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Claims  |
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What is claimed is:
1. A process for the amplification of a specific nucleic acid sequence, at
a relatively constant temperature and without serial addition of reagents,
comprising the steps of:
(A) providing a single reaction medium containing reagents comprising
(i) a first oligonucleotide primer,
(ii) a second oligonucleotide primer comprising an antisense sequence of an
RNA polymerase promoter,
(iii) a DNA-directed RNA polymerase that recognizes said promoter,
(iv) an RNA-directed DNA polymerase,
(v) a DNA-directed DNA polymerase,
(vi) a ribonuclease that hydrolyzes RNA of an RNA-DNA hybrid without
hydrolyzing single- or double-stranded RNA or DNA, and
(vii) ribonucleoside and deoxyribonucleoside triphosphates;
then
(B) providing in said reaction medium RNA comprising an RNA first template
which comprises said specific nucleic acid sequence or a sequence
complementary to said specific nucleic acid sequence, under conditions
such that a cycle ensues wherein
(i) said first oligonucleotide primer hybridizes to said RNA first
template,
(ii) said RNA-directed DNA polymerase uses said RNA first template to
synthesize a DNA second template by extension of said first
oligonucleotide primer and thereby forms an RNA-DNA hybrid intermediate,
(iii) said ribonuclease hydrolyzes RNA which comprises said RNA-DNA hybrid
intermediate,
(iv) said second oligonucleotide primer hybridizes to said DNA second
template,
(v) said DNA-directed DNA polymerase uses said second oligonucleotide
primer as template to synthesize a functional RNA polymerase promoter by
extension of said DNA second template;
and
(vi) said DNA-directed RNA polymerase recognizes said functional promoter
and transcribes said DNA second template, thereby providing copies of said
RNA first template;
and thereafter
(C) maintaining said conditions for a time sufficient to achieve a desired
amplification of said specific nucleic acid sequence.
2. A process according to claim 1, wherein said RNA first template
comprises said specific nucleic acid sequence and wherein step (B)
comprises providing single-stranded RNA in said reaction medium such that
(i) said first oligonucleotide primer hybridizes to said single-stranded
RNA,
(ii) said RNA-directed DNA polymerase uses said single-stranded RNA as a
template to synthesize a DNA second template by extension of said first
oligonucleotide primer and thereby forms an RNA-DNA hybrid,
(iii) said ribonuclease hydrolyses RNA which comprises said RNA-DNA hybrid,
(iv) said second oligonucleotide primer hybridizes to said DNA second
template,
(v) said DNA-directed DNA polymerase uses said second oligonucleotide
primer as template to synthesize a functional RNA polymerase promoter by
extension of said DNA second template; and
(vi) said DNA-directed RNA polymerase recognizes said functional promoter
and transcribes said DNA second template, thereby providing copies of said
RNA first template.
3. A process according to claim 1, wherein said RNA first template
comprises a sequence complementary to said specific nucleic acid sequence
and wherein step (B) comprises providing single-stranded RNA in said
reaction medium such that
(i) said complementary DNA hybridizes to said single-stranded RNA,
(ii) said RNA-directed DNA polymerase uses said RNA as a template to
synthesize a complementary DNA by extension of said second oligonucleotide
primer and thereby forms an RNA-DNA hybrid,
(iii) said ribonuclease hydrolyses RNA which comprises said RNA-DNA hybrid,
(iv) said first oligonucleotide primer hybridizes to said complementary
DNA,
(v) said DNA-directed DNA polymerase uses said second oligonucleotide
primer as template to synthesize said DNA second template and a functional
RNA polymerase promoter by extension of said first oligonucleotide primer
(vi) said DNA-directed RNA polymerase recognizes said functional promoter
and transcribes said DNA second template, thereby providing copies of said
RNA first template.
4. A process according to claim 1, wherein step (B) comprises adding to
said reaction medium single-stranded DNA which comprises an antisense
sequence of a promoter, such that
(i) said first oligonucleotide primer hybridizes to said single-stranded
DNA,
(ii) said DNA-directed DNA polymerase uses said single stranded DNA as
template to synthesize said DNA second template and a functional RNA
polymerase promoter by extension of said first oligonucleotide primer,
and
(iii) said DNA-directed RNA polymerase recognizes said functional promoter
and transcribes said DNA second template, thereby providing copies of said
RNA first template.
5. A process according to claim 4, wherein step (B) comprises adding to
said reaction medium an RNA-DNA hybrid comprising said single-stranded
DNA, such that said ribonuclease hydrolyzes RNA which comprises said
RNA-DNA hybrid.
