Terminal repeat amplification method

Claims

We claim:

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) an RNA polymerase;

(iii) a DNA polymerase;

(iv) a ribonuclease that hydrolyses RNA of an RNA-DNA hybrid without hydrolysing single- or double-stranded RNA or DNA;

(v) ribonucleoside and deoxyribonucleoside triphosphates;

(vi) a DNA ligase;

(vii) a second oligonucleotide primer comprising a 3'-terminal priming sequence that is complementary to the specific nucleic acid sequence, minus-sense sequences of a promoter and an initiation site that are recognized by said RNA polymerase, a 5'-terminal sequence that is self-complementary to said minus-sense sequence of