U.S. patent number RE39,007 [Application Number 10/164,833] was granted by the patent office on 2006-03-07 for isothermal strand displacement nucleic acid amplification.
This patent grant is currently assigned to Gen-Probe Incorporated. Invention is credited to Nanibhushan Dattagupta, Daniel Louis Kacian, Marc Spingola, Paul Douglas Stull.
United States Patent |
RE39,007 |
Dattagupta , et al. |
March 7, 2006 |
Isothermal strand displacement nucleic acid amplification
Abstract
Methods for amplifying target nucleic acid sequences using a
nucleic acid polymerase lacking 5' exonuclease activity and a set
of oligonucleotide primers. Preferably, a primer array is used. The
primer array contains two sets of primers. One set contains at
least two complementary primers. The other set contains at least
two sense primers. Using the described methods amplification can be
carried out under essentially constant environmental conditions
without the requirement for exonuclease activity or restriction
endonu- clease activity.
Inventors: |
Dattagupta; Nanibhushan (San
Diego, CA), Stull; Paul Douglas (San Diego, CA),
Spingola; Marc (Albuquerque, NM), Kacian; Daniel Louis
(San Diego, CA) |
Assignee: |
Gen-Probe Incorporated (San
Diego, CA)
|
Family
ID: |
22801567 |
Appl.
No.: |
10/164,833 |
Filed: |
June 7, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
08215081 |
Mar 16, 1994 |
|
|
|
Reissue of: |
08972799 |
Nov 18, 1997 |
06087133 |
Jul 11, 2000 |
|
|
Current U.S.
Class: |
435/91.1;
435/6.1; 435/6.11; 536/23.1; 536/24.33; 435/91.2 |
Current CPC
Class: |
C12Q
1/6844 (20130101); C12Q 1/6844 (20130101); C12Q
2565/537 (20130101); C12Q 2537/1376 (20130101); C12Q
2527/101 (20130101) |
Current International
Class: |
C12P
19/34 (20060101); C07H 21/02 (20060101); C07H
21/04 (20060101); C12Q 1/68 (20060101) |
Field of
Search: |
;435/6,91.1,91.2
;536/23.1,24.3,24.33 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
0200362 |
|
Mar 1986 |
|
EP |
|
0300796 |
|
Jul 1988 |
|
EP |
|
0329822 |
|
Aug 1988 |
|
EP |
|
0320308 |
|
Dec 1988 |
|
EP |
|
0395398 |
|
Apr 1990 |
|
EP |
|
0408295 |
|
Jul 1990 |
|
EP |
|
0427073 |
|
Oct 1990 |
|
EP |
|
0427074 |
|
Oct 1990 |
|
EP |
|
0497272 |
|
Jan 1992 |
|
EP |
|
0500224 |
|
Jan 1992 |
|
EP |
|
0543612 |
|
May 1993 |
|
EP |
|
8706270 |
|
Apr 1986 |
|
WO |
|
8810315 |
|
Jun 1987 |
|
WO |
|
8901050 |
|
Jul 1987 |
|
WO |
|
9102/818 |
|
Aug 1989 |
|
WO |
|
9102818 |
|
Aug 1989 |
|
WO |
|
9110746 |
|
Jan 1990 |
|
WO |
|
WO 94/03635 |
|
Feb 1994 |
|
WO |
|
Other References
Feinburg & Vogelstein, "A Technique for Radiolabeling DNA
Restriction Endonuclease Fragments to High Specific Activity,"
Analytical Biochemistry, 132:6-13 (1983). cited by examiner .
Murakawa et al., "Direct detection of HIV-1 RNA from AIDS and ARC
patient samples," DNA, 7:287-295 (1988). cited by examiner .
Shaw et al., "Modified deoxyoligonucleotides stable to exonuclease
degradation in serum," Nucleic Acids Research, 19(4):747-750
(1991). cited by examiner .
Titeeva et al., "Complexing of Single-Stranded Phage DNA with
Synthetic Oligodeoxynucleotides and the Reaction of the Complexes
with DNA Polymerase and Restriction Endonucleases,"
Bioorganicheskaya Khimiya, 12(11):1484-1491 (1986). English
Translation. cited by other .
Walker et al., "Isothermal in vitro amplification of DNA by a
restriction enzyme/DNA polymerase system," Proc. Natl. Acad. Sci.
U.S.A., 89:392-396 (1992). cited by other .
Walker et al., "Strand displacement amplification--an isothermal,
in vitro DNA amplification technique," Nucleic Acids Research,
20:1691-1696 (1992). cited by other .
Chien et al., "DNA polymerase from the extreme thermophile Thermus
aquaticus," J. Bacteriology, 127:1550-1557 (1976). cited by other
.
Kaledin et al., "Isolation and properties of DNA polymerase from
extremely thermophilic bacterium Thermus aquaticus YT1,"
Biokhimiya, (45)4:644-651 (1980). English translation. cited by
other.
|
Primary Examiner: Sitton; Jehanne
Attorney, Agent or Firm: Cappellari; Charles B. Fisher;
Carlos A.
Parent Case Text
This application .Iadd.is a reissue application of U.S. Pat. No.
6,087,133, which issued from application Ser. No. 08/972,799, which
.Iaddend.is a continuation of application Ser. No. 08/215,081,
filed Mar. 16, 1994, now abandoned. .Iadd.Application 10/165,281 is
a reissue application of U.S. Patent No. 6,214,587, which issued
from application Ser. No. 09/506,282, which is a continuation of
application Ser. No. 08/972,799, now U.S. Patent 6,087,133, of
which this application is a reissue application..Iaddend.
Claims
What is claimed is:
1. A method for amplifying a specific nucleic acid target sequence
preferentially over non-target sequences, compris- ing the steps
of: contacting a nucleic acid strand containing said target
sequence with a nucleic acid polymerase lacking 5' exonuclease
activity, at least one nucleotide triphos- phate selected from
adenosine, thymidine, guanosine and cytidine triphosphate, and at
least three different complementary primers under primer extension
condi- tions at essentially constant temperature, wherein each said
primer contains a nucleotide base sequence which is designed to be
complementary to a nucleic acid sequence on said nucleic acid
strand located 3' to said target sequence, and wherein said
nucleotide base sequence of each said primer is able to hybridize
to a nucleic acid sequence on said nucleic acid strand under said
conditions; and amplifying said target sequence.Iadd., wherein said
amplifying step is performed in the absence of a complementary or
sense primer containing a promoter site for an RNA
polymerase.Iaddend..
2. A method for amplifying a specific nucleic acid target sequence
preferentially over non-target sequences, compris- ing the steps
of: contacting a nucleic acid strand containing said target
sequence with a nucleic acid polymerase lacking 5' exonuclease
activity, at least one nucleotide triphos- phate selected from
adenosine, thymidine, guanosine and cytidine triphosphate, and at
least two different complementary primers, under primer extension
con- ditions at essentially constant temperature, wherein each said
primer contains a nucleotide base sequence which is designed to be
complementary to a nucleic acid sequence on said nucleic acid
strand located 3' to said target sequence, and wherein said
nucleotide base sequence of each said primer is able to hybridize
to a nucleic acid sequence on said nucleic acid strand under said
conditions; and amplifying said target sequence in the absence of
any restriction endonuclease active on a product of said
amplification.Iadd., wherein said amplifying step is performed in
the absence of a complementary or sense primer containing a
promoter site for an RNA polymerase.Iaddend..
3. A method for amplifying a specific nucleic acid target sequence
preferentially over non-target sequences, compris- ing the steps
of: contacting a nucleic acid strand containing said target
sequence with a nucleic acid polymerase, at least one nucleotide
triphosphate selected from adenosine, thymidine, guanosine and
cytidine triphosphate, and at least three different complementary
primers, under primer extension conditions at essentially constant
temperature, wherein each said primer contains a nucleotide base
sequence which is designed to be complementary to a nucleic acid
sequence on said nucleic acid strand located 3' to said target
sequence, and wherein said nucleotide base sequence of each said
primer is able to hybridize to a nucleic acid sequence on said
nucleic acid strand under said conditions, and wherein at least one
of said primers is modified to be resistant to nucleolytic
degradation by a 5'-exonuclease; and amplifying said target
sequence.Iadd., wherein said amplifying step is performed in the
absence of a complementary or sense primer containing a promoter
site for an RNA polymerase.Iaddend..
4. A method for amplifying a specific nucleic acid target sequence
preferentially over non-target sequences, comprising the steps of:
contacting a nucleic acid strand containing said target sequence
with a nucleic acid polymerase, at least one nucleotide
triphosphate selected from adenosine, thymidine, guanosine and
cytidine triphosphate, and at least two different complementary
primers, under primer extension conditions at essentially constant
temperature, wherein each said primer contains a nucleotide base
sequence which is designed to be complementary to a nucleic acid
sequence on said nucleic acid strand located 3' to said target
sequence, and wherein said nucleotide base sequence of each said
primer is able to hybridize to a nucleic acid sequence on said
nucleic acid strand under said conditions, and wherein at least one
of said primers is modified to be resistant to nucleolytic
degradation by a 5'-exonuclease; and amplifying said target
sequence in the absence of any restriction endonuclease active on a
product of said amplification.Iadd., wherein said amplifying step
is performed in the absence of a complementary or sense primer
containing a promoter site for an RNA polymerase.Iaddend..