6. A process according to claim 1, wherein step (B) comprises adding to
said reaction medium single-stranded DNA which comprises said DNA second
template, such that
(i) said second oligonucleotide primer hybridizes to said single-stranded
DNA,
(ii) said DNA-directed DNA polymerase uses said second oligonucleotide
primer as template to synthesize a functional RNA polymerase promoter by
extension of said DNA second template;
and
(iii) said DNA-directed RNA polymerase recognizes said functional RNA
polymerase promoter and transcribes said DNA second template, thereby
providing copies of said RNA first template.
7. A process according to claim 6, wherein step (B) comprises adding to
said reaction medium an RNA-DNA hybrid comprising said single-stranded
DNA, such that said ribonuclease hydrolyzes RNA which comprises said
RNA-DNA hybrid.
8. A process according to claim 2, wherein step (B) comprises adding to
said reaction medium a DNA comprising a functional promoter, such that
said DNA-directed RNA polymerase transcribes said DNA, thereby
synthesizing said single-stranded RNA.
9. A process according to claim 3, wherein step (B) comprises adding to
said reaction medium a DNA comprising a functional promoter, such that
said DNA-directed RNA polymerase transcribes said DNA, thereby
synthesizing said single-stranded RNA.
10. A process according to claim 1, wherein said second oligonucleotide
primer further comprises an antisense sequence of a transcription
initiation site for said DNA-directed RNA polymerase, said antisense
sequence of said transcription initiation site being operatively linked to
said antisense sequence of said promoter.
11. A process according to claim 10, wherein said DNA-directed RNA
polymerase is bacteriophage T7 RNA polymerase and wherein said antisense
sequence of a transcription initiation site and said antisense sequence of
a functional promoter together comprise the nucleotide sequence
AATTCTAATACGACTCACTATAGGGAG.
12. A process according to claim 1, wherein step (B) further comprises
adding a sample to said reaction medium under conditions such that, if
said sample thereby provides RNA comprising an RNA first template which
comprises said specific nucleic acid sequence or a sequence complementary
to said specific nucleic acid sequence, said cycle ensues, and wherein
said process further comprises, after step (C), a step (D) of monitoring
said reaction medium for consumption of any of said reagents (i), (ii) and
(vii) or for accumulation of any product of said cycle.
13. A process according to claim 12, wherein step (D) comprises detecting a
nucleic acid product of said cycle.
14. A process according to claim 13, wherein step (D) comprises detecting
said nucleic acid product using a nucleic acid probe.
15. A process according to claim 13, wherein step (D) comprises detecting
said nucleic acid product using restriction endonucleases and
electrophoretic separation.
16. A process according to claim 13, wherein step (D) comprises monitoring
the accumulation of said RNA first template.
17. A process according to claim 13, wherein step (D) comprises monitoring
the accumulation of said DNA second template.
18. A process according to claim 13, wherein step (D) comprises monitoring
DNA containing a functional promoter.
19. A process according to claim 13, wherein step (D) comprises monitoring
the accumulation of said RNA-DNA hybrid intermediate.
20. A process according to claim 13, wherein step (D) further comprises
comparing consumption of any reagent of said reagents (i), (ii) and (vii)
or accumulation of any product of said cycle with a value representing
consumption of said reagent or accumulation of said product in said
reaction medium in the absence of said specific nucleic acid sequence and
said sequence complementary thereto.
21. A process according to claim 1, wherein said ribonuclease comprises
Escherichia coli ribonuclease H and ribonuclease H of avian myeloblastosis
virus polymerase.
22. A process according to claim 1, wherein said ribonuclease comprises
calf thymus ribonuclease H.
23. A process according to claim 1, wherein said first oligonucleotide
primer or said second oligonucleotide primer is bound reversibly to an
immobilized support.
24. A process according to claim 1, wherein said DNA-directed RNA
polymerase is a bacteriophage RNA polymerase.
25. A process according to claim 24, wherein said DNA-directed RNA
polymerase is bacteriophage T7 RNA polymerase.
26. A process according to claim 24, wherein said DNA-directed RNA
polymerase is bacteriophage T3 polymerase.
27. A process according to claim 24, wherein said DNA-directed RNA
polymerase is bacteriophage .phi.II polymerase.
28. A process according to claim 24, wherein said DNA-directed RNA
polymerase is Salmonella bacteriophage sp6 polymerase.
29. A process according to claim 24, wherein said DNA-directed RNA
polymerase is Pseudomonas bacteriophage gh-1 polymerase.
30. A process according to claim 1, wherein said RNA-directed DNA
polymerase is a retrovirus reverse transcriptase.
31. A process according to claim 30, wherein said retrovirus reverse
transcriptase is avian myeloblastosis virus polymerase.
32. A process according to claim 30, wherein said retrovirus reverse
transcriptase is a Moloney murine leukemia virus polymerase.
33. A process according to claim 1, wherein said DNA-directed DNA
polymerase lacks exonuclease activity.
