extension product and a
second primer extension product, wherein the first primer extension
product is generated by extension of a first primer hybridized to
template RNA strand with an RNA-dependent DNA polymerase, wherein
the first primer is a composite primer comprising an RNA portion
and a 3' DNA portion, wherein the first primer is capable of
hybridizing to a multiplicity of sites on template RNA; wherein the
second primer extension product is generated by extension of a
second primer hybridized to the first primer extension product; and
wherein RNA from the complex of first and second primer extension
product is cleaved (using e.g., an enzyme that cleaves RNA from an
RNA/DNA hybrid or conditions permitting cleavage, such as heat
and/or alkaline conditions); (b) a composite primer that is
hybridizable to the second primer extension product, wherein the
composite primer comprises an RNA portion and a 3' DNA portion; (c)
a DNA-dependent DNA polymerase; and (d) an enzyme that cleaves RNA
from an RNA/DNA hybrid; wherein the incubation is under conditions
that permit primer hybridization, primer extension, RNA cleavage
from an RNA/DNA heteroduplex, and displacement of composite primer
from the complex of step (a) when its RNA is cleaved and another
composite primer binds to the second primer extension product and
is extended, whereby multiple copies of polynucleotide
amplification product are generated. In some embodiments, the
complex of step (a) is generated in the presence of auxiliary
primers. In some embodiments, the second primer comprises
fragment(s) of cleaved RNA template.
[0015] In another aspect, the invention provides methods for
amplification of a template polynucleotide by (a) randomly priming
a template polynucleotide with a first primer, wherein said first
primer is a composite primer that is hybridizable to a multiplicity
of template polynucleotide sites, wherein the composite primer
comprises a RNA portion and a 3' DNA portion; (b) extending the
first primer with a DNA polymerase; (c) cleaving RNA from the first
primer with an agent that cleaves RNA from a RNA/DNA heteroduplex;
(d) hybridizing an amplification primer to the template
polynucleotide, wherein said amplification primer is a composite
primer comprising a RNA portion and a 3' DNA portion; (e) extending
the hybridized amplification primer by strand displacement DNA
synthesis; and (f) cleaving RNA from the amplification primer with
an agent that cleaves RNA from a RNA/DNA heteroduplex, such that
another amplification primer can hybridize and be extended, whereby
multiple copies of a polynucleotide amplification product are
generated.
[0016] In another aspect, the invention provides methods for
amplification of a template polynucleotide by incubating a reaction
mixture including: (a) a polynucleotide template strand; (b) a
first primer, wherein said first primer is a composite primer
comprising a RNA portion and a 3' DNA portion, and wherein the
first primer is capable of hybridizing to a multiplicity of
template polynucleotide sites; (c) a DNA-dependent DNA polymerase;
(d) a RNA-dependent DNA polymerase; and (e) an agent that cleaves
RNA from a RNA/DNA heteroduplex, whereby multiple copies of
polynucleotide amplification product are generated by primer
extension and strand displacement. In some embodiments (generally
those embodiments in which the polynucleotide template is DNA), the
RNA-dependent DNA polymerase is omitted from the reaction
mixture.
[0017] As is clear to one skilled in the art, reference to
production of copies of a polynucleotide (e.g., DNA or RNA)
template or copies of a polynucleotide sequence complementary to a
polynucleotide template refers to products that may contain,
comprise or consist of such sequences. As is evident to one skilled
in the art, aspects that refer to combining and incubating the
resultant mixture also encompasses method embodiments which
comprise incubating the various mixtures (in various combinations
and/or subcombinations) so that the desired products are
formed.
[0018] Various embodiments of the composite primer(s) used in the
methods of the invention are described herein. For example, in some
embodiments, the RNA portion of a composite primer is 5' with
respect to the 3' DNA portion. In still other embodiments, the 5'
RNA portion is adjacent to the 3' DNA portion. In other
embodiments, the RNA portion of the composite primer consists of 7
to about 20 nucleotides and the DNA portion of the composite primer
consists of about 5 to about 20 nucleotides. In still other
embodiments, the RNA portion of the composite primer consists of
about 10 to about 20 nucleotides and the DNA portion of the
composite primer consists of about 7 to about 20 nucleotides. In
some embodiments the composite primer is selected from the
following composite primers: 5'-GACGGAUGCGGUCUdCdCdAdGdTdGdT-3 (SEQ
ID NO:1); and 5'-CGUAUUCUGACGACGUACUCdTdCdAdGdCdCdT-3' (SEQ ID
NO:2), wherein italics denote ribonucleotides and "d" denotes
deoxyribonucleotides.
[0019] In some embodiments, the composite primer comprises random
sequence or partially randomized sequence, although certain
embodiments (such as certain embodiments wherein the template
polynucleotide is RNA) exclude the use of primers comprising random
or partially random sequence. In embodiments utilizing a composite
primer with random or partially random sequence, the composite
primer may be a population or pool of different primers comprising
at least 2, at least 3, at least 4, at least 5, at least 10, at
least 15, at least 20, at least 30, at least 40, at least 50, or at
least 100 different sequences. In other embodiments, the composite
primer contains one or more "degenerate" nucleotides that is able
to hybridize to multiple different nucleotide bases (e.g., inosine,
which is able to hybridize to all four canonical bases).
[0020] In some embodiments, the composite primer that hybridizes to
target polynucleotide (such as mRNA or genomic DNA) and the
composite primer used during single primer isothermal amplification
(i.e., phase (b) of the methods) are the same. In some embodiments,
the composite primer that hybridizes to target polynucleotide (such
as mRNA or genomic DNA) and the composite primer used during single
primer isothermal amplification are different. In some embodiments,
two (or more) different composite primers that hybridize to target
polynucleotide are used in the methods of the invention.
[0021] The methods are applicable to amplifying any target
polynucleotide, including, for example, DNA (such as genomic DNA,
including human and other mammalian genomic DNA) and RNA (such as
total RNA, mRNA, noncoding RNA and ribosomal RNA). One or more
steps may be combined and/or performed sequentially (often in any
order, as long as the requisite product(s) are able to be formed),
and, as is evident, the invention includes various combinations of
the steps described herein. It is also evident, and is described
herein, that the invention encompasses methods in which the
initial, or first, step is any of the steps described herein. For
example, the methods of the invention do not require that the first
step be random hybridization of composite primer. Methods of the
invention encompass embodiments in which later, "downstream" steps
are an initial step.
[0022] The enzymes which may be used in the methods and
compositions are described herein. For example, the agent (such as
an enzyme) that cleaves RNA may be an RNase H, and the
RNA-dependent DNA polymerase may be reverse transcriptase. The
RNA-dependent DNA polymerase may comprise an RNase H enzyme
activity, or separate enzymes may be used. Similarly, a DNA
polymerase may comprise both RNA-dependent and DNA-dependent DNA
polymerase enzyme activities, or separate enzymes may be used. A
DNA-dependent DNA polymerase, an RNA-dependent DNA polymerase, and
the enzyme that cleaves RNA can also be the same enzyme, or
separate enzymes comprising each of these activities may be
used.
[0023] In some embodiments, methods of the invention are used to
generate labeled polynucleotide products (generally DNA products).
In some embodiments of methods for generating labeled DNA products,
at least one type of dNTP used is a labeled dNTP. In other
embodiments of methods for generating labeled DNA products, a
labeled composite primer is used.
[0024] The invention also provides methods which employ (usually,
analyze) the products of the amplification methods of the
invention, such as detection of sequence alteration(s) (e.g.,
genotyping, nucleic acid mutation detection, analysis of splice
variants, and the like); determining presence or absence of a
sequence of interest; quantifying a sequence of interest; gene
expression profiling; subtractive hybridization; preparation of
subtractive hybridization probe; differential amplification;
preparation of libraries (including genomic, cDNA and differential
expression libraries); preparation of an immobilized nucleic acid
(which can be a nucleic acid immobilized on a microarray, preparing
labeled probes for analysis on arrays (including high density
arrays) for the detection and quantification of sequences of
interest, including, for example, sequence determination, detecting
sequence variation and genotyping; comparative genome
hybridization; detection and/or identification of novel RNAs; and
characterizing nucleic acids using the amplification nucleic acid
products generated by the methods of the invention.
[0025] Any of the methods of the invention can be used to generate
polynucleotide products that are suitable for characterization of a
polynucleotide sequence of interest in a sample. In one embodiment,
the invention provides methods for characterizing (for example,
detecting (presence or absence) and/or quantifying) a
polynucleotide sequence of interest comprising: (a) amplifying a
target polynucleotide by any of the methods described herein; and
(b) analyzing the amplification products. Step (b) of analyzing the
amplification products can be performed by any method known in the
art or described herein, for example by detecting and/or
quantifying amplification products that are hybridized to a probe.
These amplification products may or may not be labeled. Any of the
methods of the invention can be used to generate polynucleotide
(such as DNA) products that are labeled by incorporating labeled
nucleotides and/or labeled composite primers into appropriate
step(s) of the methods. These labeled products are particularly
suitable for quantification and/or identification by methods known
in the art, which include the use of arrays such as cDNA
microarrays and oligonucleotide arrays. In one aspect, the
invention provides a method of characterizing a polynucleotide
sequence of interest, comprising (a) amplifying a target
polynucleotide by a method described herein to generate labeled
polynucleotide products; and (b) analyzing the labeled
polynucleotide products. In some embodiments, the step of analyzing
polynucleotide products comprises determining amount of said
products, whereby the amount of the polynucleotide sequence of
interest present in a sample is quantified.
[0026] The amplification products can also serve as template for
further analysis such as sequence analysis, polymorphism detection
(including multiplex SNP detection) using, e.g., oligonucleotide
ligation-based assays, analysis using Invader, Cleavase or limited
primer extension, and other methods known in the art. For methods
that generally require larger volumes of input material, the
methods of the invention may be used to "pre" amplify a pool of
polynucleotides to generate sufficient input material for
subsequent analysis.
[0027] In another embodiment, the polynucleotide products can be
analyzed by, for example, contacting them with at least one probe.
In some embodiments, the at least one probe is provided as a
microarray. The microarray can comprise at least one probe
immobilized on a solid or semi-solid substrate fabricated from a
material selected from the group consisting of paper, glass,
ceramics, plastic, polypropylene, polystyrene, nylon,
polyacrylamide, nitrocellulose, silicon, other metals, and optical
fiber. A probe can be immobilized on the solid or semi-solid
substrate in a two-dimensional configuration or a three-dimensional
configuration comprising pins, rods, fibers, tapes, threads, beads,
particles, microtiter wells, capillaries, and cylinders.
[0028] In another aspect, the invention provides methods of
determining a gene expression profile in a sample, the methods
comprising (a) amplifying RNA template in a sample using any of the
methods described herein; and (b) determining an amount of
amplification products of each RNA sequence of interest in the
sample, whereby the gene expression profile of the sample is
determined. The invention further provides methods of determining a
gene expression profile by determining an amount of amplification
products of each RNA sequence of interest in a sample, the sample
comprising multiple copies of RNA template amplified by any of the
methods described herein, whereby the gene expression profile of
the sample is determined.
[0029] Additionally, the invention also provides methods for
archiving polynucleotide templates. Because the amplification
methods of the invention provide representative amplification of
the sequences of the template polynucleotide, amplified product
produced by the instant methods may be used as an archival source
for the original template polynucleotide. Accordingly, the
invention provides methods for archiving a polynucleotide template
by storing the amplification products produced by the methods of
the invention. The archived amplification products may be analyzed
as described herein, or may be subjected to further amplification
in accordance with the methods of the invention.
[0030] In another aspect, the invention provides products (e.g.,
multiple copies of a template polynucleotide) produced by the
methods disclosed herein.
[0031] The invention also provides compositions, kits, complexes,
reaction mixtures and systems comprising various components (and
various combinations of the components) used in the amplification
methods described herein.
[0032] In another aspect, the invention provides compositions
comprising any of the complexes (which are generally considered as
intermediates with respect to the final amplification products)
described herein.
[0033] In another aspect, the invention includes any one or more
products (including intermediates) and compositions comprising the
products (including intermediates) produced by any aspect of the
methods of the invention.
[0034] In another aspect, the invention provides reaction mixtures
(or compositions comprising reaction mixtures) which contain
various combinations of components described herein.
[0035] In another aspect, the invention provides kits for
conducting the methods described herein. These kits, in suitable
packaging and generally (but not necessarily) containing suitable
instructions, contain one or more components used in the
amplification methods.
[0036] In another aspect, the invention provides systems for
effecting the amplification methods described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] FIG. 1 illustrates one embodiment of a composite primer
useful in the methods of the present invention. As illustrated in
the Figure, the composite primer comprises a DNA portion at its 3'
end and an RNA portion at its 5' end. As discussed herein, it is
also possible to employ a composite primer in which the 3' DNA
portion is followed, in the direction of its 5', by an RNA portion,
which is followed by a portion which is DNA.
[0038] FIG. 2 illustrates a composite primer that hybridizes to
multiple sites on a template polynucleotide where differing
portions of the composite primer are hybridized to template
polynucleotide depending on the site at which it is hybridized.
[0039] FIG. 3 illustrates primer extension from composite primers
that are hybridized at multiple sites on a template strand, where a
composite primer extension products is being displaced by primer
extension from a composite primer hybridized at a downstream site
on the template strand.
[0040] FIG. 4 shows a collection of composite primer extension
products comprising composite primer 1 linked (via extension) to
sequences corresponding to a multiplicity of target polynucleotide
sequences.
[0041] FIG. 5 shows generation of second primer extension product
that is randomly primed by the composite primer.
[0042] FIG. 6 shows single primer isothermal amplification using
the complex comprising a RNA/DNA partial heteroduplex as a template
for further composite-primer dependent amplification.
[0043] FIG. 7 illustrates primer extension from composite primers
and auxiliary primers that are hybridized at multiple sites on a
template strand.
[0044] FIG. 8 illustrates generation of a second primer extension
product primed by auxiliary primers hybridized to composite primer
extension product.
[0045] FIG. 9 shows a photograph of a gel showing amplified
reaction product generated using a single randomly primed composite
primer to amplify a multiplicity of template polynucleotide
sequences from human genomic DNA.
MODES FOR CARRYING OUT THE INVENTION
Overview of the Invention and its Advantages
[0046] The invention discloses novel methods, compositions and kits
for global amplification. The methods provide for amplification
using a composite primer that is capable of binding to multiple
sites within template polynucleotide (e.g., mRNA or genomic DNA),
whereby a large multiplicity of template polynucleotide sequences
(for example, essentially all genomic DNA) is amplified. The
methods are suitable for use with either DNA or RNA template. They
generate polynucleotide (generally, DNA) products, which are
readily suitable for a variety of uses including comparative genome
hybridization, expression profiling, and multiple genotype
determinations, e.g., by multiplex analysis by microarray
technologies. The methods are amenable to automation and do not
require thermal cycling. Thus, one of the major advantages of the
methods of the invention is the ability to amplify an entire pool
of sequences (or a subset thereof, depending on the desired extent
of amplification), which is essential for application such as
comparative genome hybridization, generation of cDNA libraries,
generation of subtractive hybridization probes, and array based
assays, including multiple genotype determinations.
[0047] The amplification methods of the invention involve composite
primer random hybridization, primer extension and displacement of
composite primer extension product by strand displacement, whereby
a complex comprising a RNA-DNA heteroduplex is generated, followed
by composite primer-dependent isothermal amplification using the
complex as a substrate for further amplification, an aspect that
permits rapid amplification and distinguishes the invention from
other strand displacement amplification methods, such as MDA.