5. The method of claim 1, wherein at least four said complementary
primers are used.
6. The method of claim 2, wherein at least four said complementary
primers are used.
7. The method of claim 5, wherein each of said comple- mentary
primers is separated by a distance of 1 to 200 nucleotides.
8. The method of claim 6, wherein each of said comple- mentary
primers is separated by a distance of 1 to 200 nucleotides.
9. A method for exponentially amplifying a specific nucleic acid
target sequence preferentially over non-target sequences,
comprising the steps of: contacting a nucleic acid strand
containing said target sequence with a nucleic acid polymerase
lacking 5' exonuclease activity, at least one nucleotide triphos-
phate selected from adenosine, thymidine, guanosine and cytidine
triphosphate, and a primer array of at least five different primers
comprising two or three different complementary primers and two or
three different sense primers, under [oligonucleotide] primer
extension con- ditions at essentially constant temperature, wherein
each said complementary primer contains a nucleotide base sequence
which is designed to be complementary to a nucleic acid sequence on
said nucleic acid strand located 3' to said target sequence, and
wherein said nucleotide base sequence of each said complementary
primer is able to hybridize to a nucleic acid sequence on said
nucleic acid strand under said conditions, wherein each said sense
primer contains a nucleotide base sequence which is designed to be
analogous to a nucleic acid sequence of said nucleic acid strand
located 5' to said target sequence, and wherein said nucleotide
base sequence of each said sense primer is able to hybridize to a
nucleic acid sequence comple- mentary to a nucleic acid sequence on
said nucleic acid strand under said conditions; and exponentially
amplifying said target sequence in the absence of any restriction
endonuclease active on a product of said amplification.Iadd.,
wherein said amplifying step is performed in the absence of a
complementary or sense primer containing a promoter site for an RNA
polymerase.Iaddend..
10. A method for amplifying a specific nucleic acid target sequence
preferentially over non-target sequences, compris- ing the steps
of: contacting a nucleic acid strand containing said target
sequence with a nucleic acid polymerase, at least one-nucleotide
triphosphate selected from adenosine, thymidine, guanosine and
cytidine triphosphate, and a primer array of at least five
different primers compris- ing two or three different complementary
primers and two or three different sense primers, under primer
extension conditions at essentially constant temperature, wherein
at least one of said primers is modified to be resistant to
nucleolytic degradation by a 5' exonuclease, wherein each said
complementary primer contains a nucleotide base sequence which is
designed to be complementary to a nucleic acid sequence on said
nucleic acid strand located 3' to said target sequence, and wherein
said nucleotide base sequence of each said complementary primer is
able to hybridize to a nucleic acid sequence on said nucleic acid
strand under said conditions, wherein each said sense primer
contains a nucleotide base sequence which is designed to be
analogous to a nucleic acid sequence on said nucleic acid strand
located 5' to said target sequence, and wherein said nucleotide
base sequence of each said sense primer is able to hybridize to a
nucleic acid complementary to a nucleic acid sequence on said
nucleic acid strand under said conditions; and amplifying said
target sequence.Iadd., wherein said amplifying step is performed in
the absence of a complementary or sense primer containing a
promoter site for an RNA polymerase.Iaddend..
11. A method for exponentially amplifying a specific DNA target
sequence preferentially over non-target sequences, comprising the
steps of: contacting a single stranded DNA containing said target
sequence with a DNA polymerase lacking 5' exonu- clease activity,
at least one nucleotide triphosphate selected from adenosine,
thymidine, guanosine and cytidine triphosphate, and a primer array
of at least five different primers comprising two or three
different complementary primers and two or three different sense
primers, under primer extension conditions at essen- tially
constant temperature, wherein each said complementary primer
contains a nucleotide base sequence which is designed to be
complementary to a nucleic acid sequence on said nucleic acid
strand located 3' to said target sequence, and wherein said
nucleotide base sequence of each said complementary primer is able
to hybridize to a nucleic acid sequence on said nucleic acid strand
under said conditions, wherein each said sense primer contains a
nucleotide base sequence which is designed to be analogous to a
nucleic acid sequence on said nucleic acid strand located 5' to
said target sequence, and wherein said nucleotide base sequence of
each said sense primer is able to hybridize to a nucleic acid
sequence comple- mentary to a nucleic acid sequence on said nucleic
acid strand under said conditions; and exponentially amplifying
said target sequence in the absence of any restriction endonuclease
active on a product of said amplification.Iadd., wherein said
amplifying step is performed in the absence of a complementary or
sense primer containing a promoter site for an RNA
polymerase.Iaddend..
12. A method for amplifying a specific DNA target sequence
preferentially over non-target sequences, compris- ing the steps
of: contacting a single stranded DNA containing said target
sequence with a DNA polymerase, at least one nucle- otide
triphosphate selected from adenosine, thymidine, guanosine and
cytidine triphosphate, and a primer array of at least five
different primers comprising two to three different complementary
primers and two to three dif- ferent sense primers, under primer
extension conditions at essentially constant temperature, wherein
at least one of said primers is modified to be resistant to
nucleolytic degradation by a 5' exonuclease, wherein each said
complementary primer contains a nucleotide base sequence which is
designed to be complementary to a nucleic acid sequence on said
nucleic acid strand located 3' to said target sequence, and wherein
said nucleotide base sequence of each said complementary primer is
able to hybridize to a nucleic acid sequence on said nucleic acid
strand under said conditions, wherein each said sense primer
contains a nucleotide base sequence which is designed to be
analogous to a nucleic acid sequence on said nucleic acid strand
located 5' to said target sequence, and wherein said nucleotide
base sequence of each said sense primer is able to hybridize to a
nucleic acid sequence comple- mentary to a nucleic acid sequence on
said nucleic acid strand under said conditions; and amplifying said
target sequence.Iadd., wherein said amplifying step is performed in
the absence of a complementary or sense primer containing a
promoter site for an RNA polymerase.Iaddend..
13. The method of claim 9, wherein at least 4 said sense primers
and at least 4 said complementary primers are used.
14. The method of claim 9, wherein at least 7 said sense primers
and at least 7 said complementary primers are used.
15. The method of claim 9, wherein at least 10 said sense primers
and at least 10 said complementary primers are used.
16. The method of claim 9, wherein each adjacent said sense primer
is separated by a distance of 1 to 200 nucleotides, and each
adjacent said complementary primer is separated by a distance of 1
to 200 nucleotides.
17. The method of claim 13, wherein each adjacent said sense primer
is separated by a distance of 1 to 200 nucleotides, and each
adjacent said complementary primer is separated by a distance of 1
to 200 nucleotides.
18. The method of claim 14, wherein each adjacent said sense primer
is separated by a distance of 1 to 200 nucleotides, and each
adjacent said complementary primer is separated by a distance of 1
to 200 nucleotides.
19. The method of claim 15, wherein each adjacent said sense primer
is separated by a distance of 1 to 200 nucleotides, and each
adjacent said complementary primer is separated by a distance of 1
to 200 nucleotides.
20. The method of claim 16, wherein two adjacent sense primers are
separated by a distance of up to 10 nucleotides, and two adjacent
complementary primer are separated by a distance of up to 10
nucleotides.
21. A method for exponentially amplifying a specific nucleic acid
target sequence preferentially over non-target sequences,
comprising the steps of: contacting a nucleic acid strand
containing said target sequence with a nucleic acid polymerase
lacking 5' exonuclease activity, at least one nucleotide triphos-
phate selected from adenosine, thymidine, guanosine and cytidine
triphosphate, and at least two different complementary primers and
at lease two different sense primers under primer extension
conditions at essen- tially constant temperature, wherein each said
complementary primer contains a nucleotide base sequence which is
designed to be complementary to a nucleic acid sequence on said
nucleic acid strand located 3' to said target sequence, and wherein
said nucleotide base sequence of each said complementary primer is
able to hybridize to a nucleic acid sequence on said nucleic acid
strand under said conditions, wherein each said sense primer
contains a nucleotide base sequence which is designed to be
analogous to a nucleic acid sequence on said nucleic acid strand
located 5' to said target sequence, and wherein said nucleotide
base sequence of each said sense primer is able to hybridize to a
nucleic acid sequence comple- mentary to a nucleic acid sequence on
said nucleic acid strand under said conditions; and exponentially
amplifying said target sequence in the absence of any restriction
endonuclease active on a product of said amplification.Iadd.,
wherein said amplifying step is performed in the absence of a
complementary or sense primer containing a promoter site for an RNA
polymerase.Iaddend..
