U.S. patent application number 10/700018 was filed with the patent office on 2004-04-01 for multiple displacement amplification.
Invention is credited to Lizardi, Paul M..
Application Number | 20040063144 10/700018 |
Document ID | / |
Family ID | 25484902 |
Filed Date | 2004-04-01 |
United States Patent
Application |
20040063144 |
Kind Code |
A1 |
Lizardi, Paul M. |
April 1, 2004 |
Multiple displacement amplification
Abstract
Disclosed are compositions and a method for amplification of
nucleic acid sequences of interest. The method is based on stand
displacement replication of the nucleic acid sequences of interest
by multiple primers. In one preferred form of the method, referred
to as multiple strand displacement amplification, two sets of
primers are used, a right set and a left set. The primers in the
right set are complementary to one strand of the nucleic acid
molecule to be amplified and the primers in the left set are
complementary to the opposite strand. The 5' end of primers in both
sets are distal to the nucleic acid sequence of interest when the
primers have hybridized to the nucleic acid sequence molecule to be
amplified. Amplification proceeds by replication initiated at each
primer and continuing through the nucleic acid sequence of
interest. A key feature of this method is the displacement of
intervening primers during replication by the polymerase. In
another preferred form of the method, referred to as whole genome
strand displacement amplification, a random set of primers is used
to randomly prime a sample of genomic nucleic acid (or another
sample of nucleic acid of high complexity). By choosing a set of
primers which are sufficiently random, the primers in the set will
be collectively, and randomly, complementary to nucleic acid
sequences distributed throughout nucleic acid in the sample.
Amplification proceeds by replication with a highly processive
polymerase initiated at each primer and continuing until
spontaneous termination. A key feature of this method is the
displacement of intervening primers during replication by the
polymerase. In this way, multiple overlapping copies of the entire
genome to be synthesized in a short time.
Inventors: |
Lizardi, Paul M.; (Hamden,
CT) |
Correspondence
Address: |
NEEDLE & ROSENBERG, P.C.
SUITE 1000
999 PEACHTREE STREET
ATLANTA
GA
30309-3915
US
|
Family ID: |
25484902 |
Appl. No.: |
10/700018 |
Filed: |
November 3, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10700018 |
Nov 3, 2003 |
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09911226 |
Jul 23, 2001 |
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6642034 |
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09911226 |
Jul 23, 2001 |
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09397915 |
Sep 17, 1999 |
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6280949 |
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09397915 |
Sep 17, 1999 |
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08946732 |
Oct 8, 1997 |
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6124120 |
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Current U.S.
Class: |
435/6.12 ;
435/91.2 |
Current CPC
Class: |
C12Q 1/6844 20130101;
C12Q 1/6844 20130101; C12Q 2525/179 20130101; C12Q 2537/143
20130101; C12Q 2531/119 20130101; C12Q 2531/125 20130101; C12Q
1/6844 20130101; C12Q 2531/119 20130101 |
Class at
Publication: |
435/006 ;
435/091.2 |
International
Class: |
C12Q 001/68; C12P
019/34 |
Claims
I claim:
1. A method of amplifying a target nucleic acid sequence, the
method comprising, (a) mixing a set of primers with a target
sample, to produce a primer-target sample mixture, and incubating
the primer-target sample mixture under conditions that promote
hybridization between the primers and the target sequence in the
primer-target sample mixture, (b) mixing DNA polymerase with the
primer-target sample mixture, to produce a polymerase-target sample
mixture, and incubating the polymerase-target sample mixture under
conditions that promote replication of the target sequence, wherein
replication of the target sequence results in replicated strands,
wherein during replication at least one of the replicated strands
is displaced from the target sequence by strand displacement
replication of another replicated strand.
2. The method of claim 1 wherein the target sequence comprises an
amplification target and a hybridization target, wherein the
hybridization target flanks the amplification target, wherein the
set of primers comprises a plurality of primers, wherein each
primer comprises a complementary portion, wherein the complementary
portions of the primers are each complementary to a different
portion of the hybridization target.
3. The method of claim 1 wherein step (b) further comprises
incubating the polymerase-target sample mixture under conditions
that promote strand displacement.
4. The method of claim 1 wherein the set of primers has 3 or more
primers.
5. The method of claim 4 wherein the set of primers has 4 or more
primers.
6. The method of claim 5 wherein the set of primers has 5 or more
primers.
7. The method of claim 1 wherein the conditions that promote
replication of the target sequence are substantially
isothermic.
8. The method of claim 1 wherein the conditions that promote
replication of the target sequence do not involve thermal
cycling.
9. The method of claim 1 wherein step (b) does not include thermal
cycling.
10. The method of claim 2 wherein the set of primers comprises a
right set of primers and a left set of primers, wherein the target
sequence is double-stranded, having a first and a second strand,
wherein the hybridization target comprises a right and left
hybridization target, wherein the right hybridizaiton target flanks
the amplification target on one end and the left hybridization
target flanks the amplification target on the other end, wherein
the complementary portions of the right set primers are (i) all
complementary to the first strand of the target sequence and (ii)
each complementary to a different portion of the right
hybridization target, and wherein the complementary portions of the
left set primers are (i) all complementary to the second strand of
the target sequence and (ii) each complementary to a different
portion of the left hybridization target.
11. The method of claim 10 wherein the right and left set of
primers each have 3 or more primers.
12. The method of claim 11 wherein the right and left set of
primers each have 4 or more primers.
13. The method of claim 12 wherein the right and left set of
primers each have 5 or more primers.
14. The method of claim 10 wherein the right and left set of
primers each have the same number of primers.
15. The method of claim 1 wherein the target sequence is a nucleic
acid sample of substantial complexity, and wherein the set of
primers comprises primers having random nucleotide sequences.
16. The method of claim 15 wherein the target sequence is a sample
of genomic nucleic acid.
17. The method of claim 15 wherein the primers are from 12 to 60
nucleotides in length.
18. The method of claim 17 wherein the primers are from 12 to 40
nucleotides in length.
19. The method of claim 18 wherein the primers are from 15 to 40
nucleotides in length.
20. The method of claim 19 wherein the primers are from 15 to 25
nucleotides in length.
21. The method of claim 15 wherein the primers are all of the same
length.
22. The method of claim 15 wherein each primer comprises a constant
portion and a random portion, wherein the constant portion of each
primer has the same nucleotide sequence and the random portion of
each primer has a random nucleotide sequence.
23. The method of claim 1 wherein the target sequence is
concatenated DNA.
24. The method of claim 23 wherein the concatenated DNA is
concatenated with linkers.
25. The method of claim 24 wherein each linker comprises a primer
complement portion, wherein each primer comprises a complementary
portion, wherein the complementary portion of each primer is
complementary to the complementary portion of the linkers.
26. The method of claim 23 wherein the set of primers comprises
primers having random nucleotide sequences.
27. The method of claim 26 wherein each primer comprises a constant
portion and a random portion, wherein the constant portion of each
primer has the same nucleotide sequence and the random portion of
each primer has a random nucleotide sequence.
28. The method of claim 23 wherein the concatenated DNA is formed
by ligating DNA fragments together.
29. The method of claim 28 wherein the DNA fragments are cDNA made
from mRNA.
30. The method of claim 29 wherein the mRNA comprises a mixture of
mRNA isolated from cells.
31. The method of claim 1 wherein the target sequence is not a
nucleic acid molecule made up of multiple tandem repeats of a
single sequence that was synthesized by rolling circle
replication.
32. A kit for amplifying a target nucleic acid sequence wherein the
target sequence comprises an amplification target and a
hybridization target, wherein the hybridization target flanks the
amplification target, the kit comprising a set of primers wherein
the set of primers comprises a plurality of primers, wherein each
primer comprises a complementary portion, wherein the complementary
portions of the primers are each complementary to a different
portion of the hybridization target, and a strand displacing DNA
polymerase or a DNA polymerase and a compatible strand displacement
factor.
33. The kit of claim 32 wherein the target sequence is
double-stranded, having a first and a second strand, wherein the
hybridization target comprises a right and left hybridization
target, wherein the right hybridization target flanks the
amplification target on one end and the left hybridization target
flanks the amplification target on the other end, wherein the set
of primers comprises a right set of primers and a left set of
primers, wherein the complementary portions of the right set
primers are (i) all complementary to the first strand of the target
sequence and (ii) each complementary to a different portion of the
right hybridization target, and wherein the complementary portions
of the left set primers are (i) all complementary to the second
strand of the target sequence and (ii) each complementary to a
different portion of the left hybridization target.
34. A kit for amplifying a target nucleic acid sequence wherein the
target sequence is a nucleic acid sample of substantial complexity,
the kit comprising a set of primers wherein the set of primers
comprises primers having random nucleotide sequences, and a strand
displacing DNA polymerase or a DNA polymerase and a compatible
strand displacement factor.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of copending application
Ser. No. 09/911,226, filed Jul. 23, 2001, entitled "Multiple
Displacement Amplification," by Paul M. Lizardi, which is a
continuation of application Ser. No. 09/397,915, filed Sep. 17,
1999, entitled "Multiple Displacement Amplification," by Paul M.
Lizardi, which is a continuation of application Ser. No.
08/946,732, filed Oct. 8, 1997, entitled "Multiple Displacement
Amplification," by Paul M. Lizardi, which applications are hereby
incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
[0002] The disclosed invention is generally in the field of nucleic
acid amplification.
BACKGROUND OF THE INVENTION
[0003] A number of methods have been developed for exponential
amplification of nucleic acids. These include the polymerase chain
reaction (PCR), ligase chain reaction (LCR), self-sustained
sequence replication (3SR), nucleic acid sequence based
amplification (NASBA), strand displacement amplification (SDA), and
amplification with Q.beta. replicase (Birkenmeyer and Mushahwar, J.
Virological Methods, 35:117-126 (1991); Landegren, Trends Genetics
9:199-202 (1993)).
[0004] Current methods of PCR amplification involve the use of two
primers which hybridize to the regions flanking a nucleic acid
sequence of interest such that DNA replication initiated at the
primers will replicate the nucleic acid sequence of interest. By
separating the replicated strands from the template strand with a
denaturation step, another round of replication using the same
primers can lead to geometric amplification of the nucleic acid
sequence of interest. PCR amplification has the disadvantage that
the amplification reaction cannot proceed continuously and must be
carried out by subjecting the nucleic acid sample to multiple
cycles in a series of reaction conditions.
[0005] A variant of PCR amplification, termed whole genome PCR,
involves the use of random or partially random primers to amplify
the entire genome of an organism in the same PCR reaction. This
technique relies on having a sufficient number of primers of random
or partially random sequence such that pairs of primers will
hybridize throughout the genomic DNA at moderate intervals.
Replication initiated at the primers can then result in replicated
strands overlapping sites where another primer can hybridize. By
subjecting the genomic sample to multiple amplification cycles, the
genomic sequences will be amplified. Whole genome PCR has the same
disadvantages as other forms of PCR.