34. A process according to claim 1, wherein all DNA polymerases in said
reaction medium lack DNA exonuclease and DNA endonuclease activity.
35. A process according to claim 1, wherein said DNA-directed DNA
polymerase is avian myeloblastosis virus polymerase.
36. A process according to claim 1, wherein said DNA-directed DNA
polymerase is DNA polymerase .alpha. or DNA polymerase .beta..
37. A process according to claim 1, wherein said DNA-directed DNA
polymerase is calf thymus DNA polymerase.
38. A process according to claim 1, wherein step (C) comprises maintaining
said conditions for a time between 30 minutes and 4 hours.
39. A process according to claim 1, further comprising the steps of
ligating a DNA product of said cycle into a cloning vector and then
cloning said DNA product.
40. A process according to claim 39, further comprising the step of
expressing a product encoded by said DNA product of said cycle in an
expression system. |
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Claims |
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Description |
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FIELD OF THE INVENTION
This invention relates to a process for amplifying a specific nucleic acid
sequence.
BACKGROUND OF THE INVENTION
The detection of a specific nucleic acid sequence present in a sample by
probing the sample with a complementary sequence of nucleic acids is a
known diagnostic technique. Nucleic acids are highly specific in binding
to complementary nucleic acids and are thus useful to determine whether a
specific nucleic acid is present in a sample. One must know the sequence
of the specific nucleic acid to be detected and then construct a probe
having a complementary nucleic acid sequence to the specific nucleic acid
sequence.
In this application, the phrase "specific nucleic acid sequence" means a
single stranded or double stranded nucleic acid which one wishes to
amplify; "sample" means a mixture containing nucleic acids; "sufficiently
complementary" means that two nucleic acids, a primer and a template, are
capable of specific interaction which allows efficient, primer-dependent
and template-directed synthesis of DNA, under given conditions of ionic
strength and temperature.
Since nucleic acid probes are highly specific, it is preferable in some
situations to probe the nucleic acid sequence itself rather than the
protein produced by the nucleic acid sequence. As a particular example, a
diagnostic method based solely on protein detection would be unreliable
for determining the presence of infectious particles of hepatitis B virus,
due to the presence of significant levels of non-infectious antigen
particles which lack the DNA genome. In another example, the various
subtypes of human papilloma virus found in either pre-cancerous or benign
cervical tumors can be distinguished only by the use of nucleic acid probe
hybridization. Also, the microbiology of AIDS makes it certain that an
assay based on the presence of AIDS specific nucleic acid sequence would
be superior as a diagnostic.
The greatest difficulty with applying existing nucleic acid probe
technology, and the reason that the utility of the existing probe
technology is limited, is the copy number problem. In a virus or cell, for
example, there is usually a single copy of a particular gene. This one
copy may give rise to many copies of gene product, either RNA or protein.
For this reason, diagnostic techniques have often involved probing the
protein, since the specific sequence of nucleic acid to be detected may
give rise to many thousand copies of protein.
The naturally-occurring high number of ribosomal RNA, up to 100,000 copies
per cell, has been used by GenProbe to facilitate diagnosis of certain
bacterial pathogens, such as Legionella and Mycoplasma, using nucleic acid
probes. However, this strategy cannot be used with non-cellular pathogens,
such as viruses. Copy number is a particular problem with the development
of a nucleic acid probe method for the detection of AIDS virus, where the
integrated provirus may be present in less than one of ten thousand
peripheral blood lymphocytes. Thus, if the particular nucleic acid
sequence suspected to be present in a sample could be amplified, the copy
number problem could be circumvented and probe assays could be more
readily used.
In a normal biological sample, containing only a few cells, and
consequently only a few copies of a particular gene, it is necessary to
utilize an amplification process in order to overcome the copy number
problem.
One method to amplify is to `grow out` the sample, that is, to arrange
conditions so that the living biological material present in the sample
can replicate itself. Replication increases the quantity of nucleic acid
sequences to detectable levels. In the food industry, for example, in
order to test processed food for the food-poisoning bacteria Salmonella,
food samples must be incubated for a number of days to increase the
quantity of nucleic acids. In clinical samples, pathogens must also be
allowed to increase their number by growing out over some considerable
time.
U.S. Pat. No. 4,683,195 issued on Jul. 28, 1987 to Cetus Corporation and
U.S. Pat. No. 4,683,202 issued on Jul. 28, 1987 to Cetus Corporation are
each directed to a process for amplifying a target nucleic acid sequence
contained in a sample. U.S. Pat. No. 4,683,195 relates to a process in
which a sample suspected of containing a target nucleic acid sequence is
treated with oligonucleotide primers such that a primer extension product
is synthesized which in turn serves as a template, resulting in
amplification of the target nucleic acid sequence. The primer extension
product is separated from the template in the preferred embodiment using
heat denaturation. Similarly, U.S. Pat. No. 4,683,202 relates to a process
for amplifying a target nucleic acid sequence having two separate
complementary strands. The process includes treating the strands with
primers to synthesize extension products, separating the primer extension
products from the templates, and in turn using the primer extension
products as templates.