22. A method for amplifying a specific nucleic acid target sequence
preferentially over non-target sequences, compris- ing the steps
of: contacting a nucleic acid strand containing said target
sequence with a nucleic acid polymerase, at least one nucleotide
triphosphate selected from adenosine, thymidine, guanosine and
cytidine triphosphate, and at least two different complementary
primers and at least two different sense primers under primer
extension conditions at essentially constant temperature, wherein
at least one of said primers is modified to be resistant to
nucleolytic degradation by a 5'-exonuclease, wherein each said
complementary primer contains a nucleotide base sequence which is
designed to be complementary to a nucleic acid sequence on said
nucleic acid strand located 3' to said target sequence, and wherein
said nucleotide base sequence of each said complementary primer is
able to hybridize to a nucleic acid sequence on said nucleic acid
strand under said conditions, wherein each said sense primer
contains a nucleotide base sequence which is designed to be
analogous to a nucleic acid sequence on said nucleic acid strand
located 5' to said target sequence, and wherein said nucleotide
base sequence of each said sense primer is able to hybridize to a
nucleic acid sequence comple- mentary to a nucleic acid sequence on
said nucleic acid strand under said conditions; and amplifying said
target sequence in the absence of any restriction endonuclease
active on a product of said amplification.Iadd., wherein said
amplifying step is performed in the absence of a complementary or
sense primer containing a promoter site for an RNA
polymerase.Iaddend..
23. A method for exponentially amplifying a specific DNA target
sequence preferentially over non-target sequences, comprising the
steps of: contacting a single stranded DNA containing said target
sequence with a DNA polymerase lacking 5' exonu- clease activity,
at least one nucleotide triphosphate selected from adenosine,
thymidine, guanosine and cytidine triphosphate, at lease two
different comple- mentary primers and at least two different sense
prim- ers under primer extension conditions at essentially constant
temperature, wherein each said complementary primer contains a
nucleotide base sequence which is designed to be complementary to a
nucleic acid sequence on said nucleic acid strand located 3' to
said target sequence, and wherein said nucleotide base sequence of
each said complementary primer is able to hybridize to a nucleic
acid sequence on said nucleic acid strand under said conditions,
wherein each said sense primer contains a nucleotide base sequence
which is designed to be analogous to a nucleic acid sequence on
said nucleic acid strand located 5' to said target sequence, and
wherein said nucleotide base sequence of each said sense primer is
able to hybridize to a nucleic acid sequence comple- mentary to a
nucleic acid sequence on said nucleic acid strand under said
conditions; and exponentially amplifying said target sequence in
the absence of any restriction endonuclease active on a product of
said amplification.Iadd., wherein said amplifying step is performed
in the absence of a primer containing a promoter site for an RNA
polymerase.Iaddend..
24. A method for amplifying a specific DNA target sequence
preferentially over non-target sequences, compris- ing the steps
of: contacting a single stranded DNA containing said target
sequence with a DNA polymerase, at least one nucle- otide
triphosphate selected from adenosine, thymidine, guanosine and
cytidine triphosphate, at least two different complementary primers
and at least two dif- ferent sense primers under primer extension
conditions at essentially constant temperature, wherein at least
one of said primers is modified to be resistant to nucleolytic
degradation by a 5' -exonuclease, wherein each said complementary
primer contains a nucleotide base sequence which is designed to be
complementary to a nucleic acid sequence on said nucleic acid
strand located 3' to said target sequence, and wherein said
nucleotide base sequence of each said complementary primer is able
to hybridize to a nucleic acid sequence on said nucleic acid strand
under said conditions, wherein each said sense primer contains a
nucleotide base sequence which is designed to be analogous to a
nucleic acid sequence on said nucleic acid strand located 5' to
said target sequence, and wherein said nucleotide base sequence of
each said sense primer is able to hybridize to a nucleic acid
sequence comple- mentary to a nucleic acid sequence on said nucleic
acid strand under said conditions; and amplifying said target
sequence in the absence of any restriction endonuclease active on a
product of said amplification.Iadd., wherein said amplifying step
is performed in the absence of a complementary or sense primer
containing a promoter site for an RNA polymerase.Iaddend..
25. The method of claim 23, wherein said conditions include
provision of DMSO.
26. The method of claim 23, wherein said essentially constant
temperature is at least 43.degree. C.
27. The method of claim 26, wherein said essentially constant
temperature is between 60.degree. C. and 70.degree. C.
28. The method of claim 23, wherein said conditions include
provision of a strand-displacing agent.
29. The method of claim 28, wherein said strand-displacing agent is
selected from the group consisting of a helicase, a RecA protein,
and a single-stranded nucleic acid binding protein.
30. The method of claim 23, wherein said DNA poly- merase is Bst
DNA polymerase I or a modified Taq DNA polymerase.
31. The method of claim 23, wherein at least one of the
oligonucleotide primers further comprises a 5' non-complementary
region.
32. The method of claim 23, wherein at least 4 said complementary
primers, and at least 4 said sense primers, are used.
33. The method of claim 23, wherein at least 7 said complementary
primers, and at least 7 said sense primers, are used.
34. The method of claim 23, wherein at least 10 said sense primers,
and at least 10 said complementary primers, are used.
35. The method of claim 10, wherein at least one said sense primer
and one said complementary primer are modified to be resistant to
nucleolytic degradation by any 5'-exonuclease present in said
contacting step.
36. The method of claim 12, wherein at least one said sense primer
and one said complementary primer are modi- fied to be resistant to
nucleolytic degradation by any 5'-exonuclease present in said
contacting step.
37. The method of claim 22, wherein at least one said sense primer
and one said complementary primer are modi- fied to be resistant to
nucleolytic degradation by any 5'-exonuclease present in said
contacting step.
38. The method of claim 24, wherein at least one said sense primer
and one said complementary primer are modi- fied to be resistant to
nucleolytic degradation by any 5'-exonuclease present in said
contacting step.
39. The method of claim 10, wherein said amplifying step is an
exponential amplification.
40. The method of claim 12, wherein said amplifying step is an
exponential amplificatiion.
41. The method of claim 22, wherein said amplifying step is an
exponential amplification.
42. The method of claim 24, wherein said amplifying step is an
exponential amplification.
Description
FIELD OF INVENTION
This invention relates to methods for amplifying nucleic acid
sequences without thermal cycling.
BACKGROUND OF THE INVENTION
This invention concerns diagnostic procedures and tech- niques for
amplifying a nucleic acid sequence. The detection and quantitation
of a specific nucleic acid sequence (i.e., a target sequence) is an
increasingly important technique having numerous applications
including identifying and classifying microorganisms, diagnosing
infectious diseases, detecting and characterizing genetic
abnormalities, identi- fying genetic changes associated with
cancer, studying genetic susceptibility to disease, measuring the
response to various types of treatment, identifying criminal
suspects, and resolving paternity disputes.
A common method for detecting and quantitating target nucleic acid
sequences is nucleic acid hybridization. Nucleic acid
hybridization, typically, uses a labeled nucleic acid probe having
a nucleic acid sequence complementary to the target sequence. The
probe is mixed with a sample suspected of containing the target
sequence under conditions suitable for hybrid formation. The probe
then hybridizes to the target sequence present in the sample.
Hybridization can be detected by various techniques well known in
the art. These techniques include selectively degrading the label
present on unhybridized probe and then measuring the amount of
label associated with the remaining hybridized probe (Arnold et
al., PCT US88/02746).
Numerous methods are available for amplifying nucleic acid strands
(i.e., nucleic acid polymers) to increase the amount of target
sequence. These methods use the nucleic acid strand containing the
target sequence as a template to produce a complementary nucleic
acid strand containing the target sequence. Such methods include
the polymerase chain reaction method (PCR), as described by Mullis
et al., (See U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159 and
European Patent Application Nos. 863022-98.4, 86302299.2, and
87300203.4, and Methods in Enzymology, Volume 155, 1987, pp.
335-350). PCR involves the use of a pair of specific
oligonucleotides as primers for the two complementary strands of
the double-stranded DNA con- taining the target sequence. The
primers are chosen to hybridize at the ends of each of the
complementary target strands, 3' of the target sequence.
Template-dependent DNA synthesis, on each strand, can then be
catalyzed using a thermostable DNA polymerase in the presence of
the appro- priate reagents. A thermal cycling process is required
to form specific hybrids prior to synthesis and then denature the
double stranded nucleic acid formed by synthesis. Repeating the
cycling process geometrically amplifies the target sequence. A PCR
method may also be used with an RNA target using RNA-dependent DNA
polymerase to create a DNA template.
The PCR method has been coupled to RNA transcription by
incorporating a promoter sequence into one of the primers used in
the PCR reaction and then, after amplifica- tion by the PCR method,
using the double-stranded DNA as a template for the transcription
of single-stranded RNA (See, e.g., Murakawa et al., DNA 7:287-295
(1988)). Other amplification methods use multiple cycles of
RNA-directed DNA synthesis and transcription to amplify DNA or RNA
targets (See, e.g., Burg et al., WO 89/1050; Gingeras et al., WO
88/10315 (sometimes called transcription amplification system or
TAS); Kacian and Fultz, EPO Application No. 89313154; Davey and
Malek, EPO Application No. 88113948.9; Malek et al., W091/02818).