[0006] Another field in which amplification is relevant is RNA
expression profiling, where the objective is to determine the
relative concentration of many different molecular species of RNA
in a biological sample. Some of the RNAs of interest are present in
relatively low concentrations, and it is desirable to amplify them
prior to analysis. It is not possible to use the polymerase chain
reaction to amplify them because the mRNA mixture is complex,
typically consisting of 5,000 to 20,000 different molecular
species. The polymerase chain reaction has the disadvantage that
different molecular species will be amplified at different rates,
distorting the relative concentrations of mRNAs.
[0007] Some procedures have been described that permit moderate
amplification of all RNAs in a sample simultaneously. For example,
in Lockhart et al., Nature Biotechnology 14:1675-1680 (1996),
double-stranded cDNA was synthesized in such a manner that a strong
RNA polymerase promoter was incorporated at the end of each cDNA.
This promoter sequence was then used to transcribe the cDNAs,
generating approximately 100 to 150 RNA copies for each cDNA
molecule. This weak amplification system allowed RNA profiling of
biological samples that contained a minimum of 100,000 cells.
However, there is a need for a more powerful amplification method
that would permit the profiling analysis of samples containing a
very small number of cells.
[0008] Accordingly, there is a need for amplification methods that
are less complicated, are more reliable, and produce greater
amplification in a shorter time.
[0009] It is therefore an object of the disclosed invention to
provide a method of amplifying a target nucleic acid sequence in a
continuous, isothermal reaction.
[0010] It is another object of the disclosed invention to provide a
method of amplifying an entire genome or other highly complex
nucleic acid sample in a continuous, isothermal reaction.
[0011] It is another object of the disclosed invention to provide a
method of amplifying a target nucleic acid sequence where multiple
copies of the target nucleic acid sequence are produced in a single
amplification cycle.
[0012] It is another object of the disclosed invention to provide a
method of amplifying a concatenated DNA in a continuous, isothermal
reaction.
[0013] It is another object of the disclosed invention to provide a
kit for amplifying a target nucleic acid sequence in a continuous,
isothermal reaction.
[0014] It is another object of the disclosed invention to provide a
kit for amplifying an entire genome or other highly complex nucleic
acid sample in a continuous, isothermal reaction.
BRIEF SUMMARY OF THE INVENTION
[0015] Disclosed are compositions and a method for amplification of
nucleic acid sequences of interest. The method is based on strand
displacement replication of the nucleic acid sequences by multiple
primers. In one preferred form of the method, referred to as
multiple strand displacement amplification (MSDA), two sets of
primers are used, a right set and a left set. Primers in the right
set of primers each have a portion complementary to nucleotide
sequences flanking one side of a target nucleotide sequence and
primers in the left set of primers each have a portion
complementary to nucleotide sequences flanking the other side of
the target nucleotide sequence. The primers in the right set are
complementary to one strand of the nucleic acid molecule containing
the target nucleotide sequence and the primers in the left set are
complementary to the opposite strand. The 5' end of primers in both
sets are distal to the nucleic acid sequence of interest when the
primers are hybridized to the flanking sequences in the nucleic
acid molecule. Preferably, each member of each set has a portion
complementary to a separate and non-overlapping nucleotide sequence
flanking the target nucleotide sequence. Amplification proceeds by
replication initiated at each primer and continuing through the
target nucleic acid sequence. A key feature of this method is the
displacement of intervening primers during replication. Once the
nucleic acid strands elongated from the right set of primers
reaches the region of the nucleic acid molecule to which the left
set of primers hybridizes, and vice versa, another round of priming
and replication will take place. This allows multiple copies of a
nested set of the target nucleic acid sequence to be synthesized in
a short period of time. By using a sufficient number of primers in
the right and left sets, only a few rounds of replication are
required to produce hundreds of thousands of copies of the nucleic
acid sequence of interest. The disclosed method has advantages over
the polymerase chain reaction since it can be carried out under
isothermal conditions. No thermal cycling is needed because the
polymerase at the head of an elongating strand (or a compatible
strand-displacement protein) will displace, and thereby make
available for hybridization, the strand ahead of it. Other
advantages of multiple strand displacement amplification include
the ability to amplify very long nucleic acid segments (on the
order of 50 kilobases) and rapid amplification of shorter segments
(10 kilobases or less). In multiple strand displacement
amplification, single priming events at unintended sites will not
lead to artifactual amplification at these sites (since
amplification at the intended site will quickly outstrip the single
strand replication at the unintended site).
[0016] In another preferred form of the method, referred to as
whole genome strand displacement amplification (WGSDA), a random
set of primers is used to randomly prime a sample of genomic
nucleic acid (or another sample of nucleic acid of high
complexity). By choosing a sufficiently large set of primers of
random or partially random sequence, the primers in the set will be
collectively, and randomly, complementary to nucleic acid sequences
distributed throughout nucleic acid in the sample. Amplification
proceeds by replication with a highly processive polymerase
initiating at each primer and continuing until spontaneous
termination. A key feature of this method is the displacement of
intervening primers during replication by the polymerase. In this
way, multiple overlapping copies of the entire genome can be
synthesized in a short time. The method has advantages over the
polymerase chain reaction since it can be carried out under
isothermal conditions. Other advantages of whole genome strand
displacement amplification include a higher level of amplification
than whole genome PCR (up to five times higher), amplification is
less sequence-dependent than PCR, and there are no re-annealing
artifacts or gene shuffling artifacts as can occur with PCR (since
there are no cycles of denaturation and re-annealing).
[0017] In another preferred form of the method, referred to as
multiple strand displacement amplification of concatenated DNA
(MSDA-CD), fragments of DNA are first concatenated together,
preferably with linkers. The concatenated DNA is then amplified by
strand displacement synthesis with appropriate primers. A random
set of primers can be used to randomly prime synthesis of the DNA
concatemers in a manner similar to whole genome amplification. As
in whole genome amplification, by choosing a sufficiently large set
of primers of random or partially random sequence, the primers in
the set will be collectively, and randomly, complementary to
nucleic acid sequences distributed throughout concatenated DNA. If
linkers are used to concatenate the DNA fragments, primers
complementary to linker sequences can be used to amplify the
concatemers. Synthesis proceeds from the linkers, through a section
of the concatenated DNA to the next linker, and continues beyond.
As the linker regions are replicated, new priming sites for DNA
synthesis are created. In this way, multiple overlapping copies of
the entire concatenated DNA sample can be synthesized in a short
time.
[0018] Following amplification, the amplified sequences can be for
any purpose, such as uses known and established for PCR amplified
sequences. For example, amplified sequences can be detected using
any of the conventional detection systems for nucleic acids such as
detection of fluorescent labels, enzyme-linked detection systems,
antibody-mediated label detection, and detection of radioactive
labels. A key feature of the disclosed method is that amplification
takes place not in cycles, but in a continuous, isothermal
replication. This makes amplification less complicated and much
more consistent in output. Strand displacement allows rapid
generation of multiple copies of a nucleic acid sequence or sample
in a single, continuous, isothermal reaction. DNA that has been
produced using the disclosed method can then be used for any
purpose or in any other method desired. For example, PCR can be
used to further amplify any specific DNA sequence that has been
previously amplified by the whole genome strand displacement
method.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 is a diagram of an example of multiple strand
displacement amplification (MSDA). Diagramed at the top is a double
stranded nucleic acid molecule which contains a nucleic acid of
interest (hatched area). Hybridized to the nucleic acid molecule
are a right and left set of primers. Diagramed in the middle are
the multiple strands of replicated nucleic acid being elongated
from each primer. The polymerase at the end of each elongating
strand displaces the elongating strand of the primer ahead of it.
Diagramed at the bottom are the multiple strands of replicated
nucleic acid further elongated. Also shown are the next sets of
primers which hybridize to their complementary sites on the newly
replicated strands. The newly replicated strands are made available
for hybridization to the primers through displacement by the
polymerase elongating the following strand.
[0020] FIG. 2 is a diagram of an example of whole genome strand
displacement amplification (WGSDA). At the top is a diagrammatical
representation of genomic DNA. Hybridized to the nucleic acid
molecule are primers from a set of random or partially random
primers (the primer lengths are not intended to be to scale). For
simplicity, only a portion of one molecule of genomic DNA is
depicted. Diagramed in the middle are the multiple strands of
replicated nucleic acid being elongated from each primer. The
polymerase at the end of each elongating strand displaces the
elongating strand of any primer it encounters. Also shown
additional primers from the set of random or partially random
primers which hybridize to complementary sites on the newly
replicated strands. The newly replicated strands are made available
for hybridization to the primers through displacement by the
polymerase elongating a following strand. Diagramed at the bottom
are the multiple strands of replicated nucleic acid further
elongated. For simplicity only four of the originally synthesized
strands (two on the upper target sequence strand and two on the
lower target sequence strand) are depicted in the bottom panel.
[0021] FIG. 3 is a diagram of an example of multiple strand
displacement amplification (MSDA). Diagramed at the top is a double
stranded nucleic acid molecule which contains a nucleic acid of
interest (hatched area). Hybridized to the nucleic acid molecule
are a right set of primers (top strand in top panel) and a left set
of primers (bottom strand in top panel). Diagramed in the middle
are the multiple strands of replicated nucleic acid being elongated
from each primer. Also shown are the next sets of primers which
hybridize to their complementary sites on the newly replicated
strands. The newly replicated strands are made available for
hybridization to the primers through displacement by the polymerase
elongating the following strand. The polymerase at the end of each
elongating strand displaces the elongating strand of the primer
ahead of it. Diagramed at the bottom are the multiple strands of
replicated nucleic acid further elongated. For simplicity only four
of the originally synthesized strands (two on the upper target
sequence strand and two on the lower target sequence strand) are
depicted in the bottom panel.
[0022] FIG. 4 is a diagram of an example of multiple strand
displacement amplification of concatenated DNA (MSDA-CD). At the
top is a diagrammatical representation of DNA concatenated with
linkers. In the middle, primers complementary to linker sequences
are hybridized to denatured strands of the concatenated DNA (the
linker and primer lengths are not intended to be to scale). For
simplicity, only a portion of one molecule of concatenated DNA is
depicted. Diagramed at the bottom are the multiple strands of
replicated nucleic acid being elongated from each primer. The
polymerase at the end of each elongating strand displaces the
elongating strand of any primer it encounters. Also shown are
additional primers which hybridize to complementary sites in
replicated linker sequences on the newly replicated strands. The
newly replicated strands are made available for hybridization to
the primers through displacement by the polymerase elongating a
following strand.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The disclosed method makes use of certain materials and
procedures which allow amplification of target nucleic acid
sequences and whole genomes or other highly complex nucleic acid
samples. These materials and procedures are described in detail
below.
Materials
A. Target Sequence
[0024] The target sequence, which is the object of amplification,
can be any nucleic acid. The target sequence can include multiple
nucleic acid molecules, such as in the case of whole genome
amplification, multiple sites in a nucleic acid molecule, or a
single region of a nucleic acid molecule. For multiple strand
displacement amplification, generally the target sequence is a
single region in a nucleic acid molecule or nucleic acid sample.