Both of the above United States patents require either manual or mechanical
participation and multi-step operations by the user in the amplification
process. The steps involved in these patents require the user to heat the
sample, cool the sample, add appropriate enzymes and then repeat the
steps. The temperature changes cause the enzymes to loose their activity.
Hence, the user is required to repeatedly supplement the amplification
mixture with aliquots of appropriate enzymes during the amplification
process.
In addition, in U.S. Pat. Nos. 4,683,195 and 4,683,202 each cycle of the
amplification process takes place by the synthesis from a first template,
of a second template, the second template in turn is used to synthesize
the first template. This procedure is repeated, thus, each cycle of the
amplification process is based on the synthesis of one product from one
substrate.
Notwithstanding the amplification processes disclosed in the prior art, a
need exists for improvements to the amplification process. It would be
preferable if the amplification process required less participation and
fewer manipulations by the user. Further, it would be advantageous if the
amplification took place at a relatively constant ambient temperature so
that the activity of the enzymes involved in the process would not be
affected. It would be more expedient if a template could be used to
generate more than one product from one substrate in each cycle of the
amplification process.
SUMMARY OF THE INVENTION
This invention relates to an amplification process which is expedient and
requires less participation and fewer manipulations by the user of the
process than conventional amplification processes. The amplification takes
place at a relatively constant ambient temperature. In addition, each
cycle of the process generates a plurality of copies of product from one
substrate. The amplification process of this invention may be used to
increase the quantity of a specific nucleic acid thus circumventing the
copy number problem. Hence, probe assays may be more readily used. The
amplification process could also be used to increase the purity of a
specific nucleic acid sequence as a substitute for conventional cloning
methodology.
According to one aspect of the invention, a process for amplifying a
specific nucleic acid sequence is used. The process involves the synthesis
of single-stranded RNA, single-stranded DNA, and double stranded DNA. The
single stranded RNA is a first template for a first primer. The single
stranded DNA is a second template for a second primer. The double stranded
DNA is a third template for the synthesis of a plurality of copies of the
first template. A sequence of the first or the second primer is
sufficiently complementary to a sequence of the specific nucleic acid
sequence and a sequence of the first or the second primer is sufficiently
homologous to a sequence of the specific nucleic acid sequence. A 3' end
of the first primer is oriented towards a 3' end of the second primer on
complementary strands.
According to another aspect of the invention, a process for amplifying a
specific nucleic acid sequence is used. The process involves:
(a) hybridizing a first primer to a first template. The first primer has a
DNA sequence which is sufficiently complementary to a RNA sequence of the
first template;
(b) synthesizing a first DNA sequence covalently attached to the first
primer and complementary to the RNA sequence of the first template. The
first DNA sequence and the first primer comprise a second template;
(c) separating the first template from the second template to allow
hybridization of a second primer;
(d) hybridizing the second primer to the second template. The second primer
has a DNA sequence which is sufficiently complementary to a DNA sequence
of the second template. The second primer also has an antisense sequence
of a promoter and an antisense sequence of a transcription initiation site
for a RNA polymerase;
(e) synthesizing a second DNA sequence covalently attached to the second
primer and complementary to the DNA sequence of the second template and
synthesizing a third DNA sequence covalently attached to the second
template and complementary to the DNA sequence of the second primer. The
second and third DNA sequences, the second primer and the second template
comprise a third template;
(f) synthesizing a plurality of copies of the RNA sequence of the first
template from the third template.
A sequence of the first or the second primer is sufficiently complementary
to a sequence of the specific nucleic acid sequence and a sequence of the
first or the second primer is sufficiently homologous to a sequence of the
specific nucleic acid sequence. A 3.gtoreq. end of the first primer is
oriented towards a 3' end of the second primer on complementary strands.
In a further alternative of the invention, the second primer of DNA has a
sequence at its 3' end which is sufficiently complementary to the DNA
sequence of the second template. The second primer has at its 5' end an
antisense sequence of a promoter and an antisense sequence of a
transcription initiation site for a RNA polymerase.
In a further alternative of the invention, the third DNA sequence
covalently attached to the second template is complementary to the DNA
sequence at the 5' end of the second primer.