[0048] In one aspect, the methods of the invention involve two
phases: (a) composite primer random hybridization to template
polynucleotide, primer extension and displacement of composite
primer extension product by strand displacement DNA synthesis,
whereby a complex comprising a RNA/DNA partial heteroduplex is
generated, and (b) composite primer-dependent single primer
isothermal amplification using the complex comprising a RNA/DNA
partial heteroduplex as a substrate for further amplification.
[0049] The methods generally comprise using specially-designed
primers, generally a RNA/DNA composite primer, to randomly prime
template polynucleotide (such as genomic DNA template, mRNA or
noncoding RNA). By "randomly prime" or "random hybridization", as
used herein, it is meant that the composite primer hybridizes to
multiple sites within template polynucleotide. In some embodiments,
an aspect of the invention is displacement of primer extension
product from template polynucleotide(s) during primer extension by
strand displacement DNA synthesis (e.g., by primer extension with a
DNA polymerase having strand displacement activity) of primers
hybridized at a downstream position(s) on the template.
[0050] Thus, the invention provides methods of incubating a
reaction mixture, said reaction mixture comprising: (a) a
polynucleotide template; (b) a composite primer; wherein the
composite primer comprises an RNA portion and a 3' DNA portion, and
wherein the composite primer is capable of hybridizing to a
multiplicity of template polynucleotide sites; (c) a DNA-dependent
DNA polymerase with strand displacement activity; and (d) an
RNA-dependent DNA polymerase; wherein the incubation is under
conditions that permit composite primer random hybridization,
primer extension and displacement of the primer extension product
from template polynucleotide, whereby a population of intermediate
complexes are generated that generally includes (a) copies of
template polynucleotide and/or copies of the complement of
polynucleotide sequence appended (via extension) to composite
primer sequences; and (b) copies of template polynucleotide and
copies of the complement of template polynucleotide appended (via
extension) to the complement of composite primer sequences. The
intermediate complexes may be double-stranded or may be partially
double stranded. By virtue of the presence of composite primer
sequences in the intermediate complexes, the complexes comprise a
RNA/DNA heteroduplex partial heteroduplex. The RNA portion of the
RNA/DNA partial heteroduplex generally is introduced by the RNA
portion of the composite primer, and the DNA portion of the
heteroduplex is made of the complement of the RNA portion of the
composite primer. For simplicity, this population of intermediate
complexes is termed "a complex comprising an RNA/DNA partial
heteroduplex."
[0051] Generally, the composite primer comprises at least a 3' DNA
portion that is capable of randomly priming template
polynucleotide. Thus, and as the description makes clear, reference
to a primer that hybridizes to a sequence encompasses embodiments
in which at least a portion of the primer is hybridized,
embodiments in which two (or more portions) of the primer are
hybridized (separated by unhybridized (looped out) portions of the
primer), and embodiments in which the entire primer is
hybridized.
[0052] The complex comprising a RNA/DNA partial heteroduplex is
substrate for further amplification as follows: an enzyme which
cleaves RNA from an RNA/DNA hybrid (such as RNase H) cleaves RNA
sequence from the hybrid, leaving a 3' single stranded DNA sequence
available for binding by a composite primer (which may or may not
be the same as the first composite primer). Extension of a bound
composite primer by DNA-dependent DNA polymerase produces a primer
extension product, which displaces the previously bound cleaved
first primer extension product, whereby polynucleotide (generally,
DNA) product accumulates. It is understood that amplified product
generally is a mixture of sense and antisense copies of a given
template polynucleotide. For example, if the template
polynucleotide is double stranded DNA, the amplification product
will correspond to each strand. If the template polynucleotide is
single stranded, amplification product will generally be produced
that is the copy of template polynucleotide (sense copy) and the
complement of the template polynucleotide (antisense copy).
[0053] The methods disclosed herein are applicable to the
amplification of any target polynucleotide, including both DNA
(e.g., genomic DNA) and RNA (e.g., mRNA and ribosomal RNA) targets.
As is evident from the description and shown in the example, the
methods of the invention are composite-primer dependent. That is,
amplification is not observed in the absence of the composite
primer.
[0054] In another aspect of the invention, auxiliary primers are
present in the reaction mixture comprising template polynucleotide,
composite primer, DNA-dependent DNA polymerase and RNA-dependent
DNA polymerase. As used herein, "auxiliary primers" refers to a
population of random and/or partially randomized primers. Inclusion
of a population of random primers during the amplification is
believed to enhance the efficiency of production of and/or global
(template-wide, e.g., providing representative amplification of the
template, whether the template is DNA or RNA) coverage of the
amplification product.
[0055] In certain aspects, global amplification of genomic DNA is
exemplified herein. It is understood, however, that the
amplification methods of the invention are suitable for
amplification of any pool or subset (or polynucleotides
representing a significant proportion of a pool or subset,
depending on desired extent of amplification) of
polynucleotides.
[0056] Accordingly, in one aspect, the invention provides methods
for amplification of a template polynucleotide, said methods
comprising: (a) incubating a reaction mixture, said reaction
mixture comprising: (i) a template polynucleotide; (ii) a first
primer, wherein the first primer is a composite primer that is
hybridizable to a multiplicity of template polynucleotide sites,
wherein the composite primer comprises an RNA portion and a 3' DNA
portion; (iii) a DNA-dependent DNA polymerase; and (iv) an
RNA-dependent DNA polymerase (which may be present as a separate
enzyme or as an enzyme comprising both DNA-dependent DNA polymerase
and RNA-dependent DNA polymerase activities); wherein the
incubation is under conditions that permit composite primer random
hybridization, primer extension and displacement of the primer
extension product from template polynucleotide, whereby a complex
comprising an RNA/DNA partial heteroduplex is generated; and (b)
incubating a reaction mixture, said reaction mixture comprising (i)
the reaction products generated according to step (a) (or an
aliquot thereof); (ii) a composite primer (which may be the same as
the first primer, or may be a different primer), wherein the
composite primer comprises an RNA portion and a 3' DNA portion;
(iii) an DNA-dependent DNA polymerase; and (iv) an agent (such as
an enzyme) that cleaves RNA from an RNA/DNA hybrid; wherein the
incubation is under conditions that permit primer hybridization,
primer extension, RNA cleavage from an RNA/DNA heteroduplex, and
displacement of the primer extension product (i.e., strand
displacement DNA synthesis) from the complex of (a) when its RNA is
cleaved and another composite primer binds to the template and is
extended, whereby multiple copies of a polynucleotide (generally,
DNA) amplification product are generated. In some embodiments, the
reaction mixture of step (b) comprises the reaction mixture
according to step (a) (or an aliquot thereof). In other
embodiments, step (b) is initiated by the addition of an agent that
cleaves RNA from an RNA/DNA heteroduplex (such as RNase H), and
optionally, a DNA-dependent DNA polymerase, to the reaction mixture
of step (a). In some embodiments, the reaction mixture of step (a)
and/or (b) further comprises auxiliary primers.
[0057] In another aspect, the invention provides methods for
amplification of a template polynucleotide by incubating a reaction
mixture, said reaction mixture comprising: (a) a complex of a first
primer extension product and a second primer extension product,
wherein the first primer extension product is generated by
extension of a randomly primed first primer hybridized to target
polynucleotide with a DNA polymerase, wherein the first primer is a
composite primer comprising an RNA portion and a 3' DNA portion,
wherein the first primer is capable of hybridizing to a
multiplicity of template polynucleotide sites, and wherein the
second primer extension product is generated by extension of a
second primer hybridized to the first primer extension product; (b)
a composite primer that is hybridizable to the second primer
extension product, wherein the composite primer comprises an RNA
portion and a 3' DNA portion; (c) a DNA-dependent DNA polymerase;
and (d) an enzyme that cleaves RNA from an RNA/DNA hybrid; wherein
the incubation is under conditions that permit primer
hybridization, primer extension, RNA cleavage from an RNA/DNA
heteroduplex, and displacement of composite primer from the complex
of step (a) when its RNA is cleaved and another composite primer
binds and is extended, whereby multiple copies of a polynucleotide
amplification product are generated. In some embodiments, the first
primer extension product and/or the second primer extension product
are generated in the presence of auxiliary primers.
[0058] In another aspect-of the invention wherein the template
polynucleotide is RNA, template RNA is cleaved following random
composite primer hybridization and primer extension. Template RNA
can be cleaved using methods well-known in the art, including
cleavage with an agent (such as an enzyme, such as RNase H) that
cleaves RNA from an RNA/DNA hybrid, cleavage resulting from heat
treatment, and cleavage due to chemical treatment (e.g., treatment
under alkaline conditions). In some embodiments, the invention
provides methods of incubating a reaction mixture, said reaction
mixture comprising: (a) an RNA template; (b) a composite primer;
wherein the composite primer comprises an RNA portion and a 3' DNA
portion; and wherein the composite primer is capable of hybridizing
to a multiplicity of sites in template RNA; (c) a DNA-dependent DNA
polymerase; (d) an RNA-dependent DNA polymerase; and (e) an enzyme
capable of cleaving RNA from an RNA/DNA hybrid; wherein the
incubation is under conditions that permit composite primer random
hybridization, primer extension, template RNA cleavage from an
RNA/DNA heteroduplex, whereby a complex comprising an RNA/DNA
partial heteroduplex is generated. The complex comprising an
RNA/DNA partial heteroduplex is the substrate for further
amplification as described above (i.e., single primer isothermal
amplification). In some embodiments, an auxiliary primer is
included in the reaction mixture.
[0059] Template polynucleotide may also be prepared from RNA
template by synthesis of cDNA. cDNA synthesis may be accomplished
using standard methods, such as priming with random primers (e.g.,
random hexamer deoxyoligonucleotides) and primer extension with a
RNA-dependent DNA polymerase (e.g. reverse transcriptase) in the
presence of dNTPs and appropriate reaction conditions (e.g.,
temperature, pH and ionic conditions). Only the first strand cDNA
synthesis need be performed, as first strand cDNA synthesis will
produce a DNA polynucleotide that can be amplified in accordance
with the methods of the invention.
[0060] The methods of the invention include methods using the
amplification products (so-called "applications"). The invention
also provides methods which employ (usually, analyze) the products
of the amplification methods of the invention, such as methods of
nucleic acid mutation detection (including methods of genotyping),
determining the presence or absence of a sequence of interest,
quantitating a sequence of interest, preparation of an immobilized
nucleic acid, comparative genomic hybridization, discovery of novel
nucleic acid sequences, and characterizing nucleic acids using the
amplified nucleic acid products generated by the methods of the
invention.
[0061] Any of the methods of the invention can be used to generate
polynucleotide products that are suitable for characterization of a
polynucleotide sequence of interest in a sample. In one embodiment,
the invention provides methods for characterizing (for example,
detecting (presence or absence) and/or quantifying) a
polynucleotide sequence of interest comprising: (a) amplifying a
target polynucleotide by any of the methods described herein; and
(b) analyzing the amplification products. Step (b) of analyzing the
amplification products can be performed by any method known in the
art or described herein, for example by detecting and/or
quantifying amplification products that are hybridized to a probe.
These amplification products may or may not be labeled. Any of the
methods of the invention can be used to generate polynucleotide
(such as DNA) products that are labeled by incorporating labeled
nucleotides and/or labeled composite primers into appropriate
step(s) of the methods. These labeled products are particularly
suitable for quantification and/or identification by methods known
in the art, which include the use of arrays such as cDNA
microarrays and oligonucleotide arrays.
[0062] The invention provides methods to characterize (for example,
detect presence or absence of and/or quantify) an polynucleotide
sequence of interest by generating polynucleotide products using
amplification methods of the invention, and analyzing the products
by detection/quantification methods such as those based on array
technologies or solution phase technologies. These amplification
products may be labeled or unlabeled.
[0063] The methods of the invention may be used to amplify a pool
of polynucleotides (or polynucleotides representing a significant
proportion of a pool, depending on desired extent of amplification)
to generate sufficient input material for subsequent analysis.
Thus, and as is described herein, amplification products can also
serve as template for further analysis such as sequence,
polymorphism detection (including multiplex SNP detection) using,
e.g., oligonucleotide ligation-based assays, analysis using
Invader, Cleavase or limited primer extension, and the like.
Amplification product may also serve as template for further
amplification by the methods of the invention or other
amplification method known in the art.
[0064] In yet another embodiment, the invention provides methods
for immobilizing nucleic acids, including methods for generating
microarrays of nucleic acids, using amplification products of the
amplification methods of the invention.
[0065] In another embodiment, the invention provides methods of
generating cDNA libraries, methods of generating subtractive
hybridization probes, and methods of generating subtractive
hybridization libraries.
ADVANTAGES OF THE INVENTION
[0066] Various methods for global amplification of nucleic acids
have been developed. PCR-based methods, such as PEP, may generate
non-specific amplification artifacts, give incomplete coverage of
loci, or generate DNA product 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. Also, PCR-based methods require cumbersome
thermal cycling.
[0067] Additionally, a number of methods for the detection and
quantification of gene expression levels have been developed in
recent years. For example, microarrays, in which either defined
cDNAs or oligonucleotides are immobilized at discrete locations on,
for example, solid or semi-solid substrates, or on defined
particles, enable the detection and/or quantification of the
expression of a multitude of genes in a given specimen. Using these
previously known methods to detect presence of absence of and/or
quantify multiple RNA species in a sample, which in turn is used as
a measure of gene expression profiling, generally requires direct
labeling of cDNA, which requires a large amount of input total RNA.
Thus, when using total RNA preparations from a given cell or tissue
sample to analyze mRNA, the analysis of gene expression in the
sample using methods such as arrays requires a substantial amount
of input RNA, which generally ranges from 50 to 200 .mu.g.
Similarly, 2 to 5 .mu.g of mRNA purified from a total RNA
preparation would generally be required for a single analysis of
gene expression profiling using array technologies. This is a clear
limitation of methods such as those based on array technology,
insofar as the number of cells, or size of tissue specimen required
is very large, and these cells or tissue specimens are often
scarcely available for testing or are too precious. This limitation
is especially severe in the study of clinical specimens, where the
cells to be studied are rare and/or difficult to cultivate in
vitro, and in high throughput screening of libraries of effector
molecules. Also, previous transcription-based methods of
amplification of mRNA (described in, for example, Lockhart et al,
Nature Biotechnology (1996), 14, 1675-1680); van Gelder et al.,
U.S. Pat. No. 5,716,785), are limited to the amplification
efficiency of DNA-dependent RNA polymerases and some of these
methods require multiple steps. Moreover, the process by which the
polymerase promoter sequence is incorporated is prone to result in
non-specific amplification.
[0068] The methods of the invention offer the ability to
efficiently amplify template polynucleotides (including both RNA
and DNA). Thus, the utility of detection/quantification methods
which can be used with the amplification products of the invention,
such as those based on array technology, real time PCR,
quantitative TaqMan, quantitative PCR using molecular beacons, and
the like, should be greatly enhanced.
[0069] The methods of the invention do not require thermocycling
and all of the steps can be performed isothermally, although the
various steps may be carried out a different temperatures. This
feature provides numerous advantages, including facilitating
automation and adaptation for high through-put procedures. The
isothermal reaction is generally faster than that afforded by
thermal cycling, and is suitable for performing the methods of the
invention in miniaturized devices.