For whole genome amplification, the target sequence is the entire
genome or nucleic acid sample. A target sequence can be in any
nucleic acid sample of interest. The source, identity, and
preparation of many such nucleic acid samples are known. It is
preferred that nucleic acid samples known or identified for use in
amplification or detection methods be used for the method described
herein. The nucleic acid sample can be a nucleic acid sample from a
single cell. For multiple strand displacement amplification,
preferred target sequences are those which are difficult to amplify
using PCR due to, for example, length or composition. For whole
genome amplification, preferred target sequences are nucleic acid
samples from a single cell. For multiple strand displacement
amplification of concatenated DNA the target is the concatenated
DNA. The target sequences for use in the disclosed method are
preferably part of nucleic acid molecules or samples that are
complex and non-repetitive (with the exception of the linkers in
linker-concatenated DNA and sections of repetitive DNA in genomic
DNA).
1. Target Sequences for Multiple Strand Displacement
Amplification
[0025] Although multiple sites in a nucleic acid sample can be
amplified simultaneously in the same MSDA reaction, for simplicity,
the following discussion will refer to the features of a single
nucleic acid sequence of interest which is to be amplified. This
sequence is referred to below as a target sequence. It is preferred
that a target sequence for MSDA include two types of target
regions, an amplification target and a hybridization target. The
hybridization target includes the sequences in the target sequence
that are complementary to the primers in a set of primers. The
amplification target is the portion of the target sequence which is
to be amplified. For this purpose, the amplification target is
preferably downstream of, or flanked by the hybridization
target(s). There are no specific sequence or structural
requirements for choosing a target sequence. The hybridization
target and the amplification target within the target sequence are
defined in terms of the relationship of the target sequence to the
primers in a set of primers. The primers are designed to match the
chosen target sequence. The top panel of FIG. 1 illustrates an
example of how primers in a primer set can define the regions in a
target sequence. Although preferred, it is not required that
sequence to be amplified and the sites of hybridization of the
primers be separate since sequences in and around the sites where
the primers hybridize will be amplified. An example of this is
illustrated in FIG. 3.
[0026] In multiple strand displacement amplification of
linker-concatenated DNA, the DNA fragments joined by the linkers
are the amplification targets and the linkers are the hybridization
target. The hybridization targets (that is, the linkers) include
the sequences that are complementary to the primers used for
amplification. A preferred form of concatenated DNA for
amplification is concatenated cDNA.
B. Primers
[0027] Primers for use in the disclosed amplification method are
oligonucleotides having sequence complementary to the target
sequence. This sequence is referred to as the complementary portion
of the primer. The complementary portion of a primer can be any
length that supports specific and stable hybridization between the
primer and the target sequence. Generally this is 10 to 35
nucleotides long, but is preferably 16 to 24 nucleotides long. For
whole genome amplification, it is preferred that the primers are
from 12 to 60 nucleotides long.
[0028] It is preferred that primers also contain additional
sequence at the 5' end of the primer that is not complementary to
the target sequence. This sequence is referred to as the
non-complementary portion of the primer. The non-complementary
portion of the primer, if present, serves to facilitate strand
displacement during DNA replication. The non-complementary portion
of the primer can also include a functional sequence such as a
promoter for an RNA polymerase. The non-complementary portion of a
primer may be any length, but is generally 1 to 100 nucleotides
long, and preferably 4 to 8 nucleotides long. The use of a
non-complementary portion is not preferred when random or partially
random primers-are used for whole genome amplification.
1. Primers for Multiple Strand Displacement Amplification
[0029] In the case of multiple strand displacement amplification,
the complementary portion of each primer is designed to be
complementary to the hybridization target in the target sequence.
In a set of primers, it is preferred that the complementary portion
of each primer be complementary to a different portion of the
target sequence. It is more preferred that the primers in the set
be complementary to adjacent sites in the target sequence. It is
also preferred that such adjacent sites in the target sequence are
also adjacent to the amplification target in the target
sequence.
[0030] It is preferred that, when hybridized to a target sequence,
the primers in a set of primers are separated from each other. It
is preferred that, when hybridized, the primers in a set of primers
are separated from each other by at least 5 bases. It is more
preferred that, when hybridized, the primers in a set of primers
are separated from each other by at least 10 bases. It is still
more preferred that, when hybridized, the primers in a set of
primers are separated from each other by at least 20 bases. It is
still more preferred that, when hybridized, the primers in a set of
primers are separated from each other by at least 30 bases. It is
still more preferred that, when hybridized, the primers in a set of
primers are separated from each other by at least 40 bases. It is
still more preferred that, when hybridized, the primers in a set of
primers are separated from each other by at least 50 bases.
[0031] It is preferred that, when hybridized, the primers in a set
of primers are separated from each other by no more than about 500
bases. It is more preferred that, when hybridized, the primers in a
set of primers are separated from each other by no more than about
400 bases. It is still more preferred that, when hybridized, the
primers in a set of primers are separated from each other by no
more than about 300 bases. It is still more preferred that, when
hybridized, the primers in a set of primers are separated from each
other by no more than about 200 bases. Any combination of the
preferred upper and lower limits of separation described above are
specifically contemplated, including all intermediate ranges. The
primers in a set of primers need not, when hybridized, be separated
from each other by the same number of bases. It is preferred that,
when hybridized, the primers in a set of primers are separated from
each other by about the same number of bases.
[0032] The optimal separation distance between primers will not be
the same for all DNA polymerases, because this parameter is
dependent on the net polymerization rate. A processive DNA
polymerase will have a characteristic polymerization rate which may
range from 5 to 300 nucleotides per second, and may be influenced
by the presence or absence of accessory ssDNA binding proteins and
helicases. In the case of a non-processive polymerase, the net
polymerization rate will depend on the enzyme concentration,
because at higher concentrations there are more re-initiation
events and thus the net polymerization rate will be increased. An
example of a processive polymerase is .phi.29 DNA polymerase, which
proceeds at 50 nucleotides per second. An example of a
non-processive polymerase is Vent exo(-) DNA polymerase, which will
give effective polymerization rates of 4 nucleotides per second at
low concentration, or 16 nucleotides per second at higher
concentrations.
[0033] To obtain an optimal yield in an MSDA reaction, the primer
spacing is preferably adjusted to suit the polymerase being used.
Long primer spacing is preferred when using a polymerase with a
rapid polymerization rate. Shorter primer spacing is preferred when
using a polymerase with a slower polymerization rate. The following
assay can be used to determine optimal spacing with any polymerase.
The assay uses sets of primers, with each set made up of 5 left
primers and 5 right primers. The sets of primers are designed to
hybridize adjacent to the same target sequence with each of the
different sets of primers having a different primer spacing. The
spacing is varied systematically between the sets of primers in
increments of 25 nucleotides within the range of 25 nucleotides to
400 nucleotides (the spacing of the primers within each set is the
same). A series of reactions are performed in which the same target
sequence is amplified using the different sets of primers. The
spacing that produces the highest experimental yield of DNA is the
optimal primer spacing for the specific DNA polymerase, or DNA
polymerase plus accessory protein combination being used.
[0034] DNA replication initiated at the sites in the target
sequence where the primers hybridize will extend to and displace
strands being replicated from primers hybridized at adjacent sites.
Displacement of an adjacent strand makes it available for
hybridization to another primer and subsequent initiation of
another round of replication. The region(s) of the target sequence
to which the primers hybridize is referred to as the hybridization
target of the target sequence. The top panel of FIG. 1 illustrates
one of the preferred relationships of a set of primers to a target
sequence and to the amplification target of the target
sequence.
[0035] A set of primers can include any desired number of primers
of different nucleotide sequence. For MSDA, it is preferred that a
set of primers include a plurality of primers. It is more preferred
that a set of primers include 3 or more primers. It is still more
preferred that a set of primers include 4 or more, 5 or more, 6 or
more, or 7 or more primers. In general, the more primers used, the
greater the level of amplification that will be obtained. There is
no fundamental upper limit to the number of primers that a set of
primers can have. However, for a given target sequence, the number
of primers in a set of primers will generally be limited to number
of hybridization sites available in the target sequence. For
example, if the target sequence is a 10,000 nucleotide DNA molecule
and 20 nucleotide primers are used, there are 500 non-overlapping
20 nucleotide sites in the target sequence. Even more primers than
this could be used if overlapping sites are either desired or
acceptable. It is preferred that a set of primers include no more
than about 300 primers. It is preferred that a set of primers
include no more than about 200 primers. It is still more preferred
that a set of primers include no more than about 100 primers. It is
more preferred that a set of primers include no more than about 50
primers. It is most preferred that a set of primers include from 7
to about 50 primers. Any combination of the preferred upper and
lower limits for the number of primers in a set of primers
described above are specifically contemplated, including all
intermediate ranges.
[0036] A preferred form of primer set for use in MSDA includes two
sets of primers, referred to as a right set of primers and a left
set of primers. The right set of primers and left set of primers
are designed to be complementary to opposite strands of a target
sequence. It is preferred that the complementary portions of the
right set primers are each complementary to the right hybridization
target, and that each is complementary to a different portion of
the right hybridization target. It is preferred that the
complementary portions of the left set primers are each
complementary to the left hybridization target, and that each is
complementary to a different portion of the left hybridization
target. The right and left hybridization targets flank opposite
ends of the amplification target in a target sequence. A preferred
form of these relationships are illustrated in the top panel of
FIG. 1. It is preferred that a right set of primers and a left set
of primers each include a preferred number of primers as described
above for a set of primers. Specifically, it is preferred that a
right or left set of primers include a plurality of primers. It is
more preferred that a right or left set of primers include 3 or
more primers. It is still more preferred that a right or left set
of primers include 4 or more, 5 or more, 6 or more, or 7 or more
primers. It is preferred that a right or left set of primers
include no more than about 200 primers. It is more preferred that a
right or left set of primers include no more than about 100
primers. It is most preferred that a right or left set of primers
include from 7 to about 100 primers. Any combination of the
preferred upper and lower limits for the number of primers in a set
of primers described above are specifically contemplated, including
all intermediate ranges. It is also preferred that, for a given
target sequence, the right set of primers and the left set of
primers include the same number of primers. It is also preferred
that, for a given target sequence, the right set of primers and the
left set of primers are composed of primers of similar length
and/or hybridization characteristics.
2. Primers for Whole Genome Strand Displacement Amplification
[0037] In the case of whole genome strand displacement
amplification, it is preferred that a set of primers having random
or partially random nucleotide sequences be used. In a nucleic acid
sample of significant complexity, which is the preferred target
sequence for WGSDA, specific nucleic acid sequences present in the
sample need not be known and the primers need not be designed to be
complementary to any particular sequence. Rather, the complexity of
the nucleic acid sample results in a large number of different
hybridization target sequences in the sample which will be
complementary to various primers of random or partially random
sequence. The complementary portion of primers for use in WGSDA can
be fully randomized, have only a portion that is randomized, or be
otherwise selectively randomized.
[0038] The number of random base positions in the complementary
portion of primers are preferably from 20% to 100% of the total
number of nucleotides in the complementary portion of the primers.
More preferably the number of random base positions are from 30% to
100% of the total number of nucleotides in the complementary
portion of the primers. Most preferably the number of random base
positions are from 50% to 100% of the total number of nucleotides
in the complementary portion of the primers. Sets of primers having
random or partially random sequences can be synthesized using
standard techniques by allowing the addition of any nucleotide at
each position to be randomized. It is also preferred that the sets
of primers are composed of primers of similar length and/or
hybridization characteristics.