In another alternative of the invention, a process for amplifying a
specific nucleic acid sequence is used. The process involves combining a
first primer, a second primer, ribonuclease H, a RNA-directed DNA
polymerase, a DNA-directed DNA polymerase, a RNA polymerase,
ribonucleoside triphosphates and deoxyribonucleoside triphosphates with a
sample. The first primer of DNA has a sequence which is sufficiently
complementary to a first template of RNA. The second primer of DNA has a
sequence which is sufficiently complementary to a second template of DNA,
and an antisense sequence of a promoter and an antisense sequence of a
transcription initiation site which are recognized as substrate by the RNA
polymerase. A sequence of the first primer or the second primer is
sufficiently complementary to a sequence of the specific nucleic acid
sequence and a sequence of the first primer or the second primer is
sufficiently homologous to a sequence of the specific nucleic acid. A 3'
end of the first primer is oriented towards a 3' end of the second primer
on complementary strands.
In a further alternative of the invention, a process for amplifying a
specific nucleic acid sequence is used. The process involves adding a
first primer, a second primer, myeloblastosis viral polymerase, E. coli
ribonuclease H, bacteriophage T7 RNA polymerase, ribonucleoside
triphosphates and deoxyribonucleoside triphosphates to a sample. The first
primer of DNA has a sequence which is sufficiently complementary to a
first template of RNA. The second primer of DNA has a sequence which is
sufficiently complementary to a second template of DNA, and an antisense
sequence of a promoter and an antisense sequence of a transcription
initiation site which are recognized as substrate by T7 RNA polymerase. A
sequence of the first primer or the second primer is sufficiently
complementary to a sequence of the specific nucleic acid sequence and a
sequence of the first primer or the second primer is sufficiently
homologous to a sequence of the specific nucleic acid sequence. A 3' end
of the first primer is oriented towards a 3' end of the second primer on
complementary strands.
BRIEF DESCRIPTION OF THE DRAWINGS
In drawings which illustrate embodiments of the invention,
FIG. 1 is a general illustration the nucleic acid amplification process;
FIG. 2 shows the synthetic oligonucleotides DNA sequences which are used
for testing the amplification process: FIG. 2A, the gag test sequence;
FIG. 2B, the gag2 test sequence;
FIG. 3 is an autoradiogram of PAGE analysis of amplification reactions
using different primer concentrations;
FIG. 4 is an autoradiogram of PAGE analysis of amplification reactions
using different template concentrations;
FIG. 5 is an autoradiogram of Dot-blot hybridization on amplification
reactions;
FIG. 6 is an autoradiogram of PAGE analysis of amplification reaction using
restriction fragments as template.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
This invention relates to a process for amplifying a specific nucleic acid
sequence. The amplification involves the alternate synthesis of DNA and
RNA and is generally illustrated in FIG. 1. In this process,
single-stranded RNA is converted to single-stranded DNA which in turn is
converted to a functional template for the synthesis of a plurality of
copies of the original single-stranded RNA. A first primer and a second
primer are used in the amplification process. A sequence of the first
primer or the second primer is sufficiently complementary to a sequence of
the specific nucleic acid sequence and a sequence of the first or the
second primer is sufficiently homologous to a sequence of the specific
nucleic acid sequence. In some instances, both the first primer and second
primer are sufficiently complementary and sufficiently homologous to a
sequence of the specific nucleic acid sequence, for example, if the
specific nucleic acid sequence is double stranded DNA.
The RNA is converted to single-stranded DNA by hybridizing an
oligonucleotide primer (the first primer) to the RNA (the first template)
and synthesizing a complementary strand of DNA from the first primer, (the
first DNA sequence) by using a RNA-directed DNA polymerase. The resulting
single-stranded DNA (the second template) is separated from the first
template by, for example, hydrolysis of the first template and by using a
ribonuclease which is specific for RNA-DNA hybrids (for example,
ribonuclease H). The second template is converted to a form which is
capable of RNA synthesis by hybridizing a synthetic oligonucleotide (the
second primer), which contains at its 3' end a sequence which is
sufficiently complementary to the 3' end of the second template and toward
its 5' end a sequence containing the antisense strand of a promoter and
antisense sequence of a transcription initiation site, and by synthesizing
a second DNA sequence covalently attached to the 3' end of the second
primer using the second template as a template and synthesizing a third
DNA sequence covalently attached to the 3' end of the second template
using the second primer as a template, using DNA-directed DNA polymerase.
The resulting functional derivative of the second template, which is a
third template, is used for the synthesis of a plurality of copies of RNA,
the first template, by using a RNA polymerase which is specific for the
promoter and transcription initiation site defined by the second primer.
Each newly synthesized first template can be converted to further copies
of the second template and the third template by repeating the cycle. In
addition, repetition of the cycle does not require participation or
manipulation by the user.