[0070] The ability to efficiently amplify pools of template
polynucleotide sequences (such as genomic DNA) under conditions
that will generally not alter the representation of the nucleic
acid sequences in the starting sample, will greatly enhance the
utility of the detection/quantification methods such as those based
on the powerful array technology.
[0071] The ability to efficiently amplify RNA using the random
initiation of replication according the methods of the invention
provides means for representing all sequences in the pool of
sequences (or representing a significant proportion of a pool
depending on desired extent of the amplification) in the sample,
for example, sequences representing the full length of mRNA species
in a sample. The methods of the invention do not rely on oligo-dT
primers (to bind the poly-A tail of mRNA) to initiate
amplification; thus, the methods may be used to amplify non-poly-A
tailed RNAs, such as noncoding RNAs and RNAs of non-eukaryotic
species. The methods of the invention do not require prior
knowledge of the sequences in the sample and are thus suitable for
discovery of novel transcripts, even when present in low abundance
and/or representing non-coding transcripts. The ability to
efficiently amplify RNA under conditions that will generally not
alter the representation of the nucleic acid sequences in the
preparation, will greatly enhance the utility of the
detection/quantification methods such as those based on the
powerful array technology.
General Techniques
[0072] 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).
[0073] Primers, oligonucleotides and polynucleotides employed in
the invention can be generated using standard techniques known in
the art.
DEFINITIONS
[0074] A "template," "template strand," "template polynucleotide,"
"template DNA," target sequence," "target nucleic acid," or "target
DNA," "target polynucleotide," "template RNA," or "target RNA," as
used herein, is a polynucleotide for which amplification is
desired. The template polynucleotide can comprise DNA or RNA. The
template sequence may be known or not known, in terms of its actual
sequence. Generally, the terms "target," "template," and variations
thereof, are used interchangeably.
[0075] "Amplification," or "amplifying", as used herein, generally
refers to the process of producing multiple copies of a desired
sequence. "Multiple copies" mean at least 2 copies. A "copy" does
not necessarily mean perfect sequence complementarity or identity
to the template sequence. For example, copies can include
nucleotide analogs such as deoxyinosine, intentional sequence
alterations (such as sequence alterations introduced through a
primer comprising a sequence that is hybridizable, but not
complementary, to the template, or a non-target sequence introduced
through a primer), and/or sequence errors that occur during
amplification. "Amplifying" a sequence may generally refer to
making copies of a sequence or its complement, with the
understanding that, as stated above, copying does not necessarily
mean perfect sequence complementarity or identity with respect to
the template sequence.
[0076] "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, carbamates, 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, poly-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.
[0077] 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. 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.
[0078] The "type" of dNTP or rNTP, as used herein, refers to the
particular base of a nucleotide, namely adenine, cytosine, thymine,
uridine, or guanine.
[0079] "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. Oligonucleotides in the invention
include the composite primer and auxiliary primer. The terms
"oligonucleotide" and "polynucleotide" are not mutually exclusive.
The description above for polynucleotides is equally and fully
applicable to oligonucleotides.
[0080] A "primer," as used herein, refers to a nucleotide sequence,
generally with a free 3'-OH group, that hybridizes with a template
sequence (such as a target polynucleotide, target DNA, 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.
[0081] "Auxiliary primers" as used herein, are a population of
primers comprising randomized and/or partially-randomized
sequences. Auxiliary primers are a polynucleotide as described
herein, though generally, auxiliary primers are made of DNA. Random
primers are known in the art and are commercially available. An
example of auxiliary primers is the population of randomized
hexamer primers shown in Example 1.
[0082] To "inhibit" is to decrease or reduce an activity, function,
and/or amount as compared to a reference.
[0083] By "randomly prime" or "random hybridization", as used
herein, it is meant that the composite primer hybridizes to
multiple sites within the template polynucleotide.
[0084] As used herein, "complex comprising an RNA/DNA partial
heteroduplex" generally refers to a population of intermediate
complexes that generally includes (a) copies of template
polynucleotide and/or copies of the complement of template
polynucleotide sequence appended to composite primer sequences; and
(b) copies of template polynucleotide and/or copies of the
complement of template polynucleotide appended to the complement of
composite primer sequences. By virtue of the presence of composite
primer sequence in the intermediate complexes, the complexes
comprise at least a RNA/DNA partial heteroduplex. The RNA portion
of the partial heteroduplex generally is introduced (via extension)
by the RNA portion of the composite primer, and the DNA portion of
the partial heteroduplex comprises the complement of the RNA
portion of the composite primer. As discussed herein, the complex
comprising an RNA/DNA partial heteroduplex functions as a substrate
for further amplification during the single primer isothermal
amplification phase of the methods. Generally, RNA in the RNA/DNA
partial heteroduplex is cleaved, 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).
Thus, reference to "a complex comprising a 3' single stranded
portion" generally refers to the complex comprising an RNA/DNA
partial heteroduplex when its RNA is cleaved.
[0085] A "complex" is an assembly of components. A complex may or
may not be stable and may be directly or indirectly detected. For
example, as is described herein, given certain components of a
reaction, and the type of product(s) of the reaction, existence of
a complex can be inferred. For purposes of this invention, a
complex is generally an intermediate with respect to the final
amplification product(s). An example of a complex is a complex of
composite primer extension product and second composite primer
extension product, as described herein.
[0086] A "portion" or "region," used interchangeably herein, of a
polynucleotide or oligonucleotide is a contiguous sequence of 2 or
more bases. In other embodiments, a region or portion is at least
about any of 3, 5, 10, 15, 20, 25 or more contiguous
nucleotides.
[0087] A region, portion, or sequence which is "adjacent" to
another sequence directly abuts that region, portion, or sequence.
For example, an RNA portion which is adjacent to a 5' DNA portion
of a composite primer directly abuts that region.
[0088] A "reaction mixture" is an assemblage of components, which,
under suitable conditions, react to form a complex (which may be an
intermediate) and/or a product(s).
[0089] "A", "an" and "the", and the like, unless otherwise
indicated include plural forms. "A" fragment means one or more
fragments.
[0090] Conditions that "allow" an event to occur or conditions that
are "suitable" for an event to occur, such as hybridization, strand
extension, and the like, or "suitable" conditions are conditions
that do not prevent such events from occurring. Thus, these
conditions permit, enhance, facilitate, and/or are conducive to the
event. Such conditions, known in the art and described herein,
depend upon, for example, the nature of the nucleotide sequence,
temperature, and buffer conditions. These conditions also depend on
what event is desired, such as hybridization, cleavage, and/or
strand extension.
[0091] Sequence "mutation," as used herein, refers to any sequence
alteration in a sequence of interest in comparison to a reference
sequence. A reference sequence can be a wild type sequence or a
sequence to which one wishes to compare a sequence of interest. A
sequence mutation includes single nucleotide changes, or
alterations of more than one nucleotide in a sequence, due to
mechanisms such as substitution, transversion, deletion or
insertion. Single nucleotide polymorphism (SNP) is also a sequence
mutation as used herein.
[0092] "Microarray" and "array," as used interchangeably herein,
comprise a surface with an array, preferably ordered array, of
putative binding (e.g., by hybridization) sites for a biochemical
sample (target) which often has undetermined characteristics. In a
preferred embodiment, a microarray refers to an assembly of
distinct polynucleotide or oligonucleotide probes immobilized at
defined locations on a substrate. Arrays are formed on substrates
fabricated with materials such as paper, glass, plastic (e.g.,
polypropylene, nylon, polystyrene), polyacrylamide, nitrocellulose,
silicon, optical fiber or any other suitable solid or semi-solid
support, and configured in a planar (e.g., glass plates, silicon
chips) or three-dimensional (e.g., pins, fibers, beads, particles,
microtiter wells, capillaries) configuration. Probes forming the
arrays may be attached to the substrate by any number of ways
including (i) in situ synthesis (e.g., high-density oligonucleotide
arrays) using photolithographic techniques (see, Fodor et al.,
Science (1991), 251:767-773; Pease et al., Proc. Natl. Acad. Sci.
U.S.A. (1994), 91:5022-5026; Lockhart et al., Nature Biotechnology
(1996), 14:1675; U.S. Pat. Nos. 5,578,832; 5,556,752; and
5,510,270); (ii) spotting/printing at medium to low-density (e.g.,
cDNA probes) on glass, nylon or nitrocellulose (Schena et al,
Science (1995), 270:467-470, DeRisi et al, Nature Genetics (1996),
14:457-460; Shalon et al., Genome Res. (1996), 6:639-645; and
Schena et al., Proc. Natl. Acad. Sci. U.S.A. (1995),
93:10539-11286); (iii) by masking (Maskos and Southern, Nuc. Acids.
Res. (1992), 20:1679-1684) and (iv) by dot-blotting on a nylon or
nitrocellulose hybridization membrane (see, e.g., Sambrook et al.,
Eds., 1989, Molecular Cloning: A Laboratory Manual, 2nd ed., Vol.
1-3, Cold Spring Harbor Laboratory (Cold Spring Harbor, N.Y.)).
Probes may also be noncovalently immobilized on the substrate by
hybridization to anchors, by means of magnetic beads, or in a fluid
phase such as in microtiter wells or capillaries. The probe
molecules are generally nucleic acids such as DNA, RNA, PNA, and
cDNA but may also include proteins, polypeptides, oligosaccharides,
cells, tissues and any permutations thereof which can specifically
bind the target molecules.
[0093] The term "3'" generally refers to a region or position in a
polynucleotide or oligonucleotide 3' (downstream) from another
region or position in the same polynucleotide or
oligonucleotide.
[0094] The term "5'" generally refers to a region or position in a
polynucleotide or oligonucleotide 5' (upstream) from another region
or position in the same polynucleotide or oligonucleotide.
[0095] The term "3'-DNA portion," "3'-DNA region," "3'-RNA
portion," and "3'-RNA region," refer to the portion or region of a
polynucleotide or oligonucleotide located towards the 3' end of the
polynucleotide or oligonucleotide, and may or may not include the
3' most nucleotide(s) or moieties attached to the 3' most
nucleotide of the same polynucleotide or oligonucleotide. The 3'
most nucleotide(s) can be preferably from about 1 to about 50, more
preferably from about 10 to about 40, even more preferably from
about 20 to about 30 nucleotides.
[0096] The term "5'-DNA portion," "5'-DNA region," "5'-RNA
portion," and "5'-RNA region," refer to the portion or region of a
polynucleotide or oligonucleotide located towards the 5' end of the
polynucleotide or oligonucleotide, and may or may not include the
5' most nucleotide(s) or moieties attached to the 5' most
nucleotide of the same polynucleotide or oligonucleotide. The 5'
most nucleotide(s) can be preferably from about 1 to about 50, more
preferably from about 10 to about 40, even more preferably from
about 20 to about 30 nucleotides.
[0097] "Absent" or "absence" of product, and "lack of detection of
product" as used herein includes insignificant, or de minimus
levels, generally due to lack of significant accumulation of
product.
[0098] In accordance with well established principles of patent law
"comprising" means "including."
Amplification Methods of the Invention
[0099] The following are examples of the amplification methods of
the invention. It is understood that various other embodiments may
be practiced, given the general description provided above. For
example, reference to using a composite primer means that any of
the composite primers described herein may be used.
Amplification Using a Composite Primer that Hybridizes to a
Multiplicity of Template Polynucleotide Sites
[0100] The invention provides methods for global amplification
using a composite primer that is capable of binding to multiple
sites within template polynucleotide, including DNA and RNA
template polynucleotides.
[0101] Generally, the methods of the invention involve two phases.
(a) composite primer random hybridization, primer extension and
displacement of composite primer extension product by strand
displacement, whereby a complex comprising a RNA/DNA partial
heteroduplex is generated, and (b) composite-primer dependent
single primer isothermal amplification.
[0102] Thus, the methods of the invention work as follows: (a)
incubating a reaction mixture, said reaction mixture comprising:
(i) a template polynucleotide; (ii) a first primer, wherein the
first primer is a composite primer that is hybridizable to a
multiplicity of template polynucleotide sites, wherein the
composite primer comprises an RNA portion and a 3' DNA portion;
(iii) a DNA-dependent DNA polymerase; and (iv) an RNA-dependent DNA
polymerase (which may be present as a separate enzyme or as an
enzyme comprising both DNA-dependent DNA polymerase and
RNA-dependent DNA polymerase activities); wherein the incubation is
under conditions that permit composite primer random hybridization,
primer extension and, in some embodiments, displacement of the
primer extension product from template polynucleotide, whereby a
complex comprising an RNA/DNA partial heteroduplex is generated;
and (b) incubating a reaction mixture, said reaction mixture
comprising (i) the reaction products generated according to step
(a) (or an aliquot thereof); (ii) a composite primer (which may be
the same as the first primer or may different from the first
primer); (iii) an DNA-dependent DNA polymerase; and (iv) an agent
(such as an enzyme) that cleaves RNA from an RNA/DNA hybrid;
wherein the incubation is under conditions that permit primer
hybridization, primer extension, RNA cleavage from an RNA/DNA
heteroduplex, and displacement of the primer extension product when
its RNA is cleaved and another composite primer binds to the
template and is extended, whereby multiple copies of a
polynucleotide (generally, DNA) amplification product are
generated. In some embodiments, the reaction mixture of step (b)
comprises the reaction mixture according to step (a) (or an aliquot
thereof). In other embodiments, step (b) is initiated by the
addition of an agent that cleaves RNA from an RNA/DNA heteroduplex
(such as RNase H) to the reaction mixture of step (a). In
embodiments in which the template polynucleotide is RNA, the
reaction mixture of step (a) further comprises an agent (such as an
enzyme) that that cleaves RNA from an RNA/DNA heteroduplex.
[0103] (a) Composite Primer Random Hybridization, Primer Extension
and Displacement of Composite Primer Extension Product by Strand
Displacement
[0104] The methods generally comprise using specially-designed
primers, generally a RNA/DNA composite primer. In a first phase of
the amplification methods, composite primer is used to randomly
prime template polynucleotide (such as genomic DNA). By "randomly
prime" or "random hybridization", as used herein, it is meant that
the composite primer hybridizes to multiple sites within template
polynucleotide. We have discovered that certain composite primers
bind a multiplicity of sites within template polynucleotide
(generally, under conditions promoting random primer hybridization)
and are thus particularly suitable for use in the methods of the
invention. Generally, suitable composite primers show partial
homology to a multiplicity of template nucleic acid sequences,
particularly in the 3' sequences of the composite primer, and thus,
the composite primer comprises at least a 3' DNA portion that is
capable of randomly priming template polynucleotide (particularly
under conditions permitting random primer hybridization). Selection
and design of composite primers is described further below.
[0105] Various embodiments of the composite primer and used in the
methods of the invention are described herein. For example, FIG. 1
illustrates one embodiment of a composite primer useful in the
methods of the present invention. As illustrated in the Figure, the
composite primer comprises a DNA portion at its 3' end and an RNA
portion at its 5' end. As discussed herein, it is also possible to
employ a composite primer in which the 3' DNA portion is followed,
in the direction of its 5', by an RNA portion, which is followed by
a portion which is DNA. The length of each of these sections is
generally determined for maximum efficiency of the amplification.
In some embodiments, the composite primer that hybridizes to target
polynucleotide and the composite primer used during single primer
isothermal amplification are the same. In some embodiments, the
composite primer that hybridizes to target polynucleotide and the
composite primer used during single primer isothermal amplification
are different.