Urdea, W091/ 10746, describe a method that achieves signal
amplification using a T7 promoter sequence.
The ligase chain reaction (LCR) is described in European Patent
Publication 320,308. This method requires at least four separate
oligonucleotides, two of which hybridize to the same nucleic acid
template so their respective 3' and 5' ends are juxtaposed for
ligation. The hybridized oligonucleotides are then ligated forming
a complementary strand on the nucleic acid template. The
double-stranded nucleic acid is then denatured, and the third and
fourth oligonucleotides are hybridized with the first and second
oligonucleotides that were joined together. The third and fourth
oligonucleotides are then ligated together. Amplification is
achieved by further cycles of hybridization, ligation, and
denaturation. The Q.beta. replicase (Q.beta.R) method described in
PCT Pub- lication No. 87-06270 and U.S. Pat. No. 4,786,600 uses a
specific RNA probe which is capable of specific transcrip- tion by
a replicase enzyme. The method requires the design and synthesis of
RNA probes with replicase initiation sites.
Amplification methods using palindromic probes are described in EPO
Publication Nos. 0427073A and 0427074A. The palindromic probe forms
a hairpin with a nucleic acid target sequence. The probe contains a
functional promoter located in the hairpin region from which RNA
transcripts are produced.
Walker et al., Proc. Natl. Acad. Sci. U.S.A., 89:392-396 (Jan.
1992), Walker et al., Nucl. Acids Res. 20:1691-1696 (1992),
European Patent Application No. EP 0 497 272 A1, and European
Patent Application No. EP 0 500 224 A2, describe an
oligonucleotide-driven amplification method using a restriction
endonuclease. The restriction endonu- clease nicks the DNA/DNA
complex to enable an extension reaction and, therefore,
amplification.
Becker, et al., EPO Application No. 88306717.5, describe an
amplification method in which a primer is hybridized to a nucleic
acid sequence and the resulting duplex cleaved prior to the
extension reaction and amplification.
Noonan, K. E. et al., Nucl. Acids Res. 16, 10366 (1988), describe
using random hexamers to prime reverse tran- scriptase in a random,
nonspecific manner for the synthesis of cDNA from mRNA.
Amplification of the desired target is accomplished with PCR.
Feinberg, A. P. et al., Anal. Biochem. 132, 6 (1983), and Liang, W.
et al., Nucl. Acids Res. 16,3579 (1988), describe the synthesis of
DNA strands complementary to a DNA template by combining
single-stranded DNA with random hexanucleotide primers,
deoxynucleoside triphosphates, buffer, the Klenow fragment of E.
coli DNA polymerase 1, and a labeled deoxynucleoside
triphosphate.
A related process, known as Random Priming Amplifi- cation (RPA)
for amplifying nucleic acid is described by Hartley, U.S. Pat. No.
5,043,272. According to Hartley, at columns 2-3, lines 63-2: The
process includes the steps of priming single-stranded template
nucleic acid strands with an excess of random oligonucleotide
primers and incubating the single-stranded template nucleic acid
strands and excess ran- dom oligonucleotide primers in the presence
of excess amounts of an inducing agent, a strand displacement
agent, and nucleoside triphosphate substrates to ran- domly amplify
nucleic acid strands.
SUMMARY OF THE INVENTION
The present invention features methods for amplifying target
nucleic acid sequences using a nucleic acid poly- merase lacking 5'
exonuclease activity and oligonucleotide primers. Alternatively,
the method of the present invention may be performed using a
nucleic acid polymerase having 5' exonuclease activity if the
oligonucleotide primers have a 5' modification which prevents or
reduces nucleolytic diges- tion of the primers. Preferably,
amplification is carried out using a primer array comprising two
sets of primers. One set contains at least two primers
complementary to the target nucleic acid. The other set contains at
least two primers of the same sense as the target nucleic acid.
Using the described methods amplification can be carried out under
essentially constant environmental conditions without the
requirement for exonuclease activity or restriction endonuclease
activity. Preferably, amplification is carried out using a DNA
template, and a DNA polymerase such as a modified Bacil- lus
stearothermophilus (Bst) DNA polymerase which lacks 5' exonuclease
activity.
The oligonucleotide members of each primer set are used as
initiation points for template-dependent synthesis of a
complementary nucleic acid strand, and to enhance displace- ment of
the synthesized complementary strand. Thus, strand displacement and
nucleic acid synthesis are carried out in one step. This is a
surprising finding, and Applicant is unclear of the exact mechanism
by which the procedure operates.
Only one set of primers is believed necessary to effectuate strand
displacement and nucleic acid synthesis. A primer array is
preferably used to amplify both the initial template and its
complement to exponentially amplify a target sequence.
Preferably, the nucleic acid polymerase lacks 5' exonu- clease
activity, and the procedure is performed at higher temperatures
than those at which enzymes such as the Klenow fragment of E. coli
DNA polymerase I are optimally active. The reaction is more
efficient at 43.degree. C. than at 37.degree. C. Optimal efficiency
is obtained at temperatures between 50.degree. C. and 70.degree. C.
The method is preferably used on DNA. An RNA target sequence is
preferably amplified by first creating a DNA copy using, for
example, reverse transcriptase. The DNA copy is then amplified by
the methods described herein.
However, the present invention should be able to directly amplify
an RNA sequence using an enzyme able to synthe- size an RNA
complementary strand on an RNA template. Such an enzyme preferably
lacks 5' exonuclease activity. For example, QB replicase used in
conjunction with manganese should be suitable for this purpose.
Thus, in a first aspect, the invention features a method for
amplifying a target nucleic acid sequence by contacting a nucleic
acid strand containing the target sequence with a nucleic acid
polymerase lacking 5' exonuclease activity (5'-exo-minus
polymerase), four different nucleotides, and at least two
complementary oligonucleotide primers. Preferably, four different
deoxynucleotides and/or three of more primers are used. The
amplification is carried out under primer extension conditions at
essentially constant temperature.
In related aspects amplification is carried out using a nucleic
acid polymerase that has 5' exonuclease activity but under
conditions wherein such activity is inhibited, and amplification is
carried out using at least two complemen- tary oligonucleotide
primers in the absence of a restriction endonuclease active on the
products of any extension reac- tion of said contacting step.
In another related aspect, the invention features a method for
amplifying a target nucleic acid sequence by contacting a nucleic
acid strand containing the target sequence with a nucleic acid
polymerase lacking 5' exonuclease activity (5'-exo-minus
polymerase), four different nucleotides, at least two complementary
oligonucleotide primers, and at least two sense primers. The
amplification is carried out under primer extension conditions at
essentially constant temperature. Preferably, the total number of
complementary primers and sense primers is more than 5.
The nucleic acid strand present prior to amplification serves as an
initial nucleic acid template for the synthesis of a complementary
strand containing the target sequence. The complementary strand can
act as a nucleic acid template to produce strands analogous to the
initial nucleic acid tem- plate. The analogous strands can act as
nucleic acid tem- plates. Thus, the resulting amplification of
target sequence is geometric.
Primer extension conditions refer to conditions wherein
template-dependent amplification initiated at an oligonucle- otide
primer can occur. Such conditions generally include provision of an
appropriate buffer and, optionally, other chemicals that may
stabilize the polymerase, or enhance primer extension
reactions.
"Template" refers to a nucleic acid strand, such as DNA or RNA,
which can serve as a substrate for the synthesis of a complementary
nucleic acid strand.
A "target sequence" refers to a specific nucleic acid sequence
contained on a single strand of an oligonucleotide or nucleic acid
molecule, or the nucleic acid sequence complementary thereto.
A "complementary primer" refers to an oligonucleotide primer having
a nucleic acid sequence complementary to a nucleic acid sequence
present on the nucleic acid template. Complementary primers are
designed to be complementary to nucleic acid sequences, on the
nucleic acid template, in regions 3' to the target sequence.
By "complementary" is meant that the oligonucleotide primer can
hybridize to a region of the nucleic acid template under the
conditions of the contacting step so it can be extended in a primer
extension reaction. Generally, such oligonucleotides have a region
of complementarity to the template of between 15 and 100 bases,
preferably between about 20 and 50 bases. Preferably the region of
complemen- tarity is 100% complementary. That is, each nucleotide
in the primer complementary region can hydrogen bond with a
nucleotide present on the single-stranded template.
However, those of ordinary skill in the art will recognize that
primers having a complementary region less than 100% complementary
to the template will operate in this method. Generally, such
complementarity can be as few as 15 out of 18 contiguous bases.
A "sense primer" refers to an oligonucleotide analogous to a
nucleic acid sequence present on the nucleic acid template. A sense
primer is designed to be analogous to a region on the template 5'
of the target sequence.
By "analogous" is meant that the oligonucleotide has essentially
the same, or equivalent, nucleotide sequence as that in the nucleic
acid template. Thus, for example, the oligonucleotide may contain
uracil in place of a thymine present in the nucleic acid template.