The amplification process commences with the addition of a suitable
template nucleic acid to the appropriate enzymes, primers, and cofactors
under the appropriate reaction conditions. This template nucleic acid is
in a form which is capable of homogenous and continuous amplification and
can function as an intermediate in the cycle set forth in FIG. 1. The
amplification process involves the net consumption of precursors (primers,
ribonucleoside triphosphates and deoxyribonucleoside triphosphates) and
the net accumulation of products (RNA and DNA). The processes of RNA and
DNA synthesis will proceed asynchronously until sufficient levels of
nucleic acids have been synthesized to allow detection. The amplification
process may be monitored by, for example, the synthesis of a labeled
product from a labeled precursor.
It is contemplated that amplification may involve another process either in
addition to or in place of the one generally illustrated in FIG. 1. Also
possible are certain counter-productive enzymatic reactions which occur at
permissibly low rates. Included among the possible non-productive side
reactions is the synthesis of RNA and/or DNA in the absence of an added
template nucleic acid. Such RNA and/or DNA products can be discriminated
from desired products by determining whether a particular sequence which
would be found only between the two priming sites of the specific nucleic
acid sequence is present.
The first primer is an oligodeoxyribonucleotide which has at its 3' end a
sequence which is sufficiently complementary to the 3' end of the first
template. The sequence at the 3' end of the first primer has a particular
length and base composition to allow specific and efficient synthesis of
the first DNA sequence, under the given conditions of ionic strength and
temperature. The first primer may be sufficiently complementary to a
region internal to the 3' end of the first template in the first cycle. In
subsequent cycles, the 5' end of the first primer would be complementary
to the 3' end of the first template. It is contemplated that the first
primer may be composed partially or completely of nucleotides or
nucleotide analogs other than the natural deoxyribonucleotides. The 5' end
of the first primer may contain sequences which are not complementary to
the first template in the first cycle. The non-complementary sequences may
be complementary to a nucleic acid which can be immobilized, or to which
can be bound a useful non-nucleic acid component, such as a reporter to
facilitate detection. Alternatively, the non-complementary sequences may
include an antisense sequence of a promoter and an antisense sequence of a
transcription initiation site, which could be used for the synthesis of
RNA. This RNA would be complementary to the first template and could be
used as an intermediate in another amplification cycle.
The second primer is an oligodeoxyribonucleotide which contains at its 3'
end a sequence which is sufficiently complementary to the 3' end of the
second template. The second primer has a particular length and base
composition to allow specific and efficient synthesis of the second and
third DNA sequences, under the given conditions of ionic strength and
temperature. In addition, the second primer contains the antisense
sequence of a functional promoter and the antisense sequence of a
transcription initiation site. This sequence, when used as a template for
synthesis of the third DNA sequence, contains sufficient information to
allow specific and efficient binding of a RNA polymerase and initiation of
transcription at the desired site. The promoter sequence may be derived
from the antisense strand of a functional promoter. The transcription
initiation site may be derived from the 5' terminal sequence of a natural
RNA transcript. In the preferred embodiment, the 5'-terminal sequence of
the second primer is AATTCTAATACGACTCACTATAGGGAG. This sequence contains
the antisense sequence of the promoter and the antisense sequence of the
transcription initiation site for T7 RNA polymerase. Alternatively, the
transcription initiation site and promoter for another phage RNA
polymerase may be used. In addition, sequences which are unrelated to the
promoter function may be included at the 5' end of the second primer or
between the transcription initiation site and the sequence at the 3' end
which hybridizes to the second template. It is contemplated that the
second primer may be composed partially or completely of nucleotides or
nucleotide analogs other than natural deoxyribonucleotides.
All of the enzymes used in this invention should meet certain practical
specifications. Each enzyme or enzyme preparation should be free of
deleterious deoxyribonuclease ("DNase") activities, such as the 5' or 3'
exonuclease activities which are often associated with certain DNA
polymerases and single-strand or double-strand specific exonuclease or
endonucleases. Each enzyme or enzyme preparation should be free of
deleterious ribonuclease ("RNase") activities, with the exception of the
preferred addition of a ribonuclease activity which is specific for
hybrids of RNA and DNA (for example, ribonuclease H). In addition, each
enzyme should be reasonably active under the common reaction conditions
which are used for the other enzymatic processes, and non-enzymatic
processes, such as hybridizing oligonucleotide primers to the RNA or DNA
templates.
The DNA-directed RNA polymerase which is used in this invention may be any
enzyme capable of binding to a particular DNA sequence called a promoter
and specifically initiating in vitro RNA synthesis at a defined initiation
site within close proximity to the promoter. The promoter and the
initiation site form part of the second primer. In addition the RNA
polymerase should be capable of synthesizing several copies of RNA per
functional copy of template in a reasonable amount of time. In the
preferred embodiment, the bacteriophage T7 RNA polymerase is used. In
addition other bacteriophage RNA polymerases, such as phage T3, phage
.phi.II, Salmonella phage sp6, or Pseudomonas phage gh-l may be used. In
another embodiment, other prokaryotic or eukaryotic DNA-directed RNA
polymerase may be used. It should be understood that if alternative RNA
polymerases are used, then the necessary changes to the promoter and
initiation sequences of the second primer should be made according to the
template specificity of the particular RNA polymerase.