[0106] Reference to a primer that binds (hybridizes to) a sequence
(or template) encompasses embodiments in which at least a portion
of the primer is hybridized, as well as those embodiments in which
an entire primer is hybridized. Thus, and as the description makes
clear, reference to a primer that hybridizes to a sequence
encompasses embodiments in which at least a portion of the primer
is hybridized as well as embodiments in which two (or more
portions) of the primer are hybridized, separated by unhybridized
(looped out) portions of the primer, and embodiments in which the
entire primer is hybridized. For example, FIG. 2 illustrates a
single composite primer that hybridizes to multiple positions on a
template polynucleotide where differing portions of the composite
primer are hybridized to template polynucleotide depending on the
site (sequence) at which it is hybridized. Thus, according to the
methods of the invention, only a portion of the 3'-end of the
composite primer must be hybridized in order for initiation of
primer extension by DNA polymerase. In some embodiments, for
example, only 2, 3, 4, 5, 6, 7, 8 or more nucleotides of the 3' end
of the primer need to hybridize in order for primer extension to be
initiated. It is understood that hybridization of the 3'-most
portion of the composite primer may be stabilized to various
extents by further hybridization of another portion of the primer
(with or without looping out of intervening primer portions). A DNA
polymerase can be included during primer hybridization to enhance
(e.g., stabilize) hybridization of composite primer by initiation
of primer extension, and thus, stabilization of primer
hybridization.
[0107] Random hybridization of the composite primer to template
polynucleotide generally occurs under conditions permitting random
(nonspecific) primer hybridization. Such conditions are well known
in the art and include: decreased stringency during primer
hybridization and/or primer extension (including reduced
temperature and/or buffer conditions of reduced stringency);
composite primer selection and/or design (discussed further
herein); and composite primer and template concentration. It is
understood that stringency of hybridization, composite primer
selection and/or primer concentration may be used to control the
frequency of composite primer hybridization, and thus to control
coverage and/or representation of template sequences in
amplification product. As noted above, an aspect of the invention
is displacement of intervening primer extension product during
primer extension of composite primers hybridized at downstream
position(s) on the template, whereby primer extension products are
displaced from template polynucleotide. Preferably, a DNA
polymerase is used that possesses stand displacement activity.
[0108] Composite primer random hybridization, primer extension and
displacement of primer extension product by strand displacement
results in generation of a multiplicity of complexes comprising a
RNA/DNA partial heteroduplex. The complexes comprise (a) copies of
template polynucleotide and/or copies of the complement of
polynucleotide sequence appended (via extension) to composite
primer sequences; and (b) copies of template polynucleotide and
copies of the complement of template polynucleotide appended (via
extension) to the complement of composite primer sequences.
Generally, the RNA portion of the complex is introduced by the
composite primer.
[0109] In some embodiments, generation of complexes comprising a
RNA/DNA partial heteroduplex involves the following steps: (i)
formation of a composite primer extension product; and (ii)
formation of a second primer extension product by primer extension
along the first primer extension product. For example, in some
embodiments, complex comprising an RNA/DNA partial heteroduplex is
generated as follows: following random hybridization of the
composite primer at multiple sites on template polynucleotide
strands, a DNA polymerase extends the composite primer along the
template strand generating a composite primer extension product
that is complementary to the polynucleotide template strand. Primer
extension extends to and displaces strands being extended from
primers hybridized at upstream sites on the template. Thus, as
noted above, an aspect of the invention is displacement of
intervening primer extension product during primer extension of
composite primers hybridized at downstream site(s) on the template,
whereby composite primer extension products are displaced from
template polynucleotide. FIG. 3 illustrates primer extension from
composite primers that are hybridized at multiple sites on a
template strand, where a composite primer extension products is
being displaced by primer extension from a composite primer
hybridized at a downstream site(s) on template strand.
[0110] Displaced composite primer product comprises the composite
primer sequence at the 5' end, including the 5' RNA portion.
Although for convenience, reference is made only to "a" composite
primer extension product, it is understood that a multiplicity of
composite primer extension products are generated the complement of
a multiplicity of template polynucleotide sequences appended (via
extension) to the sequence of the composite primer. FIG. 4 shows a
collection of composite primer extension products comprising
composite primer 1 linked (by extension) to sequences comprising
the complement of a multiplicity of target polynucleotide
sequences.
[0111] Using displaced composite primer extension product as a
template, a second primer extension product complementary to the
first primer extension product is generated by extension by a
DNA-dependent DNA polymerase along the DNA portion of the composite
primer extension product, and extension by a RNA-dependent DNA
polymerase along the 5' RNA portion of the composite primer
extension product, generating a double stranded complex comprising
a RNA/DNA complex at the end. Generation of second primer extension
product may be random primed using the composite primer, as
depicted in FIG. 5. Alternatively, second primer extension product
may be primed by the 3' end of a different composite primer
extension product. Additional embodiments in which second strand
production is primed by exogenous (added) primers and/or by
fragments of template RNA (endogenous primers) are described
below.
[0112] (b) Single Primer Isothermal Amplification Using a Complex
Comprising an RNA/DNA Partial Heteroduplex as a Template
[0113] In a second phase of the methods, termed single primer
isothermal amplification, the 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 RNA in the
composite primer, and thus forms a specific binding site for a
composite primer (which may or may not be the same as the first
composite primer). Extension of a bound composite primer by a
DNA-dependent DNA polymerase 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. FIG. 6 shows amplification of DNA product using a
composite primer and a complex comprising a RNA/DNA partial
heteroduplex as a template for further amplification.
[0114] 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, 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. Thus, in some embodiments, the
reactions conditions permit stringent hybridization (i.e.,
hybridization of sequences that are generally complementary). As is
evident from the description herein, in other embodiments, the
reaction conditions are less stringent (i.e., permit hybridization
of sequences that are less than fully complementary).
[0115] Generally, 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 50, at least 100,
at least 200, at least 300, or more distinct sequences (such as a
gene or other subsegment of a polynucleotide, 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). Although, 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.
[0116] For convenience, reference is made to a polynucleotide
(generally, DNA) product. It is understood that amplified product
generally is a mixture of sense and antisense copies of a given
template polynucleotide. For example, if the template
polynucleotide is double stranded DNA, the amplification product
will correspond to each strand. If the template polynucleotide is
single stranded (e.g., RNA or single stranded DNA), amplification
product will generally be produced that is the copy of template
polynucleotide (sense copy) and the complement of the template
polynucleotide (antisense copy). The amplification product of
different senses can be annealed to form a double stranded (or
partially double stranded) complex, or 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.
[0117] As is evident from the description and shown in the example,
the methods of the invention are composite-primer dependent. That
is, amplification is not observed in the absence of the composite
primer.
[0118] 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).
[0119] For simplicity, the 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). In other embodiments, step
(b) may be initiated by addition of an enzyme that cleaves RNA from
an RNA/DNA hybrid (e.g., ribonuclease, such as RNase H), and
optionally, a DNA-dependent DNA polymerase, as shown in Example 1.
In this embodiment, addition of an enzyme that cleaves RNA from an
RNA/DNA hybrid permits further amplification using the complex
comprising an RNA/DNA partial heteroduplex as a template (i.e.,
step (b), above). It is understood, however, that primer extension
(and strand displacement) along template polynucleotide strand from
random primed composite primer(s) may continue during single primer
isothermal amplification.
[0120] 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. In addition, it is understood that different composite
primers can be used in step (a) (i.e., random priming of template
polynucleotide) and step (b) (i.e., single primer isothermal
amplification). In this instance, the different composite primer
comprises sequences hybridizable to the 3' single stranded DNA
portion of the partially double stranded complex (which is
generated by cleaving RNA from the complex comprising a RNA/DNA
partial heteroduplex). Generally, the second composite primer
comprises sequences overlapping with the first composite
primer.
Amplification Using a Composite Primer that Hybridizes to a
Multiplicity of Template Polynucleotide Sites and Auxiliary
Primers
[0121] In another aspect of the invention, auxiliary primers are
present in the reaction mixture comprising template polynucleotide,
composite primer, DNA-dependent DNA polymerase and RNA-dependent
DNA polymerase. As used herein, "auxiliary primers" refers to a
population of random or partially randomized primers. An example of
auxiliary primers is the random hexamer primers used in Example 1.
Inclusion of auxiliary primers (i.e., population of different
random primers) during the amplification is believed to enhance the
efficiency of production of and/or target coverage of the
amplification product.
[0122] In some embodiments, the methods of the invention work as
follows: (a) incubating a reaction mixture, said reaction mixture
comprising a composite primer as described herein; auxiliary
primers; a template polynucleotide, DNA-dependent DNA polymerase,
and RNA-dependent DNA polymerase (which may be present as a single
enzyme comprising both activities), wherein the incubation is under
conditions suitable for random composite primer hybridization,
auxiliary primer hybridization, primer extension, and strand
displacement, whereby a complex comprising an RNA/DNA partial
heteroduplex is generated; and (b) incubating a reaction mixture,
said reaction mixture comprising the reaction products from step
(a) (or an aliquot thereof); a composite primer (which may be the
same as the composite primer of step (a) or may be a different
composite primer); a DNA-dependent DNA polymerase; optionally,
auxiliary primers; and an enzyme that cleaves RNA from a RNA/DNA
hybrid; wherein the incubation is under conditions that permit
primer hybridization, primer extension, RNA cleavage from an
RNA/DNA heteroduplex, and displacement of the primer extension
product from the complex when its RNA is cleaved and another
composite primer binds to the template and is extended, whereby
multiple copies of a polynucleotide template sequence are
generated.
[0123] Inclusion of auxiliary primers (i.e., a population of
different random primers) during the amplification is believed to
enhance the efficiency of production of and/or coverage of template
polynucleotide. Without being bound by theory, it is believed that
primer extension of auxiliary primers increases displacement of
composite primer extension product from template polynucleotide
and/or primes generation of second primer extension product. FIG. 7
illustrates primer extension from composite primers and auxiliary
primers that are hybridized at multiple sites on a template strand.
FIG. 8 illustrates generation of a second primer extension product
primed by auxiliary primers hybridized to composite primer
extension product.
[0124] Although for simplicity, use of auxiliary primers is
described only in the first phase, random composite primer
hybridization (i.e., step (a)), it is evident that auxiliary
primers may be present in the reaction mixture for the second phase
of the methods, single primer isothermal amplification (i.e., step
(b)).
[0125] As is evident from the description and shown in the example,
the methods of the invention are composite-primer dependent. That
is, amplification is not observed in the absence of the composite
primer.
Amplification Using a Composite Primer that Hybridizes to a
Multiplicity of Template RNA Sites and Auxiliary Primers
[0126] In another aspect of the invention, auxiliary primers are
present in the reaction mixture comprising template RNA, composite
primer, DNA-dependent DNA polymerase and RNA-dependent DNA
polymerase. As used herein, "auxiliary primers" refers to a
population of random or partially randomized primers. Inclusion of
auxiliary primers (i.e., population of different random primers)
during the amplification is believed to enhance the efficiency of
production of and/or target coverage of the amplification
product.
[0127] In some embodiments, the methods of the invention operate as
follows: (a) incubating a reaction mixture, said reaction mixture
comprising a composite primer as described herein; auxiliary
primers; a template RNA, DNA-dependent DNA polymerase, and
RNA-dependent DNA polymerase (which may be present as a single
enzyme comprising both activities), wherein the incubation is under
conditions suitable for random composite primer hybridization,
auxiliary primer hybridization, primer extension, and strand
displacement, whereby a complex comprising a RNA/DNA partial
heteroduplex is generated; and (b) incubating a reaction mixture,
said reaction mixture comprising the reaction products from step
(a) (or an aliquot thereof); a composite primer (which may be the
same as the composite primer of step (a) or may be a different
composite primer); a DNA-dependent DNA polymerase; optionally,
auxiliary primers; and an enzyme that cleaves RNA from an RNA/DNA
hybrid; wherein the incubation is under conditions that permit
primer hybridization, primer extension, RNA cleavage from an
RNA/DNA heteroduplex, and displacement of the primer extension
product from the complex when its RNA is cleaved and another
composite primer binds to the template and is extended, whereby
multiple copies of a polynucleotide template sequence are
generated.
[0128] Inclusion of auxiliary primers (i.e., a population of
different random primers) during the amplification is believed to
enhance the efficiency of production of and/or coverage of template
RNA. Without being bound by theory, it is believed that primer
extension of auxiliary primers increases displacement of composite
primer extension product from template RNA and/or primes generation
of second primer extension product. FIG. 7 illustrates primer
extension from composite primers and auxiliary primers that are
hybridized at multiple sites on a template strand. FIG. 8
illustrates generation of a second primer extension product primed
by auxiliary primers hybridized to composite primer extension
product.
[0129] Although for simplicity, use of auxiliary primers is
described only in the first phase, random composite primer
hybridization (i.e., step (a)), it is evident that auxiliary
primers may be present in the reaction mixture for the second phase
of the methods, single primer isothermal amplification (i.e., step
(b)).
[0130] As is evident from the description and shown in the example,
the methods of the invention are composite-primer dependent. That
is, amplification is not observed in the absence of the composite
primer.
Components and Reaction Conditions Used in the Methods of the
Invention
Template Nucleic Acid
[0131] The nucleic acid (NA) target to be amplified includes
nucleic acids from any source in purified or unpurified form, which
can be DNA (dsDNA and ssDNA) or RNA, including tRNA, mRNA, rRNA,
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. Preferred 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.
[0132] The target nucleic acid 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. Nucleic acid 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). Amplification of an RNA target may be accomplished by
initial cDNA synthesis, as known in the art, followed by
amplification from the cDNA template.
[0133] 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.
[0134] When the target polynucleotide is DNA, the initial step of
the amplification of a target nucleic acid sequence is 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 an 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.
[0135] 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) would
require 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 would require 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.
Composite Primer
[0136] The methods of the invention employ a composite primer that
is composed of RNA and DNA portions. We have observed that suitable
composite primers show partial nucleic acid sequence homology to a
multiplicity of genomic DNA sequences, particularly in the 3'
sequences of the composite primer, when analyzed using standard
nucleic acid comparison algorithms. For example, composite primer
sequence can be used as a query sequence in Blast, to search the
human genomic DNA database (or other suitable database, such as a
mammalian genomic DNA database). Generally, the search is performed
using search parameters suitable for identification of partial or
"low stringency" alignments, generally the least stringent
conditions provided by the program. Such parameters are known in
the art and include use of the NCBI Blast program for searching
"short, nearly exact matches", with word size=7 (conditions
permitting as few as 7 consecutive nucleotide perfect matches at
any position in the primer sequence). See, e.g.,
http://www.ncbi.nlm.nih.gov/blast/Blast.cgi?ALIGNMENTS=50&ALIGNMENT_VIEW=-
Pairwise&AUTO_FORMAT=Semiauto&CLIENT=web&DATABASE=nr&DESCRIPTIONS=100&ENTR-
EZ_QUERY=>(none)&EXPECT=1000&FORMAT_BLOCK_ON_RESPAGE=None&FORMAT_ENTREZ-
_QUERY=(none)&FORMAT_OBJECT=Alignment&FORMAT_TYPE=HTML&LAYOUT=TwoWindows&N-
CBI_GI=on&PAGE=Nucleotides&PROGRAM=blastn&SERVICE=plain&SET_DEFAULTS.x=16&-
SET_DEFAULTS.y=8&SHOW_OVERVIEW=on&WORD_SIZE=7&END_OF_HTTPGET=Yes.