The analogous oligo- nucleotides are preferably sufficiently
duplicative of the template so they can act as a complementary
primer when hybridized to a nucleic acid that is complementary to
the nucleic acid template, under primer extension conditions.
Thus, reference to complementary primers and sense primers is made
with respect to a particular template. The same primer can act as a
complementary primer or a sense primer depending upon whether it is
analogous to, or complementary to, a particular template.
Primer-dependent amplification of a target sequence present on a
nucleic acid template is achieved in the con- tacting step. The
degree of amplification has been found to increase with increasing
numbers of primers. Examples are provided below using as many as 13
primers (seven sense and six complementary), and as few as two
primers (two complementary primers). These examples illustrate the
abil- ity of primers to cause amplification and are not intended to
limit the invention. Preferably, the complementary primer set and
the sense primer set each contain more than two primers. In
preferred embodiments, at least 4 sense primers and 4 complementary
primers are used; at least 7 sense primers and 7 complementary
primers are used; and at least 10 sense primers and 10
complementary primers are used.
As illustrated in the FIGURE, a set of complementary primers
comprise oligonucleotides complementary to the template in regions
3' of the target sequence. A set of sense primers comprise
oligonucleotides analogous to the tem- plate in regions 5' of the
target sequence on the same strand. The oligonucleotides are
selected so a primer extension reaction is initiated on the nucleic
acid template at a site different from the other oligonucleotide
primers, and pref- erably at a site distinct from the region in
which the other oligonucleotide primers form base pairs.
Preferably, the oligonucleotides in a primer set are separated at
their closest 5' and 3' ends from one another by 1 to 200 bases,
and more preferably, are separated by between 1 and 10 bases.
By "four different nucleotides" is meant that adenosine (A),
guanosine (G), cytidine (C) thymidine (T), and uridine (U)
triphosphates, or an equivalent nucleotide which can be substituted
for an adenosine, guanosine, cytidine, thymidine, or uridine
nucleotide triphosphate, are present as substrates for nucleic acid
synthesis. Preferably, deoxynucleotides are used.
By "amplifying" is meant increasing the number of target
sequences.
By "contacting" is meant that the nucleic acid template, the
nucleic acid polymerase, the four different nucleotides, and the
oligonucleotide primer array are brought together under primer
extension conditions.
By "essentially constant temperature" is meant that the user makes
no attempt to raise or lower the temperature in any profound manner
as is used in the polymerase chain reaction to alternatively carry
out primer extension and denature the extension products. Thus, the
temperature is kept below the temperature at which the
template:primer extension product duplex dissociates. Preferably,
the tem- perature is kept below 90.degree. C. More preferably, the
reaction temperature is maintained at about the optimum temperature
for the reaction. Generally, such an optimum is between 42.degree.
C. and 60.degree. C. and is preferably between 50.degree. C. and
70.degree. C.
In preferred embodiments, the conditions include the provision of
DMSO; the essentially constant temperature is at least 43.degree.
C., and most preferably between 50.degree. C. and 70.degree. C.;
the conditions include a strand separation agent such as a
helicase, at least one nucleotide triphosphate, selected from a
protein having an activity corresponding to the RecA protein of E.
coli, or a single-stranded nucleic acid binding protein; the DNA
polymerase is a 5' exominus Bst DNA polymerase I or a modified Taq
DNA polymerase; and at least one of the oligonucleotide primers
includes a 5' non-complementary region. The RecA protein is not
necessary for amplification in this invention, but rather can be
used to enhance such amplification.
By "5' exonuclease minus" is meant that the nucleic acid polymerase
has essentially no 5' exonuclease activity asso- ciated with it, or
has been treated such that the exonuclease activity is reduced or
inactive. The preferred nucleic acid polymerase is a 5' exonuclease
minus DNA polymerase. Examples of DNA polymerases having no 5'
exonuclease activity include a proteolytic fragment of DNA
polymerase I from Bacillus stearothermophilus, and the DNA poly-
merase from Thermus aquaticus having its domain encoding 5'
exonuclease activity removed as described by Barnes, "Thermostable
DNA Polymerase," PCT/US91/07084. The exonuclease minus polymerase
of the Klenow fragment described by Walker, supra, may also be
used.
In a related aspect, the invention features a method and means for
amplifying a nucleic acid target sequence by the step of contacting
the nucleic acid containing the target sequence with a nucleic acid
polymerase that may have 5' exonuclease activity but under
conditions wherein such activity is inhibited. Specifically, at
least one oligonucleotide primer is modified to be resistant to
nucleolytic degradation by any 5'-exonuclease present in the
nucleic acid poly- merase. Preferably, at least one complementary
and one sense primer is modified to be resistant to 5'-exonuclease
activity. More preferably all primers are resistant to such
degradation.
Thus in this aspect, the contacting step contains four different
nucleotide triphosphates, at least two different complementary
oligonucleotide primers and at least two different oligonucleotide
sense primers, one or more of which are blocked. The reaction is
carried out under oligo- nucleotide primer extension conditions at
essentially con- stant temperature in the absence of a restriction
endonu- clease active on the products of any extension reaction of
the contacting step.
In related aspects, amplification is carried out using a primer
array and either a 5'-exo-minus DNA polymerase or a DNA polymerase
containing 5' exonuclease activity used in conjunction with a
5'-exonuclease resistant primers.
As noted above, the present invention does not require a
restriction endonuclease active on the products of any primer
extension reaction resulting from the contacting step. In contrast
to the description in Walker, supra, where the presence of such
activity appears to be essential, Applicant has surprisingly found
that no restriction enzyme is needed to create a single-stranded
region which can be used by oligonucleotide primers in the
contacting step.
Thus, in another aspect the invention features a method for
amplifying a target nucleic acid sequence by contacting a nucleic
acid strand containing the target sequence with a nucleic acid
polymerase lacking 5' exonuclease activity, four different
nucleotides, a set of at least two complementary oligonucleotide
primers, and a set of at least two sense primers. The amplification
is carried out under primer exten- sion conditions at essentially
constant temperature in the absence of any restriction endonuclease
active on the prod- ucts of any extension reaction. The
amplification is prefer- ably carried out as described above.
In related aspects amplification is performed without any
restriction endonuclease active on the products of any extension
reaction using a nucleic acid polymerase containing 5' exonuclease
activity in conjunction with a 5'-exonuclease- resistant primer; or
a single stranded DNA substrate and either a 5'-exo-minus DNA
polymerase or a DNA poly- merase containing 5' exonuclease activity
used in conjunc- tion with a 5'-exonuclease-resistant primer. Such
amplifica- tions are preferably carried out as described above.
In this invention, amplification can be allowed to proceed for any
desired length of time with product accumulating as a result of
amplification of the target sequence. Where the target sequence is
of analytical interest, high sensitivity detection of the target
sequence can be accomplished by amplifying the target in the manner
of the present invention and then detecting the accumulated
amplification product using techniques known in the art.
The present amplification method provides a number of significant
benefits. The present method requires only a nucleic acid
polymerase as opposed to several different enzymes, amplification
of a specific nucleic acid sequence is more efficient and requires
less reagent than a random amplification process, and there is no
need for time-consuming and cumbersome thermal cycling.
Other features and advantages of the invention will be apparent
from the following description of the preferred embodiments, and
from the claims.
BRIEF DESCRIPTION OF THE DRAWING
The FIGURE is an example illustrating the location of primers
relative to each other and a target sequence. The FIGURE shows four
complementary primers ("-"), four sense ("+") primers, and the
location of a target sequence (rectangular box).
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention features methods for amplifying a target
nucleic acid sequence present in a nucleic acid, or a mixture of
nucleic acids, by a process which includes synthesis of nucleic
acid sequences in a specifically-primed template-dependent manner
with a nucleic acid polymerase (e.g., a DNA polymerase) and
oligonucleotides primers, preferably, a primer array. A primer
array is made up of a set of complementary primers and a set of
sense primers. Nucleic acid synthesis is initiated at a
complementary primer. Without being bound to any theory, it is
believed that primer-dependent synthesis occurring from one primer
causes strand displacement of a strand synthesized from a different
primer.
Thus, the amplification can be performed without modi- fying
reaction conditions such as temperature, pH, or ionic strength
during the amplification procedure to alternatively carry out
amplification and denature the double-stranded nucleic acid
produced during amplification. In addition, amplification can be
performed without producing single-stranded substrates using
enzymes having exonuclease activity or restriction endonuclease
activity.
The process includes the step of priming template nucleic acid
strands with a primer array of sequence-specific oligo- nucleotide
primers, for each different target sequence to be amplified. The
mixture is incubated in the presence of a nucleic acid polymerase,
nucleotide substrates, and any necessary co-factors needed to
amplify the target nucleic acid sequence.
As a result of the methods described herein, multiple rounds of
template-dependent nucleic acid synthesis occur, concomitant with
strand displacement of the previously synthesized strand. The
displaced strand can act as a tem- plate in conjunction with the
"sense primers", which now act as complementary primers.