3. Primers for Multiple Strand Displacement Amplification on
Concatenated DNA
[0039] For multiple strand displacement amplification of
concatenated DNA, a set of primers having random or partially
random nucleotide sequences can be used. In a nucleic acid sample
of significant complexity, such as DNA concatenated from a mixture
of many sequences, specific nucleic acid sequences present in the
sample need not be known and the primers need not be designed to be
complementary to any particular sequence. Rather, the complexity of
the nucleic acid sample results in a large number of different
hybridization target sequences in the sample which will be
complementary to various primers of random or partially random
sequence. The complementary portion of primers for use in MSDA-CD
can be fully randomized, have only a portion that is randomized, or
be otherwise selectively-randomized.
[0040] The number of random base positions in the complementary
portion of primers are preferably from 20% to 100% of the total
number of nucleotides in the complementary portion of the primers.
More preferably the number of random base positions are from 30% to
100% of the total number of nucleotides in the complementary
portion of the primers. Most preferably the number of random base
positions are from 50% to 100% of the total number of nucleotides
in the complementary portion of the primers. Sets of primers having
random or partially random sequences can be synthesized using
standard techniques by allowing the addition of any nucleotide at
each position to be randomized. It is also preferred that the sets
of primers are composed of primers of similar length and/or
hybridization characteristics.
[0041] Where the DNA has been concatenated with linkers,
amplification can be performed using primers complementary to
sequences in the linkers. This is the preferred form of MSDA-CD. It
is preferred that the complementary portion of each primer is
designed to be complementary to sequences in the linkers. It is
preferred that primers for use with linker-concatenated DNA include
primers complementary to both strands of the linker sequence. This
is illustrated in FIG. 4. It is also preferred that the primers are
not complementary to each other. This prevents the primers from
hybridizing to each other. If the linkers used to concatenate the
DNA are sufficiently long, a set of primers complementary to
different portions of the linker sequence can be used. This is
equivalent to the situation in MSDA, and the sets of primers can be
designed and used in the same manner as discussed for MSDA primer
sets. Random primers can be used to amplify concatenated DNA
whether or not linkers have been used to concatenate the DNA.
[0042] It is preferred that the target sequences for use in MSDA,
WGSDA, and MSDA-CD are not, or are not part of, nucleic acid
molecules made up of multiple tandem repeats of a sequence. It is
more preferable that the target sequences are not, or are not part
of, nucleic acid molecules made up of multiple tandem repeats of a
single sequence. It is most preferred that the target sequences are
not, or are not part of, nucleic acid molecules made up of multiple
tandem repeats of a single sequence that were synthesized by
rolling circle replication. An example of such tandem repeat DNA
made by rolling circle replication is the tandem sequence DNA
described in WO 97/19193. DNA concatenated from identical or nearly
identical DNA fragments is not made by rolling circle replication
and so is not a nucleic acid molecule made up of multiple tandem
repeats of a single sequence that was synthesized by rolling circle
replication. Thus, although it is preferred that the target
sequences are not nucleic acid molecules made up of multiple tandem
repeats of a single sequence, some such target sequences, such as
DNA concatenated from identical or nearly identical DNA fragments,
are preferred over, and to the exclusion of, nucleic acid molecules
made up of multiple tandem repeats of a single sequence that are
synthesized by rolling circle replication (such as the tandem
sequence DNA described in WO 97/19193). It is preferred that target
sequences for the disclosed method are not produced by the methods
described in WO 97/19193.
4. Detection Tags
[0043] The non-complementary portion of a primer can include
sequences to be used to further manipulate or analyze amplified
sequences. An example of such a sequence is a detection tag, which
is a specific nucleotide sequence present in the non-complementary
portion of a primer. Detection tags have sequences complementary to
detection probes. Detection tags can be detected using their
cognate detection probes. Detection tags become incorporated at the
ends of amplified strands. The result is amplified DNA having
detection tag sequences that are complementary to the complementary
portion of detection probes. If present, there may be one, two,
three, or more than three detection tags on a primer. It is
preferred that a primer have one, two, three or four detection
tags. Most preferably, a primer will have one detection tag.
Generally, it is preferred that a primer have 10 detection tags or
less. There is no fundamental limit to the number of detection tags
that can be present on a primer except the size of the primer. When
there are multiple detection tags, they may have the same sequence
or they may have different sequences, with each different sequence
complementary to a different detection probe. It is preferred that
a primer contain detection tags that have the same sequence such
that they are all complementary to a single detection probe. For
some multiplex detection methods, it is preferable that primers
contain up to six detection tags and that the detection tag
portions have different sequences such that each of the detection
tag portions is complementary to a different detection probe. A
similar effect can be achieved by using a set of primers where each
has a single different detection tag. The detection tags can each
be any length that supports specific and stable hybridization
between the detection tags and the detection probe. For this
purpose, a length of 10 to 35 nucleotides is preferred, with a
detection tag portion 15 to 20 nucleotides long being most
preferred.
5. Address Tag
[0044] Another example of a sequence that can be included in the
non-complementary portion of a primer is an address tag. An address
tag has a sequence complementary to an address probe. Address tags
become incorporated at the ends of amplified strands. The result is
amplified DNA having address tag sequences that are complementary
to the complementary portion of address probes. If present, there
may be one, or more than one, address tag on a primer. It is
preferred that a primer have one or two address tags. Most
preferably, a primer will have one address tag. Generally, it is
preferred that a primer have 10 address tags or less. There is no
fundamental limit to the number of address tags that can be present
on a primer except the size of the primer. When there are multiple
address tags, they may have the same sequence or they may have
different sequences, with each different sequence complementary to
a different address probe. It is preferred that a primer contain
address tags that have the same sequence such that they are all
complementary to a single address probe. The address tag portion
can be any length that supports specific and stable hybridization
between the address tag and the address probe. For this purpose, a
length between 10 and 35 nucleotides long is preferred, with an
address tag portion 15 to 20 nucleotides long being most
preferred.
C. Linkers
[0045] As used herein for concatenating DNA, a linker is a small,
double-stranded DNA molecule. For MSDA-CD, linkers serve two main
purposes; facilitating concatenation of DNA fragments and
facilitating amplification. For the first purpose, linkers are
generally designed to have ends compatible with the ends of the DNA
fragments to be concatenated. For example, if the DNA fragments
have blunt ends (or the ends will be made blunt), blunt ended
linkers would be used. For DNA fragments that have been tailed with
one or more nucleotides, the linkers should have a complementary
tail. An example of such tailing is the addition of single
adenosine residues to the 3' ends of cDNA. For facilitating
amplification, linkers should have one or more sequences
complementary to primers to be used in MSDA-CD. Such sequences are
referred to as primer complement portions of the linkers. Primer
complement portions of linkers are complementary to complementary
portions of primers. A primer complement portion can have an
arbitrary sequence so long as it is complementary to the portion of
the intended primer. If there are primer complement portions on
opposite strands of the linker, they should not overlap. The primer
can also have one or more restriction enzyme cleavage sites. Such
restriction sites allow the amplified DNA to be cut into fragments,
and preferably into fragments representing the original DNA
fragments which were concatenated. For this purpose, it is
preferred that a rare restriction site be used (for example, an
eight-base recognition site). An example of the structure of a
linker of this type is illustrated below.
1 Primer 1> Restriction Site
P-NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNT
TNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN-P <Primer
2
[0046] Linkers can also contain one or more promoter sequences.
Such promoter sequences allow the amplified DNA to be further
amplified by transcription after MSDA-CD. If two promoters are
incorporated into the linker, they are preferably located on
different strands of the linker. An example of a linker, having a
single protruding thymidine residue at both 3' termini, and a
phosphate group at both 5' termini, is illustrated below (P
indicates phosphate);
2 Primer 1> Promoter 1>
P-NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNT
TNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN-P
<Promoter 2 <Primer 2
[0047] The promoter and primer sequences may be arranged in any
order, but the arrangement shown above is preferred. Any number of
primers and promoters may be used. However, it is preferred that,
where the DNA to be concatenated is cDNA, promoters be incorporated
into the cDNA as part of the primers used for cDNA synthesis
(Lockhart et al.).
D. Detection Labels
[0048] To aid in detection and quantitation of nucleic acids
amplified using the disclosed method, detection labels can be
directly incorporated into amplified nucleic acids or can be
coupled to detection molecules. As used herein, a detection label
is any molecule that can be associated with amplified nucleic acid,
directly or indirectly, and which results in a measurable,
detectable signal, either directly or indirectly. Many such labels
for incorporation into nucleic acids or coupling to nucleic acid
probes are known to those of skill in the art. Examples of
detection labels suitable for use in the disclosed method are
radioactive isotopes, fluorescent molecules, phosphorescent
molecules, enzymes, antibodies, and ligands.
[0049] Examples of suitable fluorescent labels include fluorescein
(FITC), 5,6-carboxymethyl fluorescein, Texas red,
nitrobenz-2-oxa-1,3-diazol-4-yl (NBD), coumarin, dansyl chloride,
rhodamine, 4'-6-diamidino-2-phenylinodo- le (DAPI), and the cyanine
dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7. Preferred fluorescent labels
are fluorescein (5-carboxyfluorescein-N-hydroxysuccini- mide ester)
and rhodamine (5,6-tetramethyl rhodamine). Preferred fluorescent
labels are FITC and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and
Cy7. The absorption and emission maxima, respectively, for these
fluors are: FITC (490 nm; 520 nm), Cy3 (554 nm; 568 nm), Cy3.5 (581
nm; 588 nm), Cy5 (652 nm: 672 nm), Cy5.5 (682 nm; 703 nm) and Cy7
(755 nm; 778 nm), thus allowing their simultaneous detection. The
fluorescent labels can be obtained from a variety of commercial
sources, including Molecular Probes, Eugene, Oreg. and Research
Organics, Cleveland, Ohio.
[0050] Labeled nucleotides are a preferred form of detection label
since they can be directly incorporated into the amplification
products during synthesis. Examples of detection labels that can be
incorporated into amplified DNA or RNA include nucleotide analogs
such as BrdUrd (Hoy and Schimke, Mutation Research
290:217-230,(1993)), BrUTP (Wansick et al., J. Cell Biology
122:283-293 (1993)) and nucleotides modified with biotin (Langer et
al., Proc. Natl. Acad. Sci. USA 78:6633 (1981)) or with suitable
haptens such as digoxygenin (Kerkhof, Anal. Biochem. 205:359-364
(1992)). Suitable fluorescence-labeled nucleotides are
Fluorescein-isothiocyanate-dUTP, Cyanine-3-dUTP and Cyanine-5-dUTP
(Yu et al., Nucleic Acids Res., 22:3226-3232 (1994)). A preferred
nucleotide analog detection label for DNA is BrdUrd (BUDR
triphosphate, Sigma), and a preferred nucleotide analog detection
label for RNA is Biotin-16-uridine-5'-triphosphate (Biotin-16-dUTP,
Boehringher Mannheim). Fluorescein, Cy3, and Cy5 can be linked to
dUTP for direct labelling. Cy3.5 and Cy7 are available as avidin or
anti-digoxygenin conjugates for secondary detection of biotin- or
digoxygenin-labelled probes.
[0051] Detection labels that are incorporated into amplified
nucleic acid, such as biotin, can be subsequently detected using
sensitive methods well-known in the art. For example, biotin can be
detected using streptavidin-alkaline phosphatase conjugate (Tropix,
Inc.), which is bound to the biotin and subsequently detected by
chemiluminescence of suitable substrates (for example,
chemiluminescent substrate CSPD: disodium,
3-(4-methoxyspiro-[1,2,-dioxetane-3-2'-(5'-chloro)tricyclo[3.3.-
1.1.sup.3,7]decane]-4-yl)phenyl phosphate; Tropix, Inc.).
[0052] A preferred detection label for use in detection of
amplified RNA is acridinium-ester-labeled DNA probe (GenProbe,
Inc., as described by Arnold et al., Clinical Chemistry
35:1588-1594 (1989)). An acridinium-ester-labeled detection probe
permits the detection of amplified RNA without washing because
unhybridized probe can be destroyed with alkali (Arnold et al.
(1989)).
[0053] Molecules that combine two or more of these detection labels
are also considered detection labels. Any of the known detection
labels can be used with the disclosed probes, tags, and method to
label and detect nucleic acid amplified using the disclosed method.
Methods for detecting and measuring signals generated by detection
labels are also known to those of skill in the art. For example,
radioactive isotopes can be detected by scintillation counting or
direct visualization; fluorescent molecules can be detected with
fluorescent spectrophotometers; phosphorescent molecules can be
detected with a spectrophotometer or directly visualized with a
camera; enzymes can be detected by detection or visualization of
the product of a reaction catalyzed by the enzyme; antibodies can
be detected by detecting a secondary detection label coupled to the
antibody. As used herein, detection molecules are molecules which
interact with amplified nucleic acid and to which one or more
detection labels are coupled.
E. Detection Probes
[0054] Detection probes are labeled oligonucleotides having
sequence complementary to detection tags on amplified nucleic
acids. The complementary portion of a detection probe can be any
length that supports specific and stable hybridization between the
detection probe and the detection tag. For this purpose, a length
of 10 to 35 nucleotides is preferred, with a complementary portion
of a detection probe 16 to 20 nucleotides long being most
preferred. Detection probes can contain any of the detection labels
described above. Preferred labels are biotin and fluorescent
molecules. A particularly preferred detection probe is a molecular
beacon. Molecular beacons are detection probes labeled with
fluorescent moieties where the fluorescent moieties fluoresce only
when the detection probe is hybridized (Tyagi and Kramer, Nature
Biotechnol. 14:303-309 (1995)). The use of such probes eliminates
the need for removal of unhybridized probes prior to label
detection because the unhybridized detection probes will not
produce a signal. This is especially useful in multiplex
assays.
F. Address Probes
[0055] An address probe is an oligonucleotide having a sequence
complementary to address tags on primers. The complementary portion
of an address probe can be any length that supports specific and
stable hybridization between the address probe and the address tag.
For this purpose, a length of 10 to 35 nucleotides is preferred,
with a complementary portion of an address probe 12 to 18
nucleotides long being most preferred. An address probe can contain
a single complementary portion or multiple complementary portions.
Preferably, address probes are coupled, either directly or via a
spacer molecule, to a solid-state support. Such a combination of
address probe and solid-state support are a preferred form of
solid-state detector.
G. Oligonucleotide Synthesis
[0056] Primers, detection probes, address probes, and any other
oligonucleotides can be synthesized using established
oligonucleotide synthesis methods. Methods to produce or synthesize
oligonucleotides are well known in the art. Such methods can range
from standard enzymatic digestion followed by nucleotide fragment
isolation (see for example, Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2nd Edition (Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1989) Chapters 5, 6) to purely
synthetic methods, for example, by the cyanoethyl phosphoramidite
method using a Milligen or Beckman System IPlus DNA synthesizer
(for example, Model 8700 automated synthesizer of
Milligen-Biosearch, Burlington, Mass. or ABI Model 380B). Synthetic
methods useful for making oligonucleotides are also described by
Ikuta et al., Ann. Rev. Biochem. 53:323-356 (1984),
(phosphotriester and phosphite-triester methods), and Narang et
al., Methods Enzymol., 65:610-620 (1980), (phosphotriester method).
Protein nucleic acid molecules can be made using known methods such
as those described by Nielsen et al., Bioconjug. Chem. 5:3-7
(1994).
[0057] Many of the oligonucleotides described herein are designed
to be complementary to certain portions of other oligonucleotides
or nucleic acids such that stable hybrids can be formed between
them. The stability of these hybrids can be calculated using known
methods such as those described in Lesnick and Freier, Biochemistry
34:10807-10815 (1995), McGraw et al., Biotechniques 8:674-678
(1990), and Rychlik et al., Nucleic Acids Res. 18:6409-6412
(1990).
H. Solid-State Detectors
[0058] Solid-state detectors are solid-state substrates or supports
to which address probes or detection molecules have been coupled. A
preferred form of solid-state detector is an array detector. An
array detector is a solid-state detector to which multiple
different address probes or detection molecules have been coupled
in an array, grid, or other organized pattern.
[0059] Solid-state substrates for use in solid-state detectors can
include any solid material to which oligonucleotides can be
coupled. This includes materials such as acrylamide, cellulose,
nitrocellulose, glass, polystyrene, polyethylene vinyl acetate,
polypropylene, polymethacrylate, polyethylene, polyethylene oxide,
glass, polysilicates, polycarbonates, teflon, fluorocarbons, nylon,
silicon rubber, polyanhydrides, polyglycolic acid, polylactic acid,
polyorthoesters, polypropylfumerate, collagen, glycosaminoglycans,
and polyamino acids. Solid-state substrates can have any useful
form including thin films or membranes, beads, bottles, dishes,
fibers, woven fibers, shaped polymers, particles and
microparticles. A preferred form for a solid-state substrate is a
microtiter dish. The most preferred form of microtiter dish is the
standard 96-well type.
[0060] Address probes immobilized on a solid-state substrate allow
capture of the products of the disclosed amplification method on a
solid-state detector. Such capture provides a convenient means of
washing away reaction components that might interfere with
subsequent detection steps. By attaching different address probes
to different regions of a solid-state detector, different
amplification products can be captured at different, and therefore
diagnostic, locations on the solid-state detector. For example, in
a microtiter plate multiplex assay, address probes specific for up
to 96 different amplified nucleic acids (each representing a
different target sequence amplified via a different set of primers)
can be immobilized on a microtiter plate, each in a different well.
Capture and detection will occur only in those wells corresponding
to amplified nucleic acids for which the corresponding target
sequences were present in a sample.
[0061] Methods for immobilization of oligonucleotides to
solid-state substrates are well established. Oligonucleotides,
including address probes and detection probes, can be coupled to
substrates using established coupling methods. For example,
suitable attachment methods are described by Pease et al., Proc.
Natl. Acad. Sci. USA 91(11):5022-5026 (1994), and Khrapko et al.,
Mol Biol (Mosk) (USSR) 25:718-730 (1991). A method for
immobilization of 3'-amine oligonucleotides on casein-coated slides
is described by Stimpson et al., Proc. Natl. Acad. Sci. USA
92:6379-6383 (1995). A preferred method of attaching
oligonucleotides to solid-state substrates is described by Guo et
al., Nucleic Acids Res. 22:5456-5465 (1994).
I. Solid-State Samples
[0062] Solid-state samples are solid-state substrates or supports
to which target sequences have been coupled or adhered. Target
sequences are preferably delivered in a target sample or assay
sample. A preferred form of solid-state sample is an array sample.
An array sample is a solid-state sample to which multiple different
target sequences have been coupled or adhered in an array, grid, or
other organized pattern.
[0063] Solid-state substrates for use in solid-state samples can
include any solid material to which target sequences can be coupled
or adhered. This includes materials such as acrylamide, cellulose,
nitrocellulose, glass, polystyrene, polyethylene vinyl acetate,
polypropylene, polymethacrylate, polyethylene, polyethylene oxide,
glass, polysilicates, polycarbonates, teflon, fluorocarbons, nylon,
silicon rubber, polyanhydrides, polyglycolic acid, polylactic acid,
polyorthoesters, polypropylfumerate, collagen, glycosaminoglycans,
and polyamino acids. Solid-state substrates can have any useful
form including thin films or membranes, beads, bottles, dishes,
slides, fibers, woven fibers, shaped polymers, particles and
microparticles. Preferred forms for a solid-state substrate are
microtiter dishes and glass slides. The most preferred form of
microtiter dish is the standard 96-well type.
[0064] Target sequences immobilized on a solid-state substrate
allow formation of target-specific amplified nucleic acid localized
on the solid-state substrate. Such localization provides a
convenient means of washing away reaction components that might
interfere with subsequent detection steps, and a convenient way of
assaying multiple different samples simultaneously. Amplified
nucleic acid can be independently formed at each site where a
different sample is adhered. For immobilization of target sequences
or other oligonucleotide molecules to form a solid-state sample,
the methods described above for can be used.
[0065] A preferred form of solid-state substrate is a glass slide
to which up to 256 separate target samples have been adhered as an
array of small dots. Each dot is preferably from 0.1 to 2.5 mm in
diameter, and most preferably around 2.5 mm in diameter. Such
microarrays can be fabricated, for example, using the method
described by Schena et al., Science 270:487-470 (1995). Briefly,
microarrays can be fabricated on poly-L-lysine-coated microscope
slides (Sigma) with an arraying machine fitted with one printing
tip. The tip is loaded with 1 .mu.l of a DNA sample (0.5 mg/ml)
from, for example, 96-well microtiter plates and deposited
.about.0.005 .mu.l per slide on multiple slides at the desired
spacing. The printed slides can then be rehydrated for 2 hours in a
humid chamber, snap-dried at 100.degree. C. for 1 minute, rinsed in
0.1% SDS, and treated with 0.05% succinic anhydride prepared in
buffer consisting of 50% 1-methyl-2-pyrrolidinone and 50% boric
acid. The DNA on the slides can then be denatured in, for example,
distilled water for 2 minutes at 90.degree. C. immediately before
use. Microarray solid-state samples can scanned with, for example,
a laser fluorescent scanner with a computer-controlled XY stage and
a microscope objective. A mixed gas, multiline laser allows
sequential excitation of multiple fluorophores.
J. DNA Polymerases
[0066] DNA polymerases useful in the multiple displacement
amplification must be capable of displacing, either alone or in
combination with a compatible strand displacement factor, a
hybridized strand encountered during replication. Such polymerases
are referred to herein as strand displacement DNA polymerases. It
is preferred that a strand displacement DNA polymerase lack a 5' to
3' exonuclease activity. Strand displacement is necessary to result
in synthesis of multiple copies of a target sequence. A 5' to 3'
exonuclease activity, if present, might result in the destruction
of a synthesized strand. It is also preferred that DNA polymerases
for use in the disclosed method are highly processive. The
suitability of a DNA polymerase for use in the disclosed method can
be readily determined by assessing its ability to carry out strand
displacement replication. Preferred strand displacement DNA
polymerases are Bst large fragment DNA polymerase (Exo(-) Bst;
Aliotta et al., Genet. Anal. (Netherlands) 12:185-195 (1996)) and
exo(-)Bca DNA polymerase (Walker and Linn, Clinical Chemistry
42:1604-1608 (1996)). Other useful polymerases include
bacteriophage .phi.29 DNA polymerase (U.S. Pat. Nos. 5,198,543 and
5,001,050 to Blanco et al.), phage M2 DNA polymerase (Matsumoto et
al., Gene 84:247 (1989)), phage .phi.PRD1 DNA polymerase (Jung et
al., Proc. Natl. Acad. Sci. USA 84:8287 (1987)), exo(-)VENT.RTM.
DNA polymerase (Kong et al., J. Biol. Chem. 268:1965-1975 (1993)),
Klenow fragment of DNA polymerase I (Jacobsen et al., Eur. J.
Biochem. 45:623-627 (1974)), T5 DNA polymerase (Chatterjee et al.,
Gene 97:13-19 (1991)), Sequenase (U.S. Biochemicals), PRD1 DNA
polymerase (Zhu and Ito, Biochim. Biophys. Acta. 1219:267-276
(1994));, and T4 DNA polymerase holoenzyme (Kaboord and Benkovic,
Curr. Biol. 5:149-157 (1995)). Exo(-)Bst DNA polymerase is most
preferred.
[0067] Strand displacement can be facilitated through the use of a
strand displacement factor, such as helicase. It is considered that
any DNA polymerase that can perform strand displacement replication
in the presence of a strand displacement factor is suitable for use
in the disclosed method, even if the DNA polymerase does not
perform strand displacement replication in the absence of such a
factor. Strand displacement factors useful in strand displacement
replication include BMRF1 polymerase accessory subunit (Tsurumi et
al., J. Virology 67(12):7648-7653 (1993)), adenovirus DNA-binding
protein (Zijderveld and van der Vliet, J. Virology 68(2):1158-1164
(1994)), herpes simplex viral protein ICP8 (Boehmer and Lehman, J.
Virology 67(2):711-715 (1993); Skaliter and Lehman, Proc. Natl.
Acad. Sci. USA 91(22):10665-10669 (1994)); single-stranded DNA
binding proteins (SSB; Rigler and Romano, J. Biol. Chem.
270:8910-8919 (1995)); phage T4 gene 32 protein (Villemain and
Giedroc, Biochemistry 35:14395-14404 (1996); and calf thymus
helicase (Siegel et al., J. Biol. Chem. 267:13629-13635
(1992)).
[0068] The ability of a polymerase to carry out strand displacement
replication can be determined by using the polymerase in a strand
displacement replication assay such as those described in Examples
1 and 2. The assay in the examples can be modified as appropriate.
For example, a helicase can be used instead of SSB. Such assays
should be performed at a temperature suitable for optimal activity
for the enzyme being used, for example, 32.degree. C. for .phi.29
DNA polymerase, from 46.degree. C. to 64.degree. C. for exo(-) Bst
DNA polymerase, or from about 60.degree. C. to 70.degree. C. for an
enzyme from a hyperthermophylic organism. For assays from
60.degree. C. to 70.degree. C., primer length may be increased to
20 bases for random primers, or to 22 bases for specific primers.
Another useful assay for selecting a polymerase is the primer-block
assay described in Kong et al., J. Biol. Chem. 268:1965-1975
(1993). The assay consists of a primer extension assay using an M13
ssDNA template in the presence or absence of an oligonucleotide
that is hybridized upstream of the extending primer to block its
progress. Enzymes able to displace the blocking primer in this
assay are useful for the disclosed method.
[0069] The materials described above can be packaged together in
any suitable combination as a kit useful for performing the
disclosed method.
Method
[0070] The disclosed method is based on strand displacement
replication of the nucleic acid sequences by multiple primers. The
method can be used to amplify one or more specific sequences
(multiple strand displacement amplification), an entire genome or
other DNA of high complexity (whole genome strand displacement
amplification), or concatenated DNA (multiple strand displacement
amplification of concatenated DNA). The disclosed method generally
involves hybridization of primers to a target nucleic acid sequence
and replication of the target sequence primed by the hybridized
primers such that replication of the target sequence results in
replicated strands complementary to the target sequence. During
replication, the growing replicated strands displace other
replicated strands from the target sequence (or from another
replicated strand) via strand displacement replication. Examples of
such displacement of replicated strands are illustrated in the
figures. As used herein, a replicated strand is a nucleic acid
strand resulting from elongation of a primer hybridized to a target
sequence or to another replicated strand. Strand displacement
replication refers to DNA replication where a growing end of a
replicated strand encounters and displaces another strand from the
template strand (or from another replicated strand). Displacement
of replicated strands by other replicated strands is a hallmark of
the disclosed method which allows multiple copies of a target
sequence to be made in a single, isothermic reaction.
A. Multiple Strand Displacement Amplification
[0071] In one preferred form of the method, referred to as multiple
strand displacement amplification (MSDA), two sets of primers are
used, a right set and a left set. Primers in the right set of
primers each have a portion complementary to nucleotide sequences
flanking one side of a target nucleotide sequence and primers in
the left set of primers each have a portion complementary to
nucleotide sequences flanking the other side of the target
nucleotide sequence. The primers in the right set are complementary
to one strand of the nucleic acid molecule containing the target
nucleotide sequence and the primers in the left set are
complementary to the opposite strand. The 5' end of primers in both
sets are distal to the nucleic acid sequence of interest when the
primers are hybridized to the flanking sequences in the nucleic
acid molecule. Preferably, each member of each set has a portion
complementary to a separate and non-overlapping nucleotide sequence
flanking the target nucleotide sequence. Amplification proceeds by
replication initiated at each primer and continuing through the
target nucleic acid sequence. A key feature of this method is the
displacement of intervening primers during replication. Once the
nucleic acid strands elongated from the right set of primers
reaches the region of the nucleic acid molecule to which the left
set of primers hybridizes, and vice versa, another round of priming
and replication will take place. This allows multiple copies of a
nested set of the target nucleic acid sequence to be synthesized in
a short period of time.
[0072] Multiple strand displacement amplification can be performed
by (a) mixing a set of primers with a target sample, to produce an
primer-target sample mixture, and incubating the primer-target
sample mixture under conditions that promote hybridization between
the primers and the target sequence in the primer-target sample
mixture, and (b) mixing DNA polymerase with the primer-target
sample mixture, to produce a polymerase-target sample mixture, and
incubating the polymerase-target sample mixture under conditions
that promote replication of the target sequence. Strand
displacement replication is preferably accomplished by using a
strand displacing DNA polymerase or a DNA polymerase in combination
with a compatible strand displacement factor. A preferred example
of MSDA is illustrated in FIG. 1. Another example of MSDA is
illustrated in FIG. 3.
[0073] By using a sufficient number of primers in the right and
left sets, only a few rounds of replication are required to produce
hundreds of thousands of copies of the nucleic acid sequence of
interest. For example, it can be estimated that, using right and
left primer sets of 26 primers each, 200,000 copies of a 5000
nucleotide amplification target can be produced in 10 minutes
(representing just four rounds of priming and replication). It can
also be estimated that, using right and left primer sets of 26
primers each, 200,000 copies of a 47,000 nucleotide amplification
target can be produced in 60 minutes (again representing four
rounds of priming and replication). These calculations are based on
a polymerase extension rate of 50 nucleotides per second. It is
emphasized that reactions are continuous and isothermal--no cycling
is required.
[0074] The disclosed method has advantages over the polymerase
chain reaction since it can be carried out under isothermal
conditions. No thermal cycling is needed because the polymerase at
the head of an elongating strand (or a compatible
strand-displacement factor) will displace, and thereby make
available for hybridization, the strand ahead of it. Other
advantages of multiple strand displacement amplification include
the ability to amplify very long nucleic acid segments (on the
order of 50 kilobases) and rapid amplification of shorter segments
(10 kilobases or less). Long nucleic acid segments can be amplified
in the disclosed method since there no cycling which could
interrupt continuous synthesis or allow the formation of artifacts
due to rehybridization of replicated strands. In multiple strand
displacement amplification, single priming events at unintended
sites will not lead to artifactual amplification at these sites
(since amplification at the intended site will quickly outstrip the
single strand replication at the unintended site).
B. Whole Genome Strand Displacement Amplification
[0075] In another preferred form of the method, referred to as
whole genome strand displacement amplification (WGSDA), a random or
partially random set of primers is used to randomly prime a sample
of genomic nucleic acid (or another sample of nucleic acid of high
complexity). By choosing a sufficiently large set of primers of
random or mostly random sequence, the primers in the set will be
collectively, and randomly, complementary to nucleic acid sequences
distributed throughout nucleic acid in the sample. Amplification
proceeds by replication with a processive polymerase initiated at
each primer and continuing until spontaneous termination. A key
feature of this method is the displacement of intervening primers
during replication by the polymerase. In this way, multiple
overlapping copies of the entire genome can be synthesized in a
short time. It can be estimated that, in a WGSDA on a genomic
sample, after 180 minutes of incubation each primer will have been
elongated by, on average, 55,000 bases. By using a sufficiently
high concentration of primers, additional priming events on
replicated strands will result in additional rounds of copying. It
can be estimated that after 180 minutes 400 copies of the entire
genome will have been produced.
[0076] Whole genome strand displacement amplification can be
performed by (a) mixing a set of random or partially random primers
with a genomic sample (or other nucleic acid sample of high
complexity), to produce an primer-target sample mixture, and
incubating the primer-target sample mixture under conditions that
promote hybridization between the primers and the genomic DNA in
the primer-target sample mixture, and (b) mixing DNA polymerase
with the primer-target sample mixture, to produce a
polymerase-target sample mixture, and incubating the
polymerase-target sample mixture under conditions that promote
replication of the genomic DNA. Strand displacement replication is
preferably accomplished by using a strand displacing DNA polymerase
or a DNA polymerase in combination with a compatible strand
displacement factor. WGSDA is illustrated in FIG. 2.
[0077] The method has advantages over the polymerase chain reaction
since it can be carried out under isothermal conditions. Other
advantages of whole genome strand displacement amplification
include a higher level of amplification than whole genome PCR (up
to 5 times higher), amplification is less sequence-dependent than
PCR, and there are no re-annealing artifacts or gene shuffling
artifacts as can occur with PCR (since there are no cycles of
denaturation and re-annealing).
[0078] Following amplification, the amplified sequences can be for
any purpose, such as uses known and established for PCR amplified
sequences. For example, amplified sequences can be detected using
any of the conventional detection systems for nucleic acids such as
detection of fluorescent labels, enzyme-linked detection systems,
antibody-mediated label detection, and detection of radioactive
labels. A key feature of the disclosed method is that amplification
takes place not in cycles, but in a continuous, isothermal
replication. This makes amplification less complicated and much
more consistent in output. Strand displacement allows rapid
generation of multiple copies of a nucleic acid sequence or sample
in a single, continuous, isothermal reaction.
[0079] It is preferred that the set of primers used for WGSDA be
used at concentrations that allow the primers to hybridize at
desired intervals within the nucleic acid sample. For example, by
using a set of primers at a concentration that allows them to
hybridize, on average, every 4000 to 8000 bases, DNA replication
initiated at these sites will extend to and displace strands being
replicated from adjacent sites. It should be noted that the primers
are not expected to hybridize to the target sequence at regular
intervals. Rather, the average interval will be a general function
of primer concentration.
[0080] As in multiple strand displacement amplification,
displacement of an adjacent strand makes it available for
hybridization to another primer and subsequent initiation of
another round of replication. The interval at which primers in the
set of primers hybridize to the target sequence determines the
level of amplification. For example, if the average interval is
short, adjacent strands will be displaced quickly and frequently.
If the average interval is long, adjacent strands will be displaced
only after long runs of replication.
[0081] In the disclosed method, the DNA polymerase catalyzes primer
extension and strand displacement in a processive strand
displacement polymerization reaction that proceeds as long as
desired, generating molecules of up to 60,000 nucleotides or
larger. Preferred strand displacing DNA polymerases are large
fragment Bst DNA polymerase (Exo(-) Bst), exo(-)Bca DNA polymerase,
the DNA polymerase of the bacteriophage .phi.29 and Sequenase.
During strand displacement replication one may additionally include
radioactive, or modified nucleotides such as bromodeoxyuridine
triphosphate, in order to label the DNA generated in the reaction.
Alternatively, one may include suitable precursors that provide a
binding moiety such as biotinylated nucleotides (Langer et al.
(1981)).
[0082] Genome amplification using PCR, and uses for the amplified
DNA, is described in Zhang et al., Proc. Natl. Acad. Sci. USA
89:5847-5851 (1992), Telenius et al., Genomics 13:718-725 (1992),
Cheung et al., Proc. Natl. Acad. Sci. USA 93:14676-14679 (1996),
and Kukasjaarvi et al., Genes, Chromosomes and Cancer 18:94-101
(1997). The uses of the amplified DNA described in these
publications are also generally applicable to DNA amplified using
the disclosed methods. Whole Genome Strand Displacement
Amplification, unlike PCR-based whole genome amplification, is
suitable for haplotype analysis since WGSDA yields longer fragments
than PCR-based whole genome amplification. PCR-based whole genome
amplification is also less suitable for haplotype analysis since
each cycle in PCR creates an opportunity for priming events that
result in the association of distant sequences-(in the genome) to
be put together in the same fragment.
C. Multiple Strand Displacement Amplification of Concatenated
DNA
[0083] In another preferred form of the method, referred to as
multiple strand displacement amplification of concatenated DNA
(MSDA-CD), concatenated DNA is amplified. A preferred form of
concatenated DNA is concatenated cDNA. Concatenated DNA can be
amplified using a random or partially random set of primers, as in
WGSDA, or using specific primers complementary to specific
hybridization targets in the concatenated DNA. MSDA-CD is preferred
for amplification of a complex mixture or sample of relatively
short nucleic acid samples (that is, fragments generally in the
range of 100 to 6,000 nucleotides). Messenger RNA is the most
important example of such a complex mixture. MSDA-CD provides a
means for amplifying all cDNAs in a cell is equal fashion. Because
the concatenated cDNA can be amplified up to 5,000-fold, MSDA-CD
will permit RNA profiling analysis based on just a few cells. To
perform MSDA-CD, DNA must first be subjected to a concatenation
step. If an RNA sample (such as mRNA) is to be amplified, the RNA
is first converted to a double-stranded cDNA using standard
methods. The cDNA, or any other set of DNA fragments to be
amplified, is then converted into a DNA concatenate, preferably
with incorporation of linkers.
[0084] DNA fragments can be concatenated by ligation using standard
conditions. The state of the ends of the DNA fragments, such as
blunt, staggered or ragged, should be taken into account when
concatenating DNA. For example, staggered ends, such as those
produced by digestion with restriction enzymes, can be used to
mediate concatenation if the overhanging sequences are compatible.
DNA with ragged or staggered ends can be made blunt ended prior to
ligation. All of these operations are well known and of general
use. If linkers are used, the linkers can either be ligated to
blunt ended DNA (using blunt ended linkers), or to DNA having
compatible overhanging ends, in which case the linkers can be in
the form of adaptors.
[0085] The following illustrates an example of how the MSDA-CD can
be used to amplify mRNA sequences. First, cDNA is made from the
mRNA of interest. In this example, the cDNA is made in such a way
that it contains phosphorylated 5'-ends. The cDNA is then tailed
with a single adenosine residue at both 3' ends using Taq DNA
polymerase (as described, for example, by Brownstein et al.,
Biotechniques 20:1004-1010 (1996), and in the catalog of Research
Genetics, Inc.). The A-tailed cDNA is then mixed with the T-tailed
linkers in the presence of ATP and T4 DNA ligase in standard
ligation buffer (see, for example, Holton and Graham, Nucl. Acids
Res. 19:1156 (1991), and instructions for the use of pGEM-T vectors
in the Promega Catalog (Promega Biotec, Madison, Wis., 1997) page
206), and the reaction is incubated overnight at 16.degree. C. to
generate long concatenated DNA molecules. The concatenated
molecules consist of tandemly ligated cDNAs and linkers, in
alternating order, of the
structure-linker-DNA-linker-DNA-linker-DNA-. The A-tailing and
T-tailing method is just one example of many possible methods to
obtain tandem, concatenated ligation of linkers and DNA fragments.
It is also possible to concatenate DNA fragments without linkers to
obtain concatenated molecules of the structure -DNA-DNA-DNA-DNA-.
Concatenated DNA fragments with linkers is referred to herein as
linker-concatenated DNA or linker-DNA concatenates. Concatenated
DNA fragments without linkers is referred to herein as
nonlinker-concatenated DNA or nonlinker-DNA concatenates. The terms
concatenated DNA and DNA concatenate refer to both
linker-concatenated DNA and nonlinker-concatenated DNA.
Amplification of linker-DNA concatenates is more specific and
efficient than amplification of nonlinker-DNA concatenates, because
specific primers can be directed to the linker sequence. Thus, the
linker-DNA concatenation method is the preferred form of performing
MSDA-CD.
[0086] It is preferred that the concatenated product be as long as
possible. This is so because the extent of DNA amplification
obtainable with MSDA-CD within any time period is influenced by the
length of the concatenated DNA. The longer the concatenated DNA is,
and the more linkers it contains, the more efficient the
amplification process will be. Concatenation is generally favored
by ligating the fragments at high concentration.
[0087] An example of MSDA-CD performed on linker-concatenated DNA
is illustrated in FIG. 4. Two different linker-specific primers
were used that prime on different sequences on different strands of
the linker. The two primers should not be complementary to each
other. At the top of FIG. 4 is the double-stranded DNA concatenate
with incorporated linkers. The DNA is denatured to make it
single-stranded, and the two linker-specific primers are utilized
to amplify the DNA by multiple strand displacement. It can be
estimated that MSDA-CD will amplify a DNA sample as much as
5,000-fold. In the case of the mRNA profiling (Lockhart et al.),
MSDA-CD, combined with transcriptional amplification, could be used
to improve the limit of detection, permitting profiling analysis in
samples containing only 20 cells.
[0088] When using linker-concatenated DNA, multiple strand
displacement amplification of concatenated DNA can be performed by
(a) mixing primers with a concatenated DNA sample, to produce an
primer-target sample mixture, and incubating the primer-target
sample mixture under conditions that promote hybridization between
the primers and the concatenated DNA in the primer-target sample
mixture, and (b) mixing DNA polymerase with the primer-target
sample mixture, to produce a polymerase-target sample mixture, and
incubating the polymerase-target sample mixture under conditions
that promote replication of the concatenated DNA. Strand
displacement-replication is preferably accomplished by using a
strand displacing DNA, polymerase or a DNA polymerase in
combination with a compatible strand displacement factor.
[0089] Following amplification, the amplified sequences can be for
any purpose, such as uses known and established for PCR amplified
sequences. For example, amplified sequences can be detected using
any of the conventional detection systems for nucleic acids such as
detection of fluorescent labels, enzyme-linked detection systems,
antibody-mediated label detection, and detection of radioactive
labels. A key feature of the disclosed method is that amplification
takes place not in cycles, but in a continuous, isothermal
replication. This makes amplification less complicated and much
more consistent in output. Strand displacement allows rapid
generation of multiple copies of a nucleic acid sequence or sample
in a single, continuous, isothermal reaction. Sequences in DNA
amplified by MSDA-CD performed on concatenated DNA where the
linkers or primers include promoter sequences can be further
amplified by transcriptional amplification using the promoters.
[0090] Where the linkers used for concatenation include a
restriction enzyme site, the amplified DNA can be fragmented by
restriction enzyme digestion. Cleavage of the amplified DNA can
permit or simplify further processing and analysis of the amplified
DNA. If the site used appears rarely (for example, eight-base
recognition sites), the resulting fragments will represent the
original DNA fragments that were concatenated.
[0091] When used, a random or partially random set of primers
randomly prime the concatenated DNA. By choosing a sufficiently
large set of primers of random or mostly random sequence, the
primers in the set will be collectively, and randomly,
complementary to nucleic acid sequences distributed throughout the
concatenated DNA. Amplification proceeds by replication with a
processive polymerase initiated at each primer and continuing until
spontaneous termination. A key feature of this method is the
displacement of intervening primers during replication by the
polymerase. In this way, multiple overlapping copies of the entire
concatenated DNA sample can be synthesized in a short time.
[0092] When using random or partially random primers, multiple
strand displacement amplification of concatenated DNA can be
performed by (a) mixing a set of random or partially random primers
with a concatenated DNA sample, to produce an primer-target sample
mixture, and incubating the primer-target sample mixture under
conditions that promote hybridization between the primers and the
concatenated DNA in the primer-target sample mixture, and (b)
mixing DNA polymerase with the primer-target sample mixture, to
produce a polymerase-target sample mixture, and incubating the
polymerase-target sample mixture under conditions that promote
replication of the concatenated DNA. MSDA-CD using random or
partially random primers is similar to WGSDA and proceeds generally
as illustrated in FIG. 2.
[0093] It is preferred that a set of random or partially random
primers used for MSDA-CD be used at concentrations that allow the
primers to hybridize at desired intervals within the nucleic acid
sample. For example, by using a set of primers at a concentration
that allows them to hybridize, on average, every 4000 to 8000
bases, DNA replication initiated at these sites will extend to and
displace strands being replicated from adjacent sites. It should be
noted that the primers are not expected to hybridize to the target
sequence at regular intervals. Rather, the average interval will be
a general function of primer concentration.
[0094] As in multiple strand displacement amplification,
displacement of an adjacent strand makes it available for
hybridization to another primer and subsequent initiation of
another round of replication. The interval at which primers in the
set of primers hybridize to the target sequence determines the
level of amplification. For example, if the average interval is
short, adjacent strands will be displaced quickly and frequently.
If the average interval is long, adjacent strands will be displaced
only after long runs of replication. For amplification of
linker-concatenated DNA, where the primers are complementary to
linker sequences, the size of the DNA fragments that were
concatenated determines the spacing between the primers.
D. Modifications and Additional Operations
1. Detection of Amplification Products
[0095] Amplification products can be detected directly by, for
example, primary labeling or secondary labeling, as described
below.
i. Primary Labeling
[0096] Primary labeling consists of incorporating labeled moieties,
such as fluorescent nucleotides, biotinylated nucleotides,
digoxygenin-containing nucleotides, or bromodeoxyuridine, during
strand displacement replication. For example, one may incorporate
cyanine dye UTP analogs (Yu et al. (1 994)) at a frequency of 4
analogs for every 100 nucleotides. A preferred method for detecting
nucleic acid amplified in situ is to label the DNA during
amplification with BrdUrd, followed by binding of the incorporated
BUDR with a biotinylated anti-BUDR antibody (Zymed Labs, San
Francisco, Calif.), followed by binding of the biotin moieties with
Streptavidin-Peroxidase (Life Sciences, Inc.), and finally
development of fluorescence with Fluorescein-tyramide (DuPont de
Nemours & Co., Medical Products Dept.).
ii. Secondary Labeling with Detection Probes
[0097] Secondary labeling consists of using suitable molecular
probes, referred to as detection probes, to detect the amplified
DNA or RNA. For example, a primer may be designed to contain, in
its non-complementary portion, a known arbitrary sequence, referred
to as a detection tag. A secondary hybridization step can be used
to bind detection probes to these detection tags. The detection
probes may be labeled as described above with, for example, an
enzyme, fluorescent moieties, or radioactive isotopes. By using
three detection tags per primer, and four fluorescent moieties per
each detection probe, one may obtain a total of twelve fluorescent
signals for every replicated strand.
iii. Multiplexing and Hybridization Array Detection
[0098] Detection of amplified nucleic acids can be multiplexed by
using sets of different primers, each set designed for amplifying
different target sequences. Only those primers that are able to
find their targets will give rise to amplified products. There are
two alternatives for capturing a given amplified nucleic acid to a
fixed position in a solid-state detector. One is to include within
the non-complementary portion of the primers a unique address tag
sequence for each unique set of primers. Nucleic acid amplified
using a given set of primers will then contain sequences
corresponding to a specific address tag sequence. A second and
preferred alternative is to use a sequence present in the target
sequence as an address tag.
iv. Enzyme-linked Detection
[0099] Amplified nucleic acid labeled by incorporation of labeled
nucleotides can be detected with established enzyme-linked
detection systems. For example, amplified nucleic acid labeled by
incorporation of biotin-16-UTP (Boehringher Mannheim) can be
detected as follows. The nucleic acid is immobilized on a solid
glass surface by hybridization with a complementary DNA
oligonucleotide (address probe) complementary to the target
sequence (or its complement) present in the amplified nucleic acid.
After hybridization, the glass slide is washed and contacted with
alkaline phosphatase-streptavidin conjugate (Tropix, Inc., Bedford,
Mass.). This enzyme-streptavidin conjugate binds to the biotin
moieties on the amplified nucleic acid. The slide is again washed
to remove excess enzyme conjugate and the chemiluminescent
substrate CSPD (Tropix, Inc.) is added and covered with a glass
cover slip. The slide can then be imaged in a Biorad
Fluorimager.
2. Linear Strand Displacement Amplification
[0100] A modified form of multiple strand displacement
amplification can be performed which results in linear
amplification of a target sequence. This modified method is
referred to as linear strand displacement amplification (LSDA) and
is accomplished by using a set of primers where all of the primers
are complementary to the same strand of the target sequence. In
LSDA, as in MSDA, the set of primers hybridize to the target
sequence and strand displacement amplification takes place.
However, only one of the strands of the target sequence is
replicated. LSDA requires thermal cycling between each round of
replication to allow a new set of primers to hybridize to the
target sequence. Such thermal cycling is similar to that used in
PCR. Unlike linear, or single primer, PCR, however, each round of
replication in LSDA results in multiple copies of the target
sequence. One copy is made for each primer used. Thus, if 20
primers are used in LSDA, 20 copies of the target sequence will be
made in each cycle of replication.
[0101] DNA amplified using MSDA and WGSDA can be further amplified
by transcription. For this purpose, promoter sequences can be
included in the non-complementary portion of primers used for
strand displacement amplification, or in linker sequences used to
concatenate DNA for MSDA-CD.
EXAMPLES
A. Example 1
Multiple Strand Displacement Amplification of lambda DNA
[0102] This example illustrates multiple displacement amplification
using a total of 14 primers, 7 in each of a right primer set and a
left primer set. The primers in each set are designed to hybridize
to opposite strands on each side of a region to be amplified.
[0103] 1. The first step is a ligation to close nicks, insuring
that long strands are available for copying. A total of 10 .mu.g of
Bacteriophage lambda DNA was dissolved in 100 .mu.l of T4 ligase
buffer (10 mM Tris, pH 7.5, 0.20 M NaCl, 10 mM MgCl.sub.2, 2 mM
ATP). T4 DNA ligase was added to a final concentration of 8
Units/.mu.l, and the material was incubated for 1.5 hours at
37.degree. C. in order to close any nicks in the DNA, making it
perfectly double-stranded. The DNA solution was then diluted
five-fold with distilled water, to yield a final DNA concentration
of 20 ng/.mu.l.
[0104] 2. An aliquot of 1.5 .mu.l of ligated lambda DNA (containing
30 ng of DNA).was mixed with 18.2 .mu.l of distilled water, and a
suitable multiple primer mixture (primers made by standard
phosphoramidite chemistry). The primers used in this example are
indicated below. The nomenclature is "PL" for left primers and "PR"
for right primers. Seven left primers and seven right primers were
used. For each set of 7 primers, the sequences are spaced 300 to
400 nucleotides between each other. The lambda DNA targeted by the
primers is located within the region demarcated by map positions
39500 to 22000, and includes a total of approximately 17500 bases.
This region encompasses lambda Hind II fragments of 2322 bp and
9416 bp.
3 Left Primers (5' to 3') 1 GTTGATACATCAACTGCAC PL7 (SEQ ID NO:1) 2
CAATTACCTGAAGTCTTTC PL6 (SEQ ID NO:2) 3 TTGTCATATTGTATCATGC PL5
(SEQ ID NO:3) 4 AAGATGAAATAAGAGTAGC PL4 (SEQ ID NO:4) 5
TGCATGCTAGATGCTGATA PL3 (SEQ ID NO:5) 6 TATGACTGTACGCCACTGT PL2
(SEQ ID NO:6) 7 AGAGTTTCTTTGAGTAATC PL1 (SEQ ID NO:7) Right Primers
(5' to 3') 1r TTACAACCACTAAACCCAC PR1 (SEQ ID NO:8) 2r
AATCGCCAGAGAAATCTAC PR2 (SEQ ID NO:9) 3r AGGGTTATGCGTTGTTCCA PR3
(SEQ ID NO:10) 4r TGTTAAGCAACGCACTCTC PR4 (SEQ ID NO:11) 5r
AGTCTGGCGTAACCATCAT PR5 (SEQ ID NO:12) 6r AATAGTGTCTTTTGTGTCC PR6
(SEQ ID NO:13) 7r GCTTGTTACGGTTGATTTC PR7 (SEQ ID NO:14)
[0105] Primers were added at a concentration such that in the
following step (step 3, below) the final concentration of each
primer was approximately 1 micromolar. The lambda DNA and primer
mixture was heated at 95.degree. C. for 2.5 minutes in order
denature the lambda DNA, and the tube was immediately placed in
ice.
[0106] 3. The Multiple Strand Displacement Amplification reaction
was set up at 0.degree. C., in a volume of 30 .mu.l, by adding to
the tube of step 2 the following reagents, to give the final
concentrations indicated below:
[0107] (a) 3 .mu.l of 10.times. reaction buffer, designed to yield
a final concentration of 40 mM Tris-HCl (pH 7.5), 25 mM NaCl, 8 mM
MgCl.sub.2, 6.7 mM DTT, 5% v/v DMSO (dimethylsulfoxide), and 400
.mu.M mM dATP, dGTP, dCTP, dTTP. Some MSDA reactions may work
better at different concentration of DMSO, in the range of 1% to
7%.
[0108] (b) E. coli single-strand binding protein (SSB) to a final
concentration of 1.4 .mu.M
[0109] (c) Sequenase 2.0 (Amersham Life Sciences) to a
concentration of 0.475 units/.mu.l (approximately 400 nM).
[0110] 4. The reaction was incubated at 37.degree. C. for 45
minutes. The DNA was amplified about 45-fold.
[0111] If desired, the amplified DNA can incubated anywhere from 2
to 24 hours at 55.degree. C. in a buffer containing 30 mM Tris-HCl
(pH 8.2), 150 mM NaCl, 1 mM EDTA, in order to permit most of the
remaining single-stranded material to renature. The amplification
yield can be increased by using more primers on each side of the
DNA region to be amplified. A suitable number of primers for this
may be in the range of 10 to 30 primers on each side of desired DNA
domain. Primer numbers exceeding 24 on each side may increase the
frequency of nonspecific amplification.
B. Example 2
Whole Genome Amplification of human DNA
[0112] This example is for whole genome amplification, as performed
for the amplification of the human genome using random primers.
[0113] 1. DNA was extracted from peripheral blood lymphocytes using
a standard proteinase K digestion, followed by extraction with
phenol/chloroform. The DNA was quantitated using the Pico-Green dye
method (Molecular Probes, Inc., Eugene, Oreg.; Kit P-7589) and the
material is then diluted in TE-0.2 buffer (10 mM Tris pH 8.3, 0.2
mM EDTA), to yield a final DNA concentration of 1 ng/.mu.l.
[0114] 2. Four microliters (4 nanograms) of human DNA and 20 .mu.l
of TE-0.2 buffer were mixed in a 500 .mu.l microcentrifuge tube and
denatured at 97.degree. C. for 5 minutes. The tube was then
immediately placed in ice.
[0115] 3. An amplification reaction was set up in an ice bath, in a
volume of 30 .mu.l, by adding to the tube of step 2 the following
reagents, to give the final concentrations indicated below:
[0116] (a) 3 .mu.l of 10.times. reaction buffer, designed to yield
a final concentration of 25 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM
(NH.sub.4).sub.2 SO.sub.4, 2 mM MgSO.sub.4, 0.1% Triton X-100, 5%
v/v DMSO (dimethylsulfoxide), and 400 .mu.M mM dATP, dGTP, dCTP,
dTTP.
[0117] (b) A random DNA oligonucleotide primer of 20 bases in
length to a final concentration of 4.0 .mu.Molar.
[0118] (c) Phage T4 Gene 32 protein added to a final concentration
of 30 ng/.mu.l.
[0119] (d) Bst DNA polymerase large fragment (New England Biolabs),
added last, at a final concentration of 0.35 units/.mu.l.
[0120] 4. The reaction was incubated at 48.degree. C. for 4 hours,
and stopped by addition of EDTA (final concentration 4 mM).
[0121] All publications cited herein are hereby incorporated by
reference.
[0122] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
* * * * *