Composite primers useful in the methods of the invention (i.e.,
that randomly hybridize to template polynucleotide) generally
exhibit high partial homology rate with genomic DNA sequences, for
example homology of stretches of 7 nucleotides with about 100
genomic DNA sequences, with about 70% of the hits located at the 3'
end of the composite primer. A composite primer with a very unique
sequence (i.e., low levels of homology with target genomic DNA
sequences) did not function efficiently in the methods of the
invention when used with genomic DNA template.
[0137] As is evident from the discussion above, reference to a
primer that binds (hybridizes to) a sequence (or template)
encompasses embodiments in which at least a portion of the primer
is hybridized, embodiments in which two (or more portions) of the
primer are hybridized (separated by unhybridized (looped out)
portions of the primer), and embodiments in which the entire primer
is hybridized. In certain embodiments, a 5'-portion, commonly the
5'-most portion, of the composite primer is designed such that the
particular 5'-portion it is not expected to randomly hybridize to
template polynucleotide (composite primers of this configuration
are referred to as "tailed" primers, in reference to the `tail` of
unhybridized primer). In some embodiments, the tail portion of the
composite primer is the entire 5' RNA portion of the composite
primer. Thus, according to the methods of the invention, only a
portion of the 3'-end of the composite primer must be hybridized in
order for initiation of primer extension by DNA polymerase. In some
embodiments, for example, only 2, 3, 4, 5, 6, 7 or more nucleotides
of the 3' end of the primer must hybridize in order for primer
extension to be initiated. It is understood that hybridization of
the 3'-most portion of the composite primer may be stabilized to
various extents by further hybridization of another portion of the
primer (with or without looping out of intervening primer
portions). A DNA polymerase can be included during primer
hybridization to enhance (e.g., stabilize) hybridization of
composite primer by initiation of primer extension, and thus,
stabilization of primer hybridization.
[0138] We have also observed that composite primers that are
suitable for use in the present methods can be identified by
conducting single primer isothermal amplification as described in
Kurn, U.S. Pat. No. 6,251,639, using the composite primer under
high stringency conditions using a genomic DNA template, and
observing the presence of a smear of reaction products as
visualized, for example, on a gel. Preferably, the genomic DNA does
not contain a sequencers) that is complementary to the composite
primer. Production of a "smear" of reaction products, i.e.,
generation of a complex mixture of product of multiple molecular
weights, visible on a gel as a smear, indicates that the composite
primer is randomly priming genomic DNA amplification.
[0139] In another example, single primer isothermal amplification
of a specific synthetic target oligonucleotide (e.g., a target
oligonucleotide comprising a specific target for composite primer
hybridization) is conducted at high stringency in the presence or
absence of genomic DNA template (e.g., 1-100 ng of human genomic
DNA). Composite primers that are suitable for the methods of the
invention will demonstrate a strong effect of genomic DNA on the
efficiency of the amplification of the specific synthetic target,
resulting in about a 100-fold or greater reduction of amplification
efficiency as compared with amplification efficiency conducted in
the absence of genomic DNA.
[0140] Random composite primer hybridization and/or generation of
composite primer extension product is promoted by use of conditions
designed to permit random (non-specific) primer hybridization. Such
conditions are well known in the art, and are further discussed
below, and include: decreased stringency during primer
hybridization and/or first strand synthesis (including reduced
temperature and/or buffer conditions of reduced stringency, such as
reduced ionic strength); composite primer selection and/or design
(discussed further herein); composite primer and template
concentration, presence or absence of an agent that stabilizes a 3'
hybridized primer (such as a DNA polymerase), and presence or
absence of agents such as DMSO that lower the temperature
requirements for stable hybridization. It is understood that the
selection of reaction conditions may be used to control the
frequency of composite primer hybridization, and thus control
coverage and/or representation of template polynucleotide sequences
in amplification product.
[0141] Generally, the composite primer is also designed so that
there is no primer-dimer formation capability, as determined using
software routinely available to one of ordinary skill in the art,
e.g. Oligo Primer Analysis Software from Molecular Biology Insight,
and references therein. One of skill in the art will understand
that other factors affect nucleic acid hybridization affinities.
For example, any and all of the guanosine-cytosine content of the
primer-target and primer-primer duplexes, minor groove binders,
O-methylation or other modification of nucleotides, temperature,
and salt are potentially important factors in constructing primers
with the requisite differences in binding energies. Another factor
in designing and constructing primers is the free energy parameters
of hybridization of given sequences under a given set of
hybridization conditions. The free energy parameters for the
formation of a given hybrid may be calculated by methods known in
the art (see, e.g., Tinoco et al. Nature (1973) 246:40-41 and
Freier et al., Proc. Natl. Acad. Sci. USA (1986) 83:9373-9377;
computer programs, e.g., Oligo Primer Analysis Software from
Molecular Biology Insight, and references therein), and it is
possible to predict, for a given oligonucleotide template, primer
sequences for which the required free energy changes for formation
of various complexes will be met.
[0142] The primers should be extendable by DNA polymerase.
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). Generally, the primer should permit
high efficiency of amplification of a synthetic target that
contains a specific primer target binding site (e.g., the
complementary sequence to the primer), for example, permitting
amplification of about 10.sup.6 to 10.sup.9 using methods described
in Kurn, U.S. Pat. No. 6,251,639. The composite primer is designed
such that subsequent displacement of the primer extension product
by binding of a new (additional) composite primer and the extension
of the new primer by the polymerase can be achieved. In addition,
cleavage of the RNA portion of the primer extension product leads
to generation of amplification product which is not a substrate for
amplification by the composite primer. It is understood that, in
the following section that generally describes aspects of the
composite primers used in the methods of the invention,
characteristics described may be applicable to the primers if used
for hybridizing and initiating the polynucleotide amplification
(production of composite extension product) and/or for single
primer isothermal amplification as described herein.
[0143] In some embodiments, a first composite primer is used in the
methods of the invention, including those steps which involve
single primer isothermal amplification (i.e., phase (b)). In other
embodiments, a first and second, different, composite primer are
used in the methods of the invention. The second composite primer
is used for the single primer isothermal amplification step, and
may comprise some or all of the sequence of the first composite
primer, and the first composite primer may comprise some or all of
the sequence of the second composite primer. In some embodiments,
the second composite primer comprises a different sequence than the
first composite primer.
[0144] For use in single primer isothermal amplification and/or
composite primer extension product formation, a composite primer
comprises at least one RNA portion that is capable of (a) binding
(hybridizing) to a sequence on the single stranded portion of the
complex (formed by cleavage of RNA in the complex comprising a
RNA/DNA partial heteroduplex) (in some embodiments, on second
primer extension product) independent of hybridization of the DNA
portion(s) to a sequence on the same single stranded portion; and
(b) being cleaved with an agent such as a ribonuclease when
hybridized to the single stranded portion. The composite primers
bind to the single stranded portion, and are extended by DNA
polymerase to form a RNA/DNA partial heteroduplex in which only the
RNA portion of the primer is cleaved upon contact with an agent
which cleaves RNA in an RNA/DNA hybrid, such as an enzyme, such as
a ribonuclease (such as RNase H), while the composite primer
extension product remains intact, thus enabling annealing of
another composite primer.
[0145] When used for the single primer isothermal amplification
described herein, the composite primers also comprise a 3' DNA
portion that is capable of hybridization to a sequence on the 3'
single stranded portion of the complex such that its hybridization
to the 3' single stranded portion is favored over that of the
nucleic acid strand that is displaced from the complex 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 complex (e.g., in the second primer extension product) favored
over the hybridization of the homologous sequence in the 5' end of
the displaced strand to the composite primer extension product.
[0146] The composite primer comprises a combination of RNA and DNA
(see definition above), 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. 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).
[0147] The length of an RNA portion in a composite primer
comprising a 3'-DNA portion and an RNA portion can be preferably
from about 1 to about 50, more preferably from about 3 to about 20,
even more preferably from about 4 to about 15, and most preferably
from about 5 to about 10 nucleotides. In some embodiments of a
composite primer comprising a 3'-DNA portion and an RNA portion, an
RNA portion can be at least about any of 1, 3, 4, 5 nucleotides,
with an upper limit of about any of 10, 14, 15, 20, 25, 3, 50
nucleotides. In certain embodiments, the composite primer has an
RNA portion of about 14 or about 20 nucleotides.
[0148] The length of the 5'-RNA portion in a composite primer
comprising a 5'-RNA portion and a 3'-DNA portion can be preferably
from about 3 to about 50 nucleotides, more preferably from about 5
to about 20 nucleotides, even more preferably from about 7 to about
18 nucleotides, preferably from about 8 to about 17 nucleotides,
and most preferably from about 10 to about 15 nucleotides. In other
embodiments of a composite primer comprising a 5'-RNA portion and a
3'-DNA portion, the 5'-RNA portion can be at least about any of 3,
5, 7, 8, 10 nucleotides, with an upper limit of about any of 14,
15, 17, 18, 20, 50 nucleotides. In certain embodiments, the
composite primer has an RNA portion of about 14 or about 20
nucleotides.
[0149] In embodiments of a composite primer comprising a 5'-RNA
portion and a 3'-DNA portion further comprising non-5'-RNA
portion(s), a non-5'-RNA portion can be preferably from about 1 to
about 7 nucleotides, more preferably from about 2 to about 6
nucleotides, and most preferably from about 3 to about 5
nucleotides. In certain embodiments of a composite primer
comprising a 5'-RNA portion and a 3'-DNA portion further comprising
non-5'-RNA portion(s), a non-5'-RNA portion can be at least about
any of 1, 2, 3, 5, with an upper limit of about any of 5, 6, 7, 10
nucleotides.
[0150] In embodiments of a composite primer comprising a 5'-RNA
portion and a 3'-DNA portion, in which the 5'-RNA portion is
adjacent to the 3'-DNA portion, the length of the 5'-RNA portion
can be preferably from about 3 to about 50 nucleotides, more
preferably from about 5 to about 20 nucleotides, even more
preferably from about 7 to about 18 nucleotides, preferably from
about 8 to about 17 nucleotides, and most preferably from about 10
to about 15 nucleotides. In certain embodiments of a composite
primer comprising a 5'-RNA portion and a 3'-DNA portion, in which
the 5'-RNA portion is adjacent to the 3'-DNA portion, the 5'-RNA
portion can be at least about any of 3, 5, 7, 8, 10 nucleotides,
with an upper limit of about any of 14, 15, 17, 18, 20, 50
nucleotides. In certain embodiments, the composite primer has an
RNA portion of about 14 or about 20 nucleotides.
[0151] The length of an intervening RNA portion in a composite
primer comprising 5'- and 3'-DNA portions with at least one
intervening RNA portion can be preferably from about 1 to about 7
nucleotides, more preferably from about 2 to about 6 nucleotides,
and most preferably from about 3 to about 5 nucleotides. In some
embodiments of a composite primer comprising 5'- and 3'-DNA
portions with at least one intervening RNA portion, an intervening
RNA portion can be at least about any of 1, 2, 3, 5 nucleotides,
with an upper limit of about any of 5, 6, 7, 10 nucleotides. The
length of an intervening RNA portion in a composite primer
comprising a 3'-DNA portion and at least one intervening RNA
portion can be preferably from about 1 to about 7 nucleotides, more
preferably from about 2 to about 6 nucleotides, and most preferably
from about 3 to about 5 nucleotides. In some embodiments of a
composite primer comprising a 3'-DNA portion and at least one
intervening RNA portion, an intervening RNA portion can be at least
about any of 1, 2, 3, 5 nucleotides, with an upper limit of about
any of 5, 6, 7, 10 nucleotides. In a composite primer comprising a
3'-DNA portion and at least one intervening RNA portion, further
comprising a 5'-RNA portion, the 5'-RNA portion can be preferably
from about 3 to about 25 nucleotides, more preferably from about 5
to about 20 nucleotides, even more preferably from about 7 to about
18 nucleotides, preferably from about 8 to about 17 nucleotides,
and most preferably from about 10 to about 15 nucleotides. In some
embodiments of a composite primer comprising a 3'-DNA portion and
at least one intervening RNA portion, further comprising a 5'-RNA
portion, the 5'-RNA portion can be at least about any of 3, 5, 7,
8, 10 nucleotides, with an upper limit of about any of 15, 17, 18,
20 nucleotides.
[0152] The length of the 3'-DNA portion in a composite primer
comprising a 3'-DNA portion and an RNA portion can be preferably
from about 1 to about 20, more preferably from about 3 to about 18,
even more preferably from about 5 to about 15, and most preferably
from about 7 to about 12 nucleotides. In some embodiments of a
composite primer comprising a 3'-DNA portion and an RNA portion,
the 3'-DNA portion can be at least about any of 1, 3, 5, 7, 10
nucleotides, with an upper limit of about any of 10, 12, 15, 18,
20, 22 nucleotides. In one embodiment, the composite primer has a
3'-DNA portion of about 7 nucleotides.
[0153] The length of the 3'-DNA portion in a composite primer
comprising a 5'-RNA portion and a 3'-DNA portion can be preferably
from about 1 to about 20 nucleotides, more preferably from about 3
to about 18 nucleotides, even more preferably from about 5 to about
15 nucleotides, and most preferably from about 7 to about 12
nucleotides. In some embodiments of a composite primer comprising a
5'-RNA portion and a 3'-DNA portion, the 3' DNA portion can be at
least about any of 1, 3, 5, 7, 10 nucleotides, with an upper limit
of about any of 10, 12, 15, 18, 20, 22 nucleotides. In one
embodiment, the composite primer has a 3'-DNA portion of about 7
nucleotides.
[0154] In embodiments of a composite primer comprising a 5'-RNA
portion and a 3'-DNA portion, further comprising non-3'-DNA
portion(s), a non-3'-DNA portion can be preferably from about 1 to
about 10 nucleotides, more preferably from about 2 to about 8
nucleotides, and most preferably from about 3 to about 6
nucleotides. In some embodiments of a composite primer comprising a
5'-RNA portion and a 3'-DNA portion, further comprising non-3'-DNA
portion(s), a non-3'-DNA portion can be at least about any of 1, 2,
3, 5 nucleotides, with an upper limit of about any of 6, 8, 10, 12
nucleotides.
[0155] In embodiments of a composite primer comprising a 5'-RNA
portion and a 3'-DNA portion in which the 5'-RNA portion is
adjacent to the 3'-DNA portion, the length of the 3'-DNA portion
can be preferably from about 1 to about 20 nucleotides, more
preferably from about 3 to about 18 nucleotides, even more
preferably from about 5 to about 15 nucleotides, and most
preferably from about 7 to about 12 nucleotides. In certain
embodiments of the primer comprising a 5'-RNA portion and a 3'-DNA
portion in which the 5'-RNA portion is adjacent to the 3'-DNA
portion, the 3'-DNA portion can be at least about any of 1, 3, 5,
7, 10 nucleotides, with an upper limit of about any of 10, 12, 15,
18, 20, 22 nucleotides. In one embodiment, the composite primer has
a 3'-DNA portion of about 7 nucleotides.
[0156] The length of a non-3'-DNA portion in a composite primer
comprising 5'- and 3'-DNA portions with at least one intervening
RNA portion can be preferably from about 1 to about 10 nucleotides,
more preferably from about 2 to about 8 nucleotides, and most
preferably from about 3 to about 6 nucleotides. In some embodiments
of a primer comprising 5'- and 3'-DNA portions with at least one
intervening RNA portion, a non-3'-DNA portion can be at least about
any of 1, 2, 3, 5 nucleotides, with an upper limit of about any of
6, 8, 10, 12 nucleotides.