Oligonucleotide Primer
The term "primer" refers to an oligonucleotide (whether occurring
naturally as in a purified restriction digest, or produced
synthetically) which can act as a point of initiation of nucleic
acid synthesis on a nucleic acid template when contacted under
primer extension conditions. Appropriate conditions include the
presence of nucleotide substrates, a nucleic acid polymerase (e.g.,
a 5'-exo-minus DNA polymerase), essentially constant temperature
and a suitable pH. The nucleic acid synthesis occurs in a
template-dependent manner wherein the sequence of the newly syn-
thesized nucleic acid strand is complementary to the sequence of
the nucleic acid template.
Hybridization of the primer to the template may be inhibited by
primer secondary structure arising from intramolecular or
intermolecular hydrogen bonding. Thus the primer preferably lacks
secondary structure and is single-stranded. A primer having a
secondary structure which would inhibit hybridization to the
template is prefer- ably treated to denature or separate its
strand(s) prior to amplification.
Preferably, the primer is an oligodeoxynucleotide and is
sufficiently long to be used as a substrate for the synthesis of
extension products by a nucleic acid polymerase. The appro- priate
primer length should take into account several factors, including
temperature, structure of primer, and how the primer is used. For
example, for diagnostic applications the oligonucleotide primer
typically contains at least 15 nucle- otides depending on the
complexity of the target sequence. Other applications may require
shorter oligonucleotide primers. It may be desirable to carry out
these other appli- cation at cooler temperatures to form stable
hybrid com- plexes with the template.
A nucleic acid polymerase as used in the invention refers to a
chemical, physical, or biological agent which brings about the
synthesis of either ribo- or deoxyribonucleotides into a nucleic
acid polymer, or strand, in a template-dependent manner. Examples
of nucleic acid polymerases include DNA polymerases. DNA
polymerases bring about nucleic acid synthesis in a
template-dependent manner in a 5' to 3' direction. Because of the
complementarity between the template and the synthesized strand,
this direction would be from a 3' region on the template to a 5'
region on the template. The large fragment of DNA polymerase I from
Bacillus stearothermophilus (Bst DNA polymerase I) lack- ing 5'-3'
exonucleolytic activity is a preferred DNA poly- merase. Other
suitable DNA polymerases include Klenow polymerase and
bacteriophage T7 DNA polymerase. The use of 5' blocked primers not
susceptible to nucleolytic degra- dation should allow the use of
DNA polymerases possessing 5'-3' exonucleolytic activity.
Complementary primers are selected to have a region complementary
to the nucleic acid template. The primer sequence need not reflect
the exact sequence of the template but must be able to hybridize
with a template strand. Non-complementary bases can be interspersed
into the primer provided the primer sequence has sufficient comple-
mentarity to hybridize with its corresponding sequence in the
nucleic acid template such that the extension reaction can occur.
The newly-formed extension product, once displaced, can act as a
template for additional nucleic acid synthesis.
Preferably, the primers contain a non-complementary 5' region.
Thus, the preferred primer contains a complementary region enabling
it to hybridize to the nucleic acid template, and a
non-complementary region. The presence of a non-complementary
region has been found to enhance strand displacement.
The primer sequence can include one or more of the
deoxyribonucleotide bases A, T, C, or G; and/or, one or more of the
ribonucleotide bases A, U, C, or G and/or one or more modified
nucleotide (deoxyribonucleotide or ribonucleotide) wherein the
modification does not prevent hybridization of the primer to the
nucleic acid, or elongation of the primer, or strand
displacement.
Primers may be modified with chemical groups to enhance their
performance or to facilitate the characteriza- tion of
amplification products. For example, backbone-modified
oligonucleotides such as phosphorothioates or methylphosphonates
which are resistant to the nucleolytic activity of certain
polymerases allow the use of such enzymes. Another example of
modification involves using non-nucleotide linkers (e.g., Arnold et
al., EPA 88 308 766-0) which do not interfere with elongation of
the primer. Oligonucleotides containing mixtures of the desired
modi- fied and natural nucleotides may be used as well.
Primers may also contain reactive sites for enzymes such as
restriction endonuclease cleavage sites or promotor sites for RNA
polymerases. Examples of the use of such sites include cloning of
amplification products and transcription of amplification products
after amplification. Such sites are not necessary for
amplification, but are helpful for subse- quent manipulations of
the amplified nucleic acids.
The primers of the present invention can be prepared by methods
known in the art. Preferably, the primers are synthesized by
chemical methods. For example, Caruthers et al., in: Methods In
Enzymology, vol. 154, p. 287 (1987), describe using standard
phosphoramidite solid-phase chem- istry to join nucleotides by
phosphodiester linkages. Bhatt, U.S. Pat. No 5,449,769 entitled
"Method and reagent for sulfurization of organophosphorous
compounds" filed Mar. 6, 1989, assigned to Gen-Probe Incorporated,
the assignee of the present invention, describe a procedure for
synthesizing oligonucleotides containing phosphorothioate linkages.
Klem et al., entitled "Improved process for the synthesis of
oligomers" PCT W092/07864, describe the synthesis of
oligonucleotides having different linkages including meth-
ylphosphonate linkages. (These references are hereby incor- porated
by reference herein.) Automated synthesizers are well suited for
oligonucleotide synthesis. In an automated synthesizer, one starts
with a 5'-protected, immobilized (through the 3'-hydroxyl)
nucleoside residue. After 5'-deprotection, a phosphite ester
linkage is introduced by reaction with a 3'-phosphoramidite,
5'-protected nucleoside residue. The phosphite linkage is then
oxidized to a stable linkage and the process is cycled with the
desired nucleotide residues introduced into the synthesized
sequence. The oxidative procedure can result in a typical phosphate
ester or a phosphorothioate ester (Arnold et al., supra, EPA 88 308
766-0). Introduction of non-nucleotide phosphoramidite derivatives
can be performed during automated synthesis. Also, the synthesized
oligonucleotide can be further modi- fied by synthetic methods
(e.g., as described in U.S. Pat. No. 4,818,681).
The 5' end of the primer can be modified to be resistant to
5'-exonuclease activity present in nucleic acid polymerase. Such
modifications can be carried out by adding a non-nucleotide group
to the terminal 5' nucleotide of the primer using techniques such
as those described by Arnold et al., U.S. Pat. No. 5,585,481,
entitled "Linking Reagents for Nucleotide Probes," and the CLONTECH
product catalogue describing the use of label-ON.TM. regents,
hereby incorpo- rated by reference herein.
Test Sample
In general any source of nucleic acid, in purified or non-purified
form, can be utilized as the starting nucleic acid or acids
provided it contains or is suspected of containing the target
nucleic acid sequence. Depending on the source and purity of the
nucleic acid, the starting nucleic acid may need to be purified
prior to amplification. Purification of starting nucleic acid is
preferably carried out when the nucleic acid source contains
substances which inhibit amplification. Nucleic acid purification
can be carried out using standard techniques.
The target sequence may be present in DNA or RNA, which may be
single-stranded or double-stranded. A mixture of these nucleic
acids or the nucleic acid from a previous amplification may also be
utilized. The nucleic acid or mixture of nucleic acids may contain
more than one target nucleic acid sequence which may be the same or
different. The invention is therefore useful for simultaneously
ampli- fying one or more different target nucleic acid sequences
located on the same or different target nucleic acid strands.
The sample suspected or known to contain the intended target
nucleic acid may be obtained from a variety of sources. Such
sources include a biological sample; a food or agricultural sample;
an environmental sample; body fluid or exudate such as urine,
blood, milk, cerebrospinal fluid, sputum, saliva, stool, lung
aspirates; and throat or genital swabs.
It may be desirable to treat the test samples to obtain nucleic
acids suitable for amplification. For example, in certain
circumstances, it is desirable to treat the test sample to release,
extract, or denature, the target nucleic acid prior to
amplification. Such treatments can remove impurities which inhibit
amplification, and denature the nucleic acid to minimize the
secondary structure (i.e., double-stranded DNA and hairpin loops) .
These treatments can be carried out by means well known in the
art.
Primers are designed to hybridize to specified regions of the
target nucleic acid. Two sets of primers are preferably used. One
set of primers, complementary primers, are designed to hybridize to
a nucleic acid sequence present on the template nucleic acid 3' to
the target sequence. Hybrid- ization takes place based on hydrogen
bonding between complementary nucleotides. The other set of
primers, sense primers, are designed to be analogous to regions
present on the template nucleic acid 5' to the target sequence. The
members of each primer set are co-linearly arranged as shown
diagrammatically in the FIGURE.
Both sets of primers are positioned such that the target sequence,
present in the template and the complement to the template, will be
amplified. DNA polymerases catalyze nucleic acid synthesis in a 5'
to 3' direction (with respect to the template nucleotides are added
from a 3' template region to a 5' template region). A complementary
strand is produced from the initial template using the
complementary primer. The complementary strand can act as a
template for template-dependent nucleic acid synthesis.
If the sense primer is analogous to a region on the nucleic acid
template 5' to the target sequence, it will be comple- mentary to a
region 3' to the target sequence on the comple- mentary strand. The
sense primer can act as a complemen- tary primer with respect to
the complementary strand. Template-dependent nucleic acid synthesis
can then occur on the complementary strand thereby producing more
target sequence.
Different numbers of complementary and sense primers may be used to
amplify a target sequence. It appears that as the number of primers
increases, amplification increases. Thus, the total number of
complementary primers and sense primers is preferably more than 5.
In preferred embodiments at least 4 sense primers and 4
complementary primers are used; at least 7 sense primers and 7
complementary primers are used; and at least 10 sense primers and
10 complemen- tary primers are used. The number of complementary
prim- ers need not equal the number of sense primers. For example,
a 2 sense primer and 3 complementary primer combination may be
used.
When certain organic solvents such as DMSO are added to the
amplification reaction and elevated temperature is used,
substantial amplification as well as stringency is observed. If
elevated temperature is used to increase strand displacement, a
heat-stable nucleic acid polymerase such as a thermostable DNA
polymerase may be employed. The temperature selected depends on the
temperature range within which the enzyme is active.
The primers, nucleic acid polymerase, and nucleotides must be used
in sufficient quantity to support the degree of amplification
desired. Nucleotide analogs can be substituted or added provided
the base pairing, DNA polymerase, and strand-displacing functions
are not adversely affected to the point that the amplification does
not proceed to the desired extent.
"Strand displacement" refers to the dissociation of a nucleic acid
strand from its nucleic acid template in a 5' to 3' direction
occurring due to template-directed nucleic acid synthesis by a
nucleic acid polymerase. The newly synthe- sized and displaced
nucleic acids have substantially the same nucleotide sequence which
is complementary to the template nucleic acid sequence.
Preferably, a strand separation agent is used to enhance the strand
displacement. Strand separation agents include enzymes such as
helicases, the enzyme RecA which has helicase activity, and
single-stranded nucleic acid binding protein. The use of helicases
for separating strands of nucleic acid is described in Cold Spring
Harbor Symposia on Quantitative Biology, Vol. XLIII, "DNA:
Replication and Recombination" (New York: Cold Spring Harbor
Laboratory, 1978) and the use of RecA is reviewed by Radding, Ann.
Rev. Genetics, 16: 405 (1982). Strand sepa- ration is discussed by
Kornberg, A, DNA Replication W. H. Freeman & Co., San
Francisco, Calif., 1980. Zarling et al., U.S. Pat. No. 5,223,414
(Jun, 1993) discusses the use of RecA in nucleic acid hybridization
and amplification.
Amplification, as discussed above, refers to increasing the number
of target sequences. The degree of amplification can be increased
by using conditions or agents which increase rates or extent of
priming, or primer elongation. For example, a stand separation
agent may increase the strand displacement rate of a DNA
polymerase.
A wide variety of methods are available to detect a target
sequence. For example, the nucleotide substrates or the primers can
include a detectable label which is incorporated into newly
synthesized DNA. The resulting labeled ampli- fication product is
then separated from the unused labeled nucleotides or primers and
the label is detected in the separated product fraction.
Substances which serve as useful detectable labels are well known
in the art and include radioactive isotopes (e.g., .sup.32p,
.sup.3H, 1251, and .sup.14C), fluorescent compounds, chemilu-
minescent compounds, chromophores, as well as ligands such as
biotin and haptens which, while not directly detectable, can be
readily detected by reaction with labeled forms of their specific
binding partners, e.g., avidin and antibodies, respectively.
Another approach is to detect the amplification product by
hybridization with a detectable nucleic acid probe and measuring
the resulting hybrids in any conventional manner. In particular,
the product can be assayed by hybridizing a chemiluminescent
acridinium ester-labeled nucleic acid probe to the target sequence,
selectively hydrolyzing the acridinium ester present on
unhybridized probe, and mea- suring the chemiluminescence produced
from the remaining acridinium ester in a luminometer. (See, e.g.,
Arnold et al., supra, PCT US88/02746, and Nelson et al., in
Nonisotopic DNA Probe Techniques, p. 275 Academic Press, San Diego
(Kricka, ed., 1992) (both references hereby incorporated by
reference herein).
EXAMPLES
The present invention will now be illustrated, but is not intended
to be limited, by the following examples.
Example 1
Amplification of an RNA Target
This example illustrates the amplification of a ribosomal RNA(rRNA)
target sequence by producing a DNA template, and amplifying the DNA
template using a primer array and a 5'-exo-minus DNA polymerase.
Production of the DNA template from rRNA, and amplification of the
template, was carried out in the same reaction vessel containing
the necessary reagents for production and amplification of the DNA
template.
The primer array used in this example comprised thirteen primers.
The primers were prepared by standard phosphora- midite chemistry.
Seven of the primers were complementary primers (with respect to
the rRNA target). These primers had the following nucleic acid
sequences (written 5' to 3'): SEQ. ID. NO. 1: CGCGTCTAGT CCTACTCAGG
TGTTG SEQ. ID. NO. 2: CCCCAGATTC AGACTAGG SEQ. ID. NO. 3:
CGGTCATTGA CTAGTATTTA GCCTT SEQ. ID. NO. 4: TTTCTTCATA GTACTGGTTC
ACT SEQ. ID. NO. 5: ATTTCACTCC CTTAACAAGG GTT SEQ. ID. NO. 6:
TTGTAAGCTA CAGGTTTCAG GT SEQ. ID. NO. 7: ACGGGCAATT GGTCTGCGAC C
Six of the primers were sense primers (with respect to the rRNA
target). These primers had the following nucleic acid sequences
(written 5' to 3'): SEQ. ID. NO. 8: AGCAAAGACC CGGAGGTCCG AAT SEQ.
ID. NO. 9: GAATACATAG GTATGCAAAG CGA SEQ. ID. NO. 10: CCTGCCGAAC
TGAAACATCT TAGT SEQ. ID. NO. 11: CAGAGGAAAA GAAATCGAAG AGAT SEQ.
ID. NO. 12: TGTAGGATTG AGGATAAAGG AT SEQ. ID. NO. 13: GGACTCCTAG
TTGAACACAT CTG- GAAAGA
Target 23S rRNA was obtained from Chlamydia tra- chomatis (See,
Glisin et al., Biochemistry 13:2633 (1974)), and diluted in buffer
(50 mM Tris-HCl (PH 8.3), 37.5 mM KCl, 1.5 mM MgCl.sub.2, 10 mM
DTT) . Variable amounts of rRNA target were combined with primers
(7.times.10.sup.-12 mole) in 20 .mu.l of buffer (7% DMSO, 50 mM
Tris-HCl (pH 8.3) 75 mM KCl, 3 mM MgCl.sub.2, and 2 mM DTT) and
incubated at 60.degree. C. for 30 minutes to pre-hybridize the
primers. The pre-hybridized primers were then treated with 0.2 mM
ATP, 0.2 mM TTP, 0.2 mM GTP, 0.2 mM CTP, 200 units of Maloney
Murine Leukemia Virus reverse transcriptase (MMLV-RT), and 2 units
of Bst DNA polymerase 1. Ampli- fication was carried out at
37.degree. C. for 60 minutes, then at 60.degree. C. for 120
minutes.
The amplification products were then denatured and assayed using
acridinium ester labeled probe according to Nelson et al., supra.
Denaturation was carried out by incu- bating at 95.degree. C. for 5
minutes. Probe labeling with acri- dinium ester was carried out as
described by Nelson et al., supra. The acridinium ester labeled
probe was synthesized to have the nucleic acid sequence
corresponding to SEQ. ID. NO. 27. The acridinium ester labeled
probe (2.times.10.sup.-11 moles) was incubated with amplified
product at 60.degree.60 C. for 15 minutes in 100 .mu.l of
hybridization buffer (100 mM lithium succinate (pH 5.2), 8% lithium
dodecyl sulfate, 1.5 mM EDTA, and 1.5 mM EGTA). The acridinium
ester present on unhybridized probe was then hydrolyzed by adding a
reagent solution (0.15 M sodium tetraborate, pH 7.6, and 5% (v/v)
Triton X-100) and incubating at 60.degree. C. for 10 minutes. The
remaining chemiluminescence was measured in a lumi- nometer. The
results shown in Table 1 demonstrate a titration of the target
nucleic acid in the amplification method of the present
invention.
TABLE-US-00001 TABLE 1 Chlamydia rRNA (Moles) RLU (mean) 4 .times.
10.sup.-18 307893 4 .times. 10.sup.-19 54771 4 .times. 10.sup.-20
3644 4 .times. 10.sup.-23 2354 0 2307
Example 2
Amplification of a Clinical Specimen
This example illustrates the amplification of an rRNA target
sequence present in a clinical sample (cervical swab sample). The
oligonucleotides, amplification procedure, and detection procedure
used in this example were as described in Example 1. This example
differs from Example 1 in the type of sample used, and sample
preparation.