The RNA-directed DNA polymerase which is used in this invention may be any
enzyme capable of synthesizing DNA from an oligodeoxyribonucleotide primer
and a RNA template. In addition this enzyme may contain activities for
DNA-directed DNA polymerase and RNase H. In the preferred embodiment, the
avian myloblastosis viral polymerase ("AMV reverse transcriptase") is
used. In addition, the RNA-directed DNA polymerase could be from another
retrovirus, such as Moloney murine leukemia virus.
Alternatively, other eukaryotic RNA-directed DNA polymerases could be used.
The DNA-directed DNA polymerase which is used in this invention may be any
enzyme capable of synthesizing DNA from an oligodeoxyribonucleotide primer
and a DNA template. This enzyme should not contain either 5'- or
3'-exonuclease activities, which are associated with many types of DNA
polymerase. In the preferred embodiment, the AMV reverse transcriptase is
used. However, other DNA-directed DNA polymerases which naturally lack the
5'- or 3'-exonuclease activities could be used. These could include
certain eukaryotic DNA polymerases, such as, DNA polymerase .alpha. or
.beta. those DNA polymerases which could be isolated from a mammalian
tissue, such as calf thymus. An otherwise unsuitable DNA polymerase could
be made useful by removing the undesirable exonuclease activities either
by alteration of the DNA polymerase gene followed by expression of the
altered polymerase in a suitable host cell, or by chemical modification of
the DNA polymerase protein. Altered versions of DNA polymerase could be
made from the Klenow fragment of E. coli DNA polymerase I or the
bacteriophage T7 DNA polymerase. It should be understood that such
alternative DNA-directed DNA polymerase activities are added to supplement
the activity contributed by the RNA-directed DNA polymerase, since in the
preferred embodiment, both RNA-directed and DNA-directed DNA polymerase
activities are supplied by the same enzyme.
The RNase H which could be used in this invention may be any enzyme capable
of hydrolyzing a RNA which is annealed to a complementary DNA. This enzyme
should not be capable of hydrolyzing single or double-stranded RNA or any
DNA. In the preferred embodiment, the E. coli RNase H is used. In
addition, other RNase H enzymes could be used, such as calf thymus RNase
H. Since RNase H is an intrinsic activity of AMV reverse transcriptase,
the E. coli RNase H will be supplemented in the preferred embodiment by
the RNase H of AMV reverse transcriptase. Alternatively, any other enzyme
capable of separating the second template from the first template could be
used.
The abovementioned enzymes and primers are mixed together in a reaction
vessel which contains the necessary buffers and cofactors for both DNA and
RNA synthesis. In addition, the ionic conditions and reaction temperature
should be compatible with specific hybridization of the primers to the DNA
and RNA templates as is known to those skilled in the art. The reaction
mixture should be free of such agents which would interfere with the
amplification process, specifically substances which could greatly inhibit
the activity of the enzymes, interfere with the hybridizing of primers and
templates, or degrade non-productively the nucleic acid intermediates and
products.
The description of possible detection schemes may be useful to the
application of the amplification process. It should be understood that
schemes which may be used for detecting the nucleic acids which are
synthesized in the amplification process are not limited to those
described herein, and it is contemplated that other methods may be used.
In one embodiment, a labeled precursor may be added to the reaction
mixture. Amplification is determined by quantitive or qualitative analysis
of labeled products, which can be separated from the labeled precursor by
using methods known in the art. A labeled precursor may be a
ribonucleoside triphosphate for detecting RNA synthesis, or a
deoxynucleoside triphosphate or an oligonucleotide primer for detecting
DNA synthesis. The type of label may be a radioisotope or a useful
chemical group, such as biotin, a chromophore, a fluorophore, or a hapten
which could bind to an antibody, or possibly a protein or an enzyme. The
labeled products may be separated from the labeled precursors on the basis
of solubility, charge, or size. In addition, the labeled DNA or RNA may be
hybridized to a nucleic acid which contains a complementary sequence and
which can be immobilized.
In another embodiment, the products of the amplification process may be
bound to an immobilized support, hybridized to a nucleic acid probe
containing a complementary sequence, and separated from the unhybridized
nucleic acid probe which remains in solution. The products, DNA or RNA,
may be bound directly to a solid support by any stable interaction, such
as hydrophobic, electrostatic, or covalent interaction. In addition, the
products may contain certain chemical groups, for example, biotin, which
may be incorporated into the products during the amplification process to
allow binding to an immobilized protein, for example, avidin or
streptavidin. In addition, the products may be hybridized to a nucleic
acid which contains a complementary sequence and which can be immobilized.
The nucleic acid probe would contain a complementary sequence which forms
a sufficiently stable interaction with a product of the amplification
process to allow binding under the conditions of hybridization and
sustained binding under the conditions used for removal of the
unhybridized nucleic acid probe. In the preferred embodiment the
complementary sequence would be derived from that part of the specific
nucleic acid sequence which is between the sequences of the first primer
and the second primer. The nucleic acid probe may be a single-stranded DNA
or RNA, or a double-stranded DNA or RNA which can be made single-stranded,
or an oligonucleotide which can be composed of deoxyribonucleotides and/or
ribonucleotides. In addition, the nucleic acid probe may contain a
chemical group which could covalently bind to a product DNA or RNA under
the appropriate conditions. The nucleic acid probe may be labeled with a
radioisotope or a useful chemical group, such as biotin, a chromophore, a
fluorophore, or a hapten which could bind to an antibody. In addition, the
nucleic acid probe could be conjugated to a protein or enzyme, for
example, a phosphatase or a peroxidase. In addition, the nucleic acid
probe may contain sequences which would allow in vitro replication of the
probe.
It is contemplated that the products of the amplification process may be
analyzed by methods which are typically used for nucleic acids that have
been enriched by molecular cloning techniques. In one alternative, the
synthesis of a specific DNA sequence may be detected by digestion of the
synthesized DNA with a restriction endonuclease, followed by
electrophoretic separation and detection using methods known in the art.
In another alternative, the sequence of amplified RNA may be determined by
DNA synthesis using a RNA-directed DNA polymerase, the first primer, and
dideoxynucleoside triphosphates (Stoflet et al., 1988). In another
alternative, the sequence of the amplified third template may be
determined by RNA synthesis using the DNA-directed RNA polymerase used in
the amplification process, and 3'-deoxyribonucleoside triphosphates
(Axelrod & Kramer, 1985). In another alternative, the amplified RNA may
encode a polypeptide which could be translated, in vitro. The polypeptide
product of the in vitro translation could be analyzed by using an
antibody.
A sample suspected of containing or known to contain the specific nucleic
acid sequence is added to the reaction mixture in the form of a template
nucleic acid which is capable of homogeneous and continuous amplification
and may be any intermediate in the cycle set forth in FIG. 1. In
particular, the template nucleic acid may be a single-stranded RNA which
contains at its 5' end a sequence which is sufficiently homologous to that
which is at the 3' end of the second primer, and contains a sequence which
is sufficiently complementary to the first primer. A template nucleic acid
of this form would function as a first template in the amplification
process. Alternatively, the template nucleic acid may be a single-stranded
DNA which contains at its 3' end a sequence which is sufficiently
complementary to at least the 3' end of the second primer, and contains a
sequence which is sufficiently homologous to that which is at the 3' end
of the first primer. A template nucleic acid of this form would function
as a second template in the amplification process. Alternatively, the
template nucleic acid may be a double-stranded DNA, one strand of which
contains at its 5' end the entire sequence of the second primer and
contains a sequence which is sufficiently complementary to the first
primer. The double-stranded DNA functions as a third template in the
amplification process.
Although the preparation of a template nucleic acid is not part of the
amplification process, the description of possible schemes for generating
template nucleic acids may be useful to the application of the
amplification process. It should be understood that the schemes which may
be used for obtaining the template nucleic acid are not limited to the
alternatives which are described herein, and it is contemplated that other
methods may be used.
In one alternative, a template nucleic acid which could function as a first
template could be a naturally occurring RNA or a RNA fragment which could
be generated from a larger RNA molecule by using site specific hydrolysis
methods known in the art (Shibahara et al., 1987).
In another alternative, a template nucleic acid which could function as a
second template could be generated from a double-stranded DNA by digestion
with a restriction endonuclease which has a site immediately flanking the
sequence which is sufficiently complementary to the 3' end of the second
primer. The resulting double-stranded DNA fragments could then be made
single-stranded by using chemical or thermal denaturation methods.
In another alternative, a template nucleic acid which could function as a
second template could be generated from a single-stranded DNA or RNA to
which has been hybridized an oligonucleotide which is capable of blocking
DNA synthesis. This blocking oligonucleotide may contain a chemical group,
which could covalently bind to the template, under the appropriate
conditions. DNA synthesis from this blocked template using the first
primer could result in a synthesized DNA with the same 3' end as the
second template. If the original template is RNA, then the resulting
DNA-RNA hybrid may be used directly as a template nucleic acid. If the
original template is DNA, then the resulting copy of the second template
could then be separated from the original template by using chemical or
thermal denaturation methods.
In another alternative, a template nucleic acid which could function as a
third template could be generated from a single-stranded DNA or RNA by DNA
synthesis from the DNA or RNA template using the second primer | | |