[0157] The length of the 3'-DNA portion in a composite primer
comprising 5'- and 3'-DNA portions with at least one intervening
RNA portion can be preferably from about 1 to about 20 nucleotides,
more preferably from about 3 to about 18 nucleotides, even more
preferably from about 5 to about 15 nucleotides, and most
preferably from about 7 to about 12 nucleotides. In some
embodiments of a composite primer comprising 5'- and 3'-DNA
portions with at least one intervening RNA portion, the 3'-DNA
portion can be at least about any of 1, 3, 5, 7, 10 nucleotides,
with an upper limit of about any of 10, 12, 15, 18, 20, 22
nucleotides. In one embodiment, the composite primer has a 3'-DNA
portion of about 7 nucleotides.
[0158] The length of a non-3'-DNA portion (i.e., any DNA portion
other than the 3'-DNA portion) in a composite primer comprising a
3'-DNA portion and at least one intervening RNA portion can be
preferably from about 1 to about 10 nucleotides, more preferably
from about 2 to about 8 nucleotides, and most preferably from about
3 to about 6 nucleotides. In some embodiments of a composite primer
comprising a 3'-DNA portion and at least one intervening RNA
portion, a non-3'-DNA portion can be at least about any of 1, 3, 5,
7, 10 nucleotides, with an upper limit of about any of 6, 8, 10, 12
nucleotides. The length of the 3'-DNA portion in a composite primer
comprising a 3'-DNA portion and at least one intervening RNA
portion can be preferably from about 1 to about 20 nucleotides,
more preferably from about 3 to about 18 nucleotides, even more
preferably from about 5 to about 15 nucleotides, and most
preferably from about 7 to about 12 nucleotides. In some
embodiments of a composite primer comprising a 3'-DNA portion and
at least one intervening RNA portion, the 3'-DNA portion can be at
least about any of 1, 3, 5, 7, 10 nucleotides, with an upper limit
of about any of 10, 12, 15, 18, 20, 22 nucleotides. In one
embodiment, the composite primer has a 3'-DNA portion of about 7
nucleotides. It is understood that the lengths for the various
portions can be greater or less, as appropriate under the reaction
conditions of the methods of this invention.
[0159] In some embodiments, the 5'-DNA portion of a composite
primer includes the 5'-most nucleotide of the primer. In some
embodiments, the 5'-RNA portion of a composite primer includes the
5' most nucleotide of the primer. In other embodiments, the 3'-DNA
portion of a composite primer includes the 3' most nucleotide of
the primer. In other embodiments, the 3'-DNA portion is adjacent to
the 5'-RNA portion and includes the 3' most nucleotide of the
primer (and the 5'-RNA portion includes the 5' most nucleotide of
the primer).
[0160] The total length of the composite primer can be preferably
from about 10 to about 50 nucleotides, more preferably from about
15 to about 30 nucleotides, and most preferably from about 20 to
about 25 nucleotides. In some embodiments, the length can be at
least about any of 10, 15, 20, 25 nucleotides, with an upper limit
of about any of 25, 30, 50, 60 nucleotides. In certain embodiments,
the composite primer is about 21 or about 27 nucleotides in length.
It is understood that the length can be greater or less, as
appropriate under the reaction conditions of the methods of this
invention.
[0161] As described herein, one or more different composite primers
may be used in an amplification reaction.
Auxiliary Primers
[0162] "Auxiliary primer" as used herein, are a population of
primers comprising randomized and/or partially-randomized
sequences. Auxiliary primers are a polynucleotide as described
herein, though generally, auxiliary primers are made of DNA. Such
random primers are known in the art. An example of auxiliary
primers is the population of randomized hexamer primers shown in
Example 1. In some embodiments, the random primers may contain
natural or non-natural nucleotide(s) that permit non-specific
hybridization in order to increase the number of sequences to which
the random primers may bind. Similarly, abasic sites can be
introduced randomly within the population of random primers, which
can permit non-specific hybridization by stabilizing mismatches
between primer and template. The primers should be extendable by
DNA polymerase. Generation of primers suitable for extension by
polymerization is well known in the art, such as described in PCT
Pub. No. WO 99/42618 (and references cited therein).
[0163] In some embodiments, auxiliary primers can be at least 6, at
least 7, at least 8, at least 9, at least 10, at least 11, at least
12, at least 15, at least 18, at least 20, or more nucleotides in
length. In some embodiments, a population of primers of differing
lengths is used.
DNA Polymerase, and an Agent Capable of Cleaving an RNA-DNA
Hybrid
[0164] The 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 the RNase
H activity. Reverse transcriptase devoid 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.
[0165] 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).
[0166] 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.
[0167] 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'->3'
exonuclease activity so as to minimize degradation of primer, or
primer extension polynucleotides. 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'->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). Preferably, the DNA polymerase has little to no
proofreading activity
[0168] 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.
[0169] The ribonuclease for use in the methods and compositions of
the invention is capable of cleaving ribonucleotides in an RNA/DNA
hybrid. 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.
[0170] 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.
[0171] In general, the enzymes used in the methods and compositions
of the invention should not produce substantial degradation of the
nucleic acid components of said methods and compositions.
Reaction Conditions and Detection
[0172] Appropriate reaction media and conditions for carrying out
the methods of the invention are those that permit nucleic acid
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 or acetylated BSA, single strand binding proteins (for e.g., 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. Preferably, the
amplification reactions (particularly, primer extension other than
the composite and second primer extension product synthesis steps,
and strand displacement) are performed isothermally, which avoids
the cumbersome thermocycling process. The amplification reaction is
carried out at a temperature that permits hybridization of the
oligonucleotides (primer) of the invention to the template
polynucleotide and primer extension products, and that does not
substantially inhibit the activity of the enzymes employed. The
temperature can be in the range of 0.degree. C. to about 85.degree.
C., about 25.degree. C. to about 85.degree. C., about 30.degree. C.
to about 80.degree. C., and about 37.degree. C. to about 75.degree.
C.
[0173] Random priming and/or primer extension and/or isothermal
amplification can be conducted under conditions of reduced
stringency (i.e., permitting hybridization of sequences that are
not fully complementary). For a given set of reaction conditions,
the ability of two nucleotide sequences to hybridize with each
other is based on the degree of complementarity of the two
nucleotide sequences, which in turn is based on the fraction of
matched complementary nucleotide pairs. The more nucleotides in a
given sequence that are complementary to another sequence, the more
stringent the conditions can be for hybridization and the more
specific will be the binding of the two sequences. Conversely, the
lower the stringency of the conditions for hybridization, the lower
the complementarity necessary for binding between the hybridizing
and/or partially hybridizing composite primer and template
polynucleotide. Decreased stringency is achieved by any one or more
of the following: reducing the temperature, decreasing the ratio of
cosolvents, lowering the salt concentration, and the like.
Conditions that increase or reduce the stringency of a
hybridization reaction are widely known and published in the art.
See, for example, Sambrook et al. (1989), and in Ausubel (1987),
supra. Useful hybridization conditions are also provided in, e.g.,
Tijessen, 1993, Hybridization With Nucleic Acid Probes, Elsevier
Science Publishers B. V. and Kricka, 1992, Nonisotopic DNA Probe
Techniques, Academic Press San Diego, Calif. The hybridization
conditions chosen depend on a variety of factors known in the art,
for example the length and type (e.g., RNA, DNA, PNA) of primer and
primer binding region of the oligonucleotide template, as well as
the concentration of primer and template polynucleotides.
[0174] Insofar as it is convenient to use buffer conditions that
are compatible with DNA polymerase activity and/or ribonuclease
activity, stringency of hybridization of composite primers can be
controlled by altering temperature of the reaction. Examples of
relevant conditions include (in order of increasing stringency):
incubation temperatures of approximately 15.degree. C., 20.degree.
C., 25.degree. C., 30.degree. C., 37.degree. C., 40.degree. C.,
45.degree. C., 50.degree. C., 60.degree. C., or more. Accordingly,
in some embodiments, composite primer random hybridization occurs
at a reduced temperature, for example at 25.degree. C.-37.degree.
C., followed at incubation at increased temperature(s) suitable for
the isothermal amplification phase of the methods (such as about
50.degree. C.). In some embodiments, temperature is increased at
5.degree. C. increments. In other embodiments, temperature is
shifted from low to high temperature.
[0175] 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. In some embodiments, a
nucleotide or nucleotide analog whose presence in the primer
extension strand enhances displacement of the strand (for example,
by causing base pairing that is weaker than conventional AT, CG
base pairing) is included. Such nucleotide or nucleotide analogs
include deoxyinosine and other modified bases, all of which are
known in the art.
[0176] The oligonucleotide components of the amplification
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, 10.sup.12 times the amount
of target nucleic acid. Composite primers can each be provided at
about or at least about any of the following concentrations: 50 nM,
100 nM, 500 nM, 1 uM, 2.5 uM, 5 uM, 10 uM. Composite primer
concentration also impacts frequency and/or position of composite
primer hybridization. Generally, increased primer concentrations
increased frequency of primer hybridization. Auxiliary primers can
be provided at about or at least about any of the following
concentrations: about 25 nM, about 50 nM, about 100 nM, about 500
nM, about 1 uM, about 2.5 uM, about 5 uM, about 10 uM, or more.
[0177] In one embodiment, the foregoing components are added
simultaneously at the initiation of the 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 the
target nucleic acid denaturation step, following the denaturation
step, or following hybridization of the primer to the target
polynucleotide, as determined by their thermal stability and/or
other considerations known to the person of skill in the art. In
these embodiments, the reaction conditions and components may be
varied between the different reactions.
[0178] The amplification process 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 random composite primer hybridization. Another
timepoint is at the end of random composite primer hybridization
and composite primer extension product synthesis. Another timepoint
(in some embodiments) is following cleavage of template RNA.
Another timepoint is immediately prior to initiation of single
primer isothermal amplification (which in some embodiments, may be
initiated by addition of an enzyme (such as RNase H) that cleaves
RNA from RNA/DNA heteroduplex, and optionally, DNA polymerase).
Another timepoint is at the end of 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, replenishing a destroyed
(depleted) enzyme, or adding reagent(s) necessary for initiation of
a step (for example, addition of RNase H and/or DNA polymerase to
initiate the single primer isothermal amplification phase of the
methods). In some embodiments, one or more of the components of the
reactions is replenished prior to, at, or following the resumption
of the reactions. For example, it may be necessary to replenish the
composite primer prior to beginning the single primer isothermal
amplification reaction if the same composite primer is being used.
Alternatively, the reaction can be allowed to proceed (i.e., from
start to finish) without interruption.
[0179] 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 formation of the complex comprising an RNA/DNA partial
heteroduplex. Another timepoint is at the end of random composite
primer hybridization.
[0180] The detection of the amplification product is indicative of
the presence of the target sequence. Quantitative analysis is also
feasible. 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.
Compositions and Kits of the Invention
[0181] The invention also provides compositions and kits used in
the methods described herein. The compositions may be any
component(s), reaction mixture and/or intermediate described
herein, as well as any combination.
[0182] In one embodiment, the invention provides a composition
comprising a composite primer as described herein. In some
embodiments, the composite primer comprises an RNA portion adjacent
to the DNA portion. In another embodiment, the composite primer
comprises 5'- and 3'-DNA portions with at least one intervening RNA
portion. In other embodiments, the RNA portion of the composite
primer consists of 7 to about 20 nucleotides and the DNA portion of
the composite primer consists of about 5 to about 20 nucleotides.
In still other embodiments, the RNA portion of the composite primer
consists of about 10 to about 20 nucleotides and the DNA portion of
the composite primer consists of about 7 to about 20 nucleotides.
In some embodiments, the composite primer is selected from the
following composite primers: 5'-GACGGAUGCGGUCUdCdCdAdGdTdGdT-3 (SEQ
ID NO:1); and 5'-CGUAUUCUGACGACGUACUCdTdCdAdGdCdCdT-3' (SEQ ID
NO:2), wherein italics denote ribonucleotides and "d" denotes
deoxyribonucleotides.
[0183] In other examples, the invention provides a composition
comprising a composite primer as described herein, and auxiliary
primers (for example, a population of randomized hexamer primers).
In some embodiments, the composite primer is selected from the
following composite primers: 5'-GACGGAUGCGGUCUdCdCdAdGdTdGdT-3 (SEQ
ID NO:1); and 5'-CGUAUUCUGACGACGUACUCdTdCdAdGdCdCdT-3' (SEQ ID
NO:2), wherein italics denote ribonucleotides and "d" denotes
deoxyribonucleotides.
[0184] In other examples, the invention provides a composition
comprising a composite primer that is derivatized by attachment of
a moiety capable of effecting attachment of a polynucleotide
comprising the composite primer to a solid substrate used in
preparing nucleic acid microarrays. In some embodiments, the
composite primer is further by attachment of a positively charged
moiety such as an amine. In other embodiments, the composite primer
is labeled, for example by derivatizing the composite primer with a
detectable moiety, such as a label, or a moiety that can be
covalently or non-covalently attached to a label. Labeled composite
primers are further described herein.
[0185] In other examples, the invention provides composition
comprising a composite primer and one or more of: a DNA polymerase;
an enzyme that cleaves RNA from an RNA/DNA duplex; and auxiliary
primers (for example, a population of random hexamer primers). In
some embodiments, the composition further comprises a labeled dNTP.
In still other embodiments, the composition comprises a
non-canonical nucleotide (such as dUTP), and reagents suitable for
labeling and/or fragmenting abasic sites, as described in U.S.
Patent Application Publication No. 2004/0005614 and Kurn et al,
co-pending U.S. patent application No. 60/533,381.
[0186] The compositions are generally in lyophilized or aqueous
form, preferably in a suitable buffer.
[0187] The invention also provides compositions comprising the
amplification products described herein. Accordingly, the invention
provides a population of DNA which are copies or the complement of
a target sequence, which are produced by any of the methods
described herein (or compositions comprising the products). The
invention also includes compositions and various configurations
(such as arrays) of these populations, which may be homogeneous
(same sequence) or heterogeneous (different sequence). These
populations may be any assembly of sequences obtained from the
methods described herein.
[0188] The compositions are generally in a suitable medium,
although they can be in lyophilized form. Suitable media include,
but are not limited to, aqueous media (such as pure water or
buffers).
[0189] The invention provides kits for carrying out the methods of
the invention. Accordingly, a variety of kits are provided in
suitable packaging. The kits may be used for any one or more of the
uses described herein, and, accordingly, may contain instructions
for any one or more of the following uses: methods of
amplification; genotyping, nucleic acid mutation detection
(including methods of genotyping), determining the presence or
absence of a sequence of interest, quantitating a sequence of
interest, preparation of an immobilized nucleic acid (which can be
a nucleic acid immobilized on a microarray), comparative genomic
hybridization, and characterizing nucleic acids using the amplified
nucleic acid products generated by the methods of the invention,
methods of expression profiling, subtractive hybridization and the
preparation of probes for subtractive hybridization, and methods of
preparing libraries (which may be cDNA and/or differential
hybridization libraries).
[0190] The kits of the invention comprise one or more containers
comprising any combination of the components described herein, and
the following are examples of such kits. A kit may comprise any of
the composite primers described herein. In some embodiments, the
kit comprises one or more composite primer selected from the
following composite primers: 5'-GACGGAUGCGGUCUdCdCdAdGdTdGdT-3 (SEQ
ID NO:1); and 5'-CGUAUUCUGACGACGUACUCdTdCdAdGdCdCdT-3' (SEQ ID
NO:2), wherein italics denote ribonucleotides and "d" denotes
deoxyribonucleotides. In some embodiments, a kit further comprises
auxiliary primers, which may or may not be separately packaged. The
composite primer may be labeled or unlabeled. Kits may also
optionally further include any of one or more of the enzymes
described herein (for example, DNA-dependent DNA polymerase,
RNA-dependent DNA polymerase, a DNA polymerase that provides both
DNA-dependent and RNA-dependent DNA polymerase activities, and an
enzyme capable of cleaving RNA from an RNA/DNA hybrid, such as
RNase H), as well as deoxynucleoside triphosphates (labeled or
unlabeled or derivatized). Kits may also include one or more
suitable buffers (for example, as described herein). Kits may also
include a labeled dNTP(s) and/or a non-canonical nucleotide (such
as dUTP), as described in Kurn et al, co-pending U.S. patent
application No. 60/381,457.
[0191] One or more reagents in the kit can be provided as a dry
powder, usually lyophilized, including excipients, which on
dissolution will provide for a reagent solution having the
appropriate concentrations for performing any of the methods
described herein. Each component can be packaged in separate
containers or some components can be combined in one container
where cross-reactivity and shelf life permit.
[0192] The kits of the invention may optionally include a set of
instructions, generally written instructions, although electronic
storage media (e.g., magnetic diskette or optical disk) containing
instructions are also acceptable, relating to the use of components
of the methods of the invention for the intended nucleic acid
amplification, and/or, as appropriate, for using the amplification
products for purposes such as detection of sequence mutation. The
instructions included with the kit generally include information as
to reagents (whether included or not in the kit) necessary for
practicing the methods of the invention, instructions on how to use
the kit, and/or appropriate reaction conditions.
[0193] In another example, the kits of the invention comprise a
complex of composite primer extension product and second primer
extension product. In yet another example, any of these kits
further comprises one or more controls (which can be, for example,
template polynucleotide (e.g., DNA template such as genomic DNA or
RNA template such as total RNA or mRNA), composite primers, and/or
auxiliary primer(s).
[0194] The component(s) of the kit may be packaged in any
convenient, appropriate packaging. The components may be packaged
separately, or in one or multiple combinations.
[0195] The relative amounts of the various components in the kits
can be varied widely to provide for concentrations of the reagents
that substantially optimize the reactions that need to occur to
practice the methods disclosed herein and/or to further optimize
the sensitivity of any assay.
[0196] The invention also provides systems for effecting the
methods described herein. These systems comprise various
combinations of the components discussed above.
[0197] Any of the systems embodiments may also comprise a template
(target) sequence, as described herein. A system generally includes
one or more apparatuses for performing the amplification methods of
the invention. Such apparatuses include, for example, heating
devices (such as heating blocks or water baths) and apparatuses
which effect automation of one or more steps of the methods
described herein. The methods of the invention are particularly
suitable for use with miniaturized devices, as thermal cycling is
not required for any of the steps. A non-limiting example of
suitable devices includes the BioAnalyzer (Agilant and Caliper) and
the eSensor.
[0198] The invention also provides reaction mixtures (or
compositions comprising reaction mixtures) which contain various
combinations of components described herein. Examples of reaction
mixtures have been described. In some embodiments, the invention
provides reaction mixtures comprising (a) a target polynucleotide;
(b) a composite primer comprising a 3' DNA portion and an RNA
portion; (c) auxiliary primers; and (d) DNA polymerase. As
described herein, any of the composite primers may be in the
reaction mixture (or a plurality of composite primers), including a
composite primer that comprises a 5' RNA portion which is adjacent
to the 3' DNA portion. The reaction mixture could also further
comprise an enzyme which cleaves RNA from an RNA/DNA hybrid, such
as RNase H. In some embodiments, the composite primer is selected
from the following composite primers:
5'-GACGGAUGCGGUCUdCdCdAdGdTdGdT-3 (SEQ ID NO:1); and
5'-CGUAUUCUGACGACGUACUCdTdCdAdGdCdCdT-3' (SEQ ID NO:2), wherein
italics denote ribonucleotides and "d" denotes
deoxyribonucleotides.
[0199] Other reaction mixtures are described herein and are
encompassed by the invention.
[0200] The invention also includes compositions comprising any of
the complexes (which are intermediates in the methods described
herein) described herein. Examples of such complexes are
schematically depicted in FIGS. 1-8. As an example, one complex of
the invention is a complex comprising: (a) a target polynucleotide
strand; and (b) a composite primer, said composite primer
comprising a 3' DNA portion and an RNA portion. The composite
primer may have an RNA portion which is 5' and adjacent to the 3''
DNA portion. As another example, a complex of the invention is a
complex comprising: (a) a composite primer extension product; and
(b) a target polynucleotide.
[0201] In yet another example, a complex of the invention is a
complex comprising a RNA/DNA partial heteroduplex, prepared by any
of the methods described herein. In some embodiments, the complex
further comprises a second RNA/DNA partial heteroduplex at a second
end. In yet another example, the complex of the invention is a
complex comprising a 3' single stranded DNA portion produced by any
of the methods described herein. In some embodiments, the complex
further comprises a second 3' single stranded region. In another
example, the complex of the invention is (a) a complex comprising a
3' single stranded DNA portion, and (b) a composite primer
hybridized to the 3' single stranded portion.
Methods Using the Amplification Methods and Compositions of the
Invention
[0202] The methods and compositions of the invention can be used
for a variety of purposes. For purposes of illustration, methods of
nucleic acid mutation detection (including methods of genotyping),
determining the presence or absence of a sequence of interest,
quantitating a sequence of interest, preparation of an immobilized
nucleic acid (which can be a nucleic acid immobilized on a
microarray), comparative genomic hybridization, and characterizing
nucleic acids using the amplified nucleic acid products generated
by the methods of the invention, detecting and/or identifying novel
nucleic acid sequences (such as novel coding or non-coding
transcripts), and characterization of splice variant sequences, are
described. Methods of expression profiling, methods of subtractive
hybridization and the preparation of probes for subtractive
hybridization, and methods of preparing libraries (which can be
cDNA and/or differential hybridization libraries) are also
described.
Method of Preparing Nucleic Acids Immobilized to a Substrate,
Including a Microarray of Nucleic Acids
[0203] The products of some of the amplification methods of the
invention are suitable for immobilizing to a surface. In so far as
the amplification products of the methods of the invention
generally comprises a mixture of sequences corresponding to sense
and antisense copies of template polynucleotide, it is useful to
immobilize a population of sequences generated by amplification of
template polynucleotide from a defined source (e.g., DNA or RNA
from a defined cell population or a single cell; organism-specific
template (for example, the DNA or RNA of specific viruses or other
pathogen(s) sufficient to identify the organism); or a
disease-specific template. Immobilized amplification product may
then be probed with different probes and the hybridization signals
can be compared. For example, an immobilized array of genomic
polynucleotides (DNA or RNA) from a known pathogen or non-pathogen
(such as a virus, or group of viruses) may be used for assessment
of the presence or identity of a pathogen within a sample of
genetic material. Such arrays would be of use in disease
surveillance and in identification of a pathogenic agent in the
event of a disease outbreak. Polynucleotides may be isolated from a
suspected sample, labeled using any method known in the art, and
hybridized to such an array. The detection of signal due to
hybridization to the array provides information as to the presence
or identity of a pathogen present in sample polynucleotide.
[0204] Amplification products can be attached to a solid or
semi-solid support or surface, which may be made, e.g., from glass,
plastic (e.g., polystyrene, polypropylene, nylon), polyacrylamide,
nitrocellulose, or other materials.
[0205] Several techniques are well-known in the art for attaching
nucleic acids to a solid substrate such as a glass slide. One
method is to incorporate modified bases or analogs that contain a
moiety that is capable of attachment to a solid substrate, such as
an amine group, a derivative of an amine group or another group
with a positive charge, into the amplified nucleic acids. The
amplification product is then contacted with a solid substrate,
such as a glass slide, which is coated with an aldehyde or another
reactive group which will form a covalent link with the reactive
group that is on the amplification product and become covalently
attached to the glass slide. Microarrays comprising the
amplification products can be fabricated using a Biodot (BioDot,
Inc. Irvine, Calif.) spotting apparatus and aldehyde-coated glass
slides (CEL Associates, Houston, Tex.). Amplification products can
be spotted onto the aldehyde-coated slides, and processed according
to published procedures (Schena et al., Proc. Natl. Acad. Sci.
U.S.A. (1995) 93:10614-10619). Arrays can also be printed by
robotics onto glass, nylon (Ramsay, G., Nature Biotechnol. (1998),
16:40-44), polypropylene (Matson, et al., Anal Biochem. (1995),
224(1):110-6), and silicone slides (Marshall, A. and Hodgson, J.,
Nature Biotechnol. (1998), 16:27-31). Other approaches to array
assembly include fine micropipetting within electric fields
(Marshall and Hodgson, supra), and spotting the polynucleotides
directly onto positively coated plates. Methods such as those using
amino propyl silicon surface chemistry are also known in the art,
as disclosed at http://www.cmt.corning.com and
http://cmgm.stanford.edu/pbrown/.
[0206] One method for making microarrays is by making high-density
polynucleotide arrays. Techniques are known for rapid deposition of
polynucleotides (Blanchard et al., Biosensors & Bioelectronics,
11:687-690). Other methods for making microarrays, e.g., by masking
(Maskos and Southern, Nuc. Acids. Res. (1992), 20:1679-1684), may
also be used. In principle, and as noted above, any type of array,
for example, dot blots on a nylon hybridization membrane, could be
used. However, as will be recognized by those skilled in the art,
very small arrays will frequently be preferred because
hybridization volumes will be smaller.
[0207] The amplified polynucleotides may be spotted as a matrix on
substrates comprising paper, glass, plastic, polystyrene,
polypropylene, nylon, polyacrylamide, nitrocellulose, silicon,
optical fiber or any other suitable solid or semi-solid (e.g., thin
layer of polyacrylamide gel (Khrapko, et al., DNA Sequence (1991),
1:375-388) surface.
[0208] An array may be assembled as a two-dimensional matrix on a
planar substrate or may have a three-dimensional configuration
comprising pins, rods, fibers, tapes, threads, beads, particles,
microtiter wells, capillaries, cylinders and any other arrangement
suitable for hybridization and detection of target molecules. In
one embodiment the substrate to which the amplification products
are attached is magnetic beads or particles. In another embodiment,
the solid substrate comprises an optical fiber. In yet another
embodiment, the amplification products are dispersed in fluid phase
within a capillary which, in turn, is immobilized with respect to a
solid phase.
[0209] Arrays may also be composed of particles, such as beads. The
beads may be labeled with the amplified products alone, or may be
labeled with both the amplified products and an additional label,
such as defined dyes or other labels.
Characterization of Nucleic Acids
[0210] The amplification products obtained by the methods of the
invention are amenable to further characterization. The products of
the methods of the invention are particularly amenable to
quantitative analysis, as sufficient DNA is produced which
generally accurately reflect the representation of the various
polynucleotides in the starting material.
[0211] The amplified polynucleotide products (i.e., products of any
of the amplification methods described herein), can be analyzed
using, for example, probe hybridization techniques known in the
art, such as Southern and Northern blotting, and hybridizing to
probe arrays. They can also be analyzed by electrophoresis-based
methods, such as differential display and size characterization,
which are known in the art. In addition, the polynucleotide
products may serve as 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 U.S. Pat. Nos. 6,251,639, 6,686,156, and
6,692,918; U.S. Patent Publication Nos. 2002/0115088 A1,
2003/0186234 A1, 2003/0087251A1, 2002/0164628, and 2003/0215926,
and International Patent Application Publication WO 03/08343. Thus,
the invention includes those further analytical and/or
quantification methods as applied to any of the products of the
methods herein.
[0212] In one embodiment, the amplification methods of the
invention are utilized to generate multiple copies of
polynucleotide products, and products are analyzed by contact with
a probe.
[0213] In some embodiments, the amplification methods of the
invention are utilized to generate multiple copies of single
stranded polynucleotide (generally, DNA) products that are labeled
by using composite primers that are labeled (in the portion(s) that
is not cleaved). For example, the primer can be labeled with an
aminoallyl labeled nucleotide. In other embodiments, the
amplification methods of the invention are utilized to generate
multiple copies of polynucleotide (generally, DNA) products that
are labeled by the incorporation of labeled nucleotides during DNA.
For example, amplification according to the methods of the
invention can be carried out with suitable labeled dNTPs. 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 template polynucleotide
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. Amplification can also
be in the presence of an aminoallyl-derivatized nucleotide, such as
aminoallyl dUTP. Amplification product comprising aminoallyl dUTP
can be coupled to a label, such as Cy3 or Cy5.
[0214] In other embodiments, the methods of the amplification are
performed in the present of a non-canonical nucleotide, e.g., dUTP,
and amplification comprising a non-canonical nucleotide is labeled
and/or fragmented according to the methods disclosed in U.S. Patent
Application Publication No. 2004/0005614. Briefly, non-canonical
nucleotide (when incorporated into amplification product) is
cleaved, generating an abasic site. The abasic site is then labeled
by contacting with a reagent capable of labeling an abasic site.
The polynucleotide comprising an abasic site can also be cleaved at
the abasic site, generating fragments suitable for further
analysis, e.g., hybridization to an array. The fragments can also
be labeled as described above.
[0215] The labeled amplification products are particularly 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) an target
polynucleotide 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,
which have been described above. 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 amplification products, and thus by
extrapolation the identity of the target polynucleotide 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
target polynucleotide of interest.
[0216] 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 polynucleotide
species in the sample.
[0217] Methods of characterization include sequencing by
hybridization (see, e.g., Dramanac, U.S. Pat. No. 6,270,961) and
global genomic hybridization (also termed comparative genome
hybridization) (see, e.g., Pinkel, U.S. Pat. No. 6,159,685; Daigo
et al (2001) Am. J. Pathol. 158 (5):1623-1631. Briefly, comparative
genome hybridization comprises preparing a first population of
labeled polynucleotides (which can be polynucleotide fragments)
according to any of the methods described herein, wherein the
template from which the first population is synthesized is total
genomic DNA. A second population of labeled polynucleotides (to
which the first population is desired to be compared) is prepared
from a second genomic DNA template. The first and second
populations are labeled with different labels. The hybridized first
and second populations are mixed, and hybridized to an array or
chromosomal spread. The different labels are detected and
compared.
[0218] In another aspect, the invention provides a method of
quantitating labeled and/or fragmented nucleic acids comprising use
of an oligonucleotide (probe) of defined sequence (which may be
immobilized, for example, on a microarray).
[0219] The amplification products generated according to the
methods of the invention are also suitable for analysis for the
detection of any alteration in the target nucleic acid sequence, as
compared to a reference nucleic acid sequence which is identical to
the target nucleic acid sequence other than the sequence
alteration. When the target polynucleotide is genomic DNA or RNA,
the sequence alterations may be sequence alterations present in the
genomic sequence or may be sequence alterations which are not
reflected in the genomic sequence, for example, alterations due to
post transcriptional alterations, and/or mRNA processing, including
splice variants. Sequence alterations (interchangeably called
"mutations") include deletion, substitution, insertion and/or
transversion of one or more nucleotide.