Cervical swab samples were collected and suspended in a buffered
solution containing an anionic detergent (60 mM phosphate buffer
(pH 6.8), 6% lithium dodecyl sulfate, 2 mM EDTA, and 2 mM EGTA).
The clinical specimen was spiked with 10-18 moles of 23S Chamydia
trachomatis rRNA, hybridized with the primers, amplified, and
detected, as described in Example 1. A successful amplification of
target nucleic acid in the clinical sample was indicated by the
clinical sample with Chlamydia rRNA having an RLU over 50 fold
greater than a clinical sample without Chlamy- dia rRNA.
Example 3
Specificity of Amplification
This example illustrates the amplification of a specific target
sequence using probes directed to a target sequence and a
non-target sequence (i.e., a region of the transcript outside the
amplicon). The target nucleic acid used in this example was derived
from gene 10 of 17 bacteriophage. In particular, an XmaI and BanI
digest of pGEMX1 containing part of gene 10 of T7 bacteriophage was
sub-cloned into pUC19. Expression of the insert with T7 RNA
polymerase yielded an RNA product 863 bases in length and this
transcript was used to produce a DNA template which was then
amplified.
The primer array, used to amplify the target sequence, was made up
of four complementary primers (with respect to the RNA transcript)
having the following nucleic acid sequences (written 5' to 31'):
SEQ. ID. NO. 14 CTATAGGGAG ACCACAACGG TTT SEQ. ID. NO. 15
CTAGAAATAA TTTTGTTTAA CTT SEQ. ID. NO. 16 AGAAGGAGAT ATACATATGG CT
SEQ. ID. NO. 17 CATGACTGGT GGACAGCAAA TG and four primers having
the same sense as the RNA transcript, having the following nucleic
acid sequences (written 5' to 3'): SEQ. ID. NO. 18 GAGCGAACGC
AGTCAGGA SEQ. ID. NO. 19 GAGAAGTGGT CACGGAGGTA CGAGCGAACG CAGT SEQ.
ID. NO. 20 GGAGATGGAA CGTACCATGT SEQ. ID. NO. 21 GGGAACTGAG
CGGATTTACC GC
Hybridization, amplification, and detection were carried out as
described in Example 1. Acridinium ester-labeled probes, SEQ. ID.
NO: 22 and SEQ. ID. NO. 27 were used to detect the presence of the
target sequence. A non-target acridinium ester labeled probe (SEQ.
ID. NO. 23) was used as a control to determine non-specific
amplification occur- ring outside the primer region (e.g., 3' of
the complementary primers and 5' the sense primers) . The results
shown in Table 2 demonstrate that the amplification is
specific.
TABLE-US-00002 TABLE 2 Probe Sample RLU (mean) SEQ. ID. 22 + (4
.times. 10.sup.-19 mole) 240474 SEQ. ID. 27 SEQ. ID. 23 (4 .times.
10.sup.-19 mole) 3091 SEQ. ID. 23 no target 2720
Example 4
Enhanced Strand Displacement
This example demonstrates the ability of a non-complementary
region, attached to a primer, to enhance strand displacement and
amplification. Strand displacement was measured using two different
combinations of comple- mentary primers. Both combinations
contained a 5'-.sup.32p labeled primer (SEQ. ID. NO. 19) and an
additional primer. The additional primer in one combination
corresponded to SEQ. ID. NO. 24. The additional primer in the other
combination corresponded to SEQ. ID. NO. 25. The SEQ. ID. NO. 25
primer was similar to the SEQ. ID. NO. 24 primer, but had five
fewer complementary nucleotides and contained a 5'
non-complementary region. Amplification was carried out on an
oligodeoxynucleotide template corre- sponding to a portion of the
T7 bacteriophage gene 10. The template, corresponding to SEQ. ID.
NO: 26, was synthe- sized as described in Example 1.
The primers 4'10.sup.-32 mole) were prehybridized with template
(4.times.10.sup.-13 mole) in 20 .mu.l of buffer (50 mM Tris-HCl (PH
8.3), 37.5 mM KCl, 1.5 MM MgCl.sub.2, 10 mM DTT), by incubating at
70.degree. C. for 5 minutes, and the reaction was then allowed to
cool to room temperature (about 20-25.degree. C.) over the course
of about 2 hours. Amplification was carried out by adding 0.1 mM
ATP, 0.1 mM TTP, 0.1 mM GTP, 0.1 mM CTP, 4 units Bst DNA polymerase
I and incubating at 50.degree. C. Time points were taken at 5, 15,
and 30 minutes by trans- ferring aliquots of the reaction to a
formamide/EDTA solu- tion (50% formamide 10 mM Tris-HCl pH 8.0, 0.1
mM EDTA). The samples were loaded and run on a denaturing 15%
polyacrylamide gel. Bands corresponding to the elon- gation product
and primer were excised from the gel and analyzed by Cerenkov
counting in a scintillation counter.
The results shown in Table 3 demonstrate that strand displacement
is more efficient using primers with a 5' non-complementary region.
The "% product" refers to the amount of elongation product divided
by the sum of the amount of elongation product and primer. The
higher per- centage of product observed with the primer having a 5'
non-complementary region indicates increased amplifica- tion and
strand displacement.
TABLE-US-00003 TABLE 3 Primer (SEQ. 24 24 24 25 25 25 ID. NO).sup.1
Time 5 min 15 min 30 min 5 min 15 min 30 min % Product 4.3 5.8 5.9
18 25 35 .sup.1In addition to the primers listed in Table 4, each
reaction mixture contained a radiolabeled primer having the
nucleotide sequence of SEQ. ID. NO. 19.
The present embodiments of this invention are to be considered in
all respects as illustrative and not restrictive, the scope of the
invention being indicated by the appended claims rather than by the
foregoing description, and all changes which come within the
meaning and range of equivalency of the claims therefore are
intended to be embraced therein.
Other embodiments are within the following claims.
SEQUENCE LISTINGS
1
2725nucleic acidsinglelinear 1CGCGTCTAGT CCTACTCAGG TGTTG
2518nucleic acidsinglelinear 2CCCCAGATTC AGACTAGG 1825nucleic
acidsinglelinear 3CGGTCATTGA CTAGTATTTA GCCTT 2523nucleic
acidsinglelinear 4TTTCTTCATA GTACTGGTTC ACT 2323nucleic
acidsinglelinear 5ATTTCACTCC CTTAACAAGG GTT 2322nucleic
acidsinglelinear 6TTGTAAGCTA CAGGTTTCAG GT 2221nucleic
acidsinglelinear 7ACGGGCAATT GGTCTGCGAC C 2123nucleic
acidsinglelinear 8AGCAAAGACC CGGAGGTCCG AAT 2323nucleic
acidsinglelinear 9GAATACATAG GTATGCAAAG CGA 2324nucleic
acidsinglelinear 10CCTGCCGAAC TGAAACATCT TAGT 2424nucleic
acidsinglelinear 11CAGAGGAAAA GAAATCGAAG AGAT 2422nucleic
acidsinglelinear 12TGTAGGATTG AGGATAAAGG AT 2229nucleic
acidsinglelinear 13GGACTCCTAG TTGAACACAT CTGGAAAGA 2923nucleic
acidsinglelinear 14CTATAGGGAG ACCACAACGG TTT 2323nucleic
acidsinglelinear 15CTAGAAATAA TTTTGTTTAA CTT 2322nucleic
acidsinglelinear 16AGAAGGAGAT ATACATATGG CT 2222nucleic
acidsinglelinear 17CATGACTGGT GGACAGCAAA TG 2218nucleic
acidsinglelinear 18GAGCGAACGC AGTCAGGA 1834nucleic acidsinglelinear
19GAGAAGTGGT CACGGAGGTA CGAGCGAACG CAGT 3420nucleic
acidsinglelinear 20GGAGATGGAA CGTACCATGT 2022nucleic
acidsinglelinear 21GGGAACTGAG CGGATTTACC GC 2227nucleic
acidsinglelinear 22GAACAACGCC AGTTTGATCT CCAGCAG 2726nucleic
acidsinglelinear 23CAACCTCAAA GGCCCATAAC GTTGCG 2629nucleic
acidsinglelinear 24GTCAGGACTT CACCGCCAAA TACCTTCAA 2950nucleic
acidsinglelinear 25GAAATTAATA CGACTCACTA TAGGGAGACT TCACCGCCAA
ATACCTTCAA 50137nucleic acidsinglelinear 26CATGACTGGT GGACAGCAAA
TGGGTACTAA CCAAGGTAAA GGTGTAGTTG 50CTGCTGGAGA TAAACTGGCG TTGTTCTTGA
AGGTATTTGG CGGTGAAGTC 100CTGACTGCGT TCGCTCGTAC CTCCGTGACC ACTTCTC
13727nucleic acidsinglelinear 27CCCGTAGACG AAAGGAGAGA AAGACCG
27
* * * * *