[0220] Other art recognized methods of analysis for the detection
of any alteration in the target nucleic acid sequence, as compared
to a reference nucleic acid sequence, are suitable for use with the
nucleic acid products of the amplification methods of the
invention. Such methods are well-known in the art, and include
various methods for the detection of specific defined sequences
including methods based on allele specific primer extension, allele
specific probe ligation, differential probe hybridization, and
limited primer extension. See, for example, Kurn et al, U.S. Pat.
No. 6,251,639 B1; U.S. Pat. Nos. 5,888,819; 6,004,744; 5,882,867;
5,854,033; 5,710,028; 6,027,889; 6,004,745; 5,763,178; 5,011,769;
5,185,243; 4,876,187; 5,882,867; 5,731,146; WO US88/02746; WO
99/55912; WO 92/15712; WO 00/09745; WO 97/32040; WO 00/56925; and
5,660,988. Thus, the invention also provides methods for detection
of a mutation in a target polynucleotide comprising a mutation
(which can be a single nucleotide polymorphism), comprising: (a)
amplifying a target polynucleotide using any of the methods
described herein; and (b) analyzing the amplification products for
presence of an alteration (mutation) as compared to a reference
polynucleotide.
[0221] It is understood that the amplification products can also
serve as template for further analysis such as sequence,
polymorphism detection (including multiplex SNP detection) using,
e.g., oligonucleotide ligation-based assays, analysis using
Invader, Cleavase or limited primer extension, and the like. For
methods that generally require larger volumes of input material,
the methods of the invention may be used to "pre" amplify a pool of
polynucleotides to generate sufficient input material for
subsequent analysis.
Determination of Gene Expression Profile
[0222] The amplification methods of the invention are particularly
suitable for use in determining the levels of expression of one or
more genes in a sample since the methods described herein are
capable of amplifying a multiplicity, including a large
multiplicity of 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 mRNAs, 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.
[0223] Accordingly, the invention provides methods of determining
gene expression profile in a sample, said method comprising:
amplifying single stranded product from template RNAs in the
sample, using any of the methods described herein; and determining
amount of amplification products of each RNA, wherein each said
amount is indicative of amount of each RNA in the sample, whereby
the expression profile in the sample is determined. Generally,
labeled products are generated. In certain embodiments, the target
RNA is mRNA. It is understood that amount of amplification product
may be determined using quantitative and/or qualitative methods.
Determining 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.
[0224] 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.
Method of Preparing a Library
[0225] The DNA products of the methods of the invention are useful
in preparing libraries, including cDNA libraries and subtractive
hybridization libraries. Using the methods of the invention,
libraries may be prepared from limited amount of starting material,
for example, mRNA extracted from limited amount of tissue or even
single cells. Accordingly, in one aspect, the methods of the
invention provides preparing a library from the DNA products of the
invention. In still another aspect, the invention provides methods
for making a library, said method comprising: preparing a
subtractive hybridization probe using any of the methods described
herein.
Methods of Subtractive Hybridization
[0226] The amplification methods of the invention are particularly
suitable for use in subtractive hybridization methods, in which (at
least) a first and second target polynucleotide population is
compared, since the methods described herein are capable of
amplifying multiple target polynucleotides in the same sample, and
the methods of the invention are suitable for producing large
amounts of single stranded antisense nucleic acid suitable for use
as "driver" in subtractive hybridization. For example, two nucleic
acid populations, one sense and one antisense, can be allowed to
mix together with one population present in molar excess
("driver"). Sequence present in both populations will form hybrids,
while sequences present in only one population remain
single-stranded. Thereafter, various well known techniques are used
to separate the unhybridized molecules representing differentially
expressed sequences. See, e.g., Hamson et al., U.S. Pat. No.
5,589,339; Van Gelder, U.S. Pat. No. 6,291,170. The methods of
subtractive hybridization provided herein are particularly suited
for subtractive hybridization using amplified target RNAs.
[0227] Accordingly, the invention provides methods for performing
subtractive hybridization, said methods comprising: (a) preparing
multiple DNA copies of the complement of target polynucleotide from
a first polynucleotide population using any of the amplification
methods described herein; and (b) hybridizing the multiple copies
to a second polynucleotide population, whereby a subpopulation of
the second polynucleotide population forms a complex with DNA
copies of the first polynucleotide population. The invention also
provides methods for performing subtractive hybridization, said
methods comprising: hybridizing multiple copies of the complement
of at least one polynucleotide from a first polynucleotide
population using any of the amplification methods described herein
to a second polynucleotide population, whereby a subpopulation of
the second population forms a complex with a copy from the copies
of the first polynucleotide population. In preferred embodiments,
the polynucleotide populations utilized in subtractive
hybridization are RNA populations. In some embodiments, "driver"
single stranded anti-sense DNA product of the methods of the
invention is combined with tester (sense) RNA species. In some
embodiments, "driver" single stranded antisense nucleic acid
(generally, DNA) product is produced using the methods of the
invention described herein.
[0228] In another aspect, the invention provides methods of
differential amplification in which single stranded driver
(antisense) DNA sequences that hybridize with tester RNA sequence
are subjected to cleavage by an agent that cleaves RNA present in a
DNA/RNA hybrid, such as RNase H. Cleavage of the RNA results in the
inability to generate single stranded DNA product from the test RNA
strands. Conversely, non-cleaved tester (i.e., tester RNA that did
not hybridize to driver DNA molecules) may serve as a substrate for
subsequent amplification. Amplified differentially expressed
products have many uses, including as a differential expression
probe, to produce differential expression libraries. Accordingly,
the invention provides methods for differential amplification of
one or more RNA template sequence, said method comprising: (a)
preparing multiple polynucleotide (generally, DNA) copies of the
complement of RNA from a first RNA population using any of the
amplification methods described herein; (b) hybridizing the
multiple copies to a second RNA population, whereby a subpopulation
of the second RNA population forms a complex with a DNA copy; (c)
cleaving RNA in the complex of step (b) with an enzyme that
cleaves-RNA from an RNA/DNA hybrid; and (d) amplifying an
unhybridized subpopulation of the second RNA population, whereby
multiple copies of single stranded DNA complementary to the
unhybridized subpopulation of the second RNA population are
generated. In some embodiments, step (d) is performed using any of
the amplification methods described herein. In some embodiments,
the methods comprise hybridizing multiple polynucleotide
(generally, DNA) copies of the complement of at least one RNA
sequences of interest from a first RNA population using any of the
amplification methods described herein to a second RNA population,
whereby a subpopulation of the second RNA population forms a
complex with a DNA copy; (b) cleaving RNA in the complex of step
(a) with an enzyme that cleaves RNA from an RNA/DNA hybrid; and (c)
amplifying an unhybridized subpopulation of the second RNA
population, whereby multiple copies of single stranded DNA
complementary to the unhybridized subpopulation of the second RNA
population are generated.
[0229] The following Examples are provided to illustrate, but not
limit, the invention.
EXAMPLES
Example 1
Global Amplification of Human Genomic DNA Using a Composite Primer
and Random Hexamer Primers
[0230] Global amplification reactions were performed using
composite primer IA20 and human genomic DNA as a template. The
sequence of composite primer IA20 is as follows: IA20:
5'-GACGGAUGCGGUCUdCdCdAdGdTdGdT-3' (SEQ ID NO:1), where italics
denote ribonucleotides, and "d" denotes deoxyribonucleotides.
[0231] Human genomic DNA (Clontech, Cat. No. 6550-1) was diluted in
TE buffer and denatured by heating to 99.degree. C. In some
samples, DNA and primers were mixed in amplification buffer and
heated at 96.degree. C. for 2-4 minutes.
[0232] The following reaction mixture was used.
[0233] 2 ng of pre-denatured human genomic DNA (approximately 600
copies)
[0234] 2 .mu.l of random hexamer N6 (final concentration 2.5 .mu.L)
(Qiagen-Operon; item No.: PolyN (6-mer));
[0235] 0.5 .mu.l (100 .mu.M) composite primer IA20 (final
concentration: 2.5 .mu.M),
[0236] 0.1 .mu.l Bst DNA polymerase (0.04 U/.mu.l) (New England
BioLabs, Catalog No. M0275);
[0237] 10 .mu.l buffer (final concentration: 20 mM Tris-HCl, pH
8.5; 5 mM MgCl.sub.2; RNasin, 0.3 U/.mu.l; DTT, 0.5 mM; acetylated
BSA, 0.1 .mu.g/.mu.l; T4 gp32 protein, 0.15 .mu.g/.mu.l)), and
[0238] RNase-free water to final volume of 15 .mu.l.
[0239] The mixture was incubated at 30.degree. C. for 5 minutes,
followed by 5 minutes at 40.degree. C., and 2 minutes at 50.degree.
C.
[0240] 5 .mu.l of enzyme mixture (RNase H, final concentration of
0.025 U/.mu.l; and Bst DNA polymerase (large fragment), final
concentration 0.2 U/.mu.l) was added, and the reaction was
incubated at 50.degree. C. for 30-40 minutes. The reaction was
stopped by incubation at 80.degree. C. for 5 minutes to inactivate
the enzymes.
[0241] Control reactions were prepared in which either composite
primer, N6 random primers, or RNase H were omitted. In control
reactions, the corresponding volume (of the omitted reagent) was
replaced by water.
[0242] Amplification reaction product was analyzed as follows. 0.5
.mu.l of reaction mixture was loaded onto 4-20% gradient acrylamide
gel and electrophoresed at 200-220 V constant voltage for 30 min.
The gel was stained in 0.005 mg/ml Ethidium Bromide for 2 minutes
and washed in water for 1 minute. The gel was then visualized and
photographed on the AlphaImager 2200 system. The results are shown
in FIG. 9. Lanes correspond to the reaction mixtures containing the
following components:
[0243] Lanes #1-2: Complete reaction mixture
[0244] Lanes #3-4: Reaction lacking N6 random primer.
[0245] Lanes #5-6: Reaction lacking composite primer.
[0246] Lanes #7-8: Reaction lacking RNase H.
[0247] FIG. 9 shows that amplification product of varying molecular
weight was produced in reactions containing the complete reaction
mixture (described above), and in reaction lacking N6 random
primers. However, reactions in which composite primer or RNase H
were omitted did not show detectable reaction product.
[0248] Amplification product was quantified using the following
procedure: reactions were prepared and processed as described
above. Reaction mixture was diluted 100-fold and 2 .mu.l of the
diluted samples were used in Real Time PCR quantification with the
following primer pairs that amplify a single copy sequence on
Chromosome 7:
TABLE-US-00001 221PF2 (5'-AGTATCTGGCACATCTT-3' (SEQ TD
NO:.sub.----)) and 221PR2 (5'-GGGAGATATTATTTGGC-3'. (SEQ ID
NO:.sub.----))
[0249] Amplification with primers 221PF2 and 221 PR2 was expected
to yield a 62 base pair PCR product. The PCR reaction mixture
contained: 1 .mu.l of 10 .mu.M of each primer, 6 .mu.l of water, 10
.mu.l of 2.times.SYBR Green PCR Master Mix (Applied Biosystems),
and 2 .mu.l diluted reaction mixture (diluted as described above).
A control reaction was conducted using 2 .mu.l human genomic DNA,
instead of amplification reaction product as a template.
[0250] The thermal cycling program used was: one cycle of
94.degree. C. for 10 min., followed by 45 cycles of 94.degree. C.
for 30 sec., 55.degree. C. for 30 sec., and 72.degree. C. for 30
sec. The Real Time PCR quantification data were presented as Ct
values (threshold cycle). The values obtained for quantification of
amplification products were compared with that obtained for 2 .mu.l
human genomic DNA (labeled "non-amplified Genomic DNA" in Table 1).
The dilution factor between the diluted global amplification
products and the original human genomic DNA input into the
amplification reactions is 1000 fold. The data were summarized in
Table 1.
TABLE-US-00002 TABLE 1 Quantification of amplification products and
target human genomic DNA employing Real Time PCR with SYBR Green.
Reaction components Ct Efficiency Complete 29 4000 fold No N6
random primers 36 40 fold No Composite primer None None No RNase H
None None None (non amplified Genomic DNA) 31 None A single copy
sequence on Chromosome 7 of human genomic DNA (221) was used for
determination of amplification efficiency. Amplification efficiency
is expressed as the relative amount of this single copy sequence in
starting genomic DNA sample and following global amplification.
[0251] In a second experiment, human genomic DNA was amplified
using composite primer IA20 essentially as described above.
Following amplification, several target sequences were quantified
using Real Time PCR essentially as described above. Chromosomal
location of target sequences and PCR primer pairs are shown in
Table 2. Table 2 shows the relative amount of target sequence
following global amplification, and the amplification efficiency.
Real Time PCR quantification data were presented as Ct values
(threshold cycle).
TABLE-US-00003 TABLE 2 Target Real time Amplification location
Forward PCR primer Reverse PCR primer Delta C(t) fold chromosome #6
GGACGTGTGTTCCTGTTAA CACTTTGATCCTGAAAGACT 3.5 2000 (SEQ ID
NO.sub.----) (SEQ ID NO:.sub.----) chromosome #7 AGTATCTGGCACATCT
GGGAGATATTAATTTGGC 4 3000 (SEQ ID NO:.sub.----) (SEQ ID
NO:.sub.----) chromosome #11 AGGTTCCCAGCCTTGGTCC
TGAGGCCATGTGTGTGGAAT 2 800 (SEQ ID NO:.sub.----) (SEQ ID
NO:.sub.----) chromosome #12 AATAATGTCCAGATATCTTGGT
TCCCTACTCCAGCTACTTCT 2.5 1000 (SEQ ID NO:.sub.----) (SEQ ID
NO:.sub.----) chromosome #16 CAGCAAGAACACAAGGGAC TCTTGAGAGCGAGGGCA
2.5 1000 (SEQ ID NO:.sub.----) (SEQ ID NO:.sub.----)
[0252] In a third experiment, human genomic DNA was amplified using
composite primer BSCA-128F essentially as described above. The
sequence of composite primer BSCA-128F is:
[0253] 5'-CGUAUUCUGACGACGUACUCdTdCdAdGdCdCdT-3' (SEQ ID NO:2) where
italics denote ribonucleotides, and "d" denotes
deoxyribonucleotides.
[0254] Amplification reaction product was analyzed as described
above. Amplification product of varying molecular weights was
generated, suggesting that the composite primer permitted
amplification from a multiplicity of template sequences.
[0255] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be apparent to those skilled in the art
that certain changes and modifications may be practiced. Therefore,
the descriptions and examples should not be construed as limiting
the scope of the invention.
Sequence CWU 1
1
12121DNAArtificial SequenceDescription of Combined DNA/RNA Molecule
Synthetic primer 1gacggaugcg gucuccagtg t 21227DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic primer
2cguauucuga cgacguacuc tcagcct 27317DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
3agtatctggc acatctt 17417DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 4gggagatatt atttggc
17519DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 5ggacgtgtgt tcctgttaa 19620DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
6cactttgatc ctgaaagact 20719DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 7aggttcccag ccttggtcc
19820DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 8tgaggccatg tgtgtggaat 20922DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
9aataatgtcc agatatcttg gt 221020DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 10tccctactcc agctacttct
201119DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 11cagcaagaac acaagggac 191217DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
12tcttgagagc gagggca 17
* * * * *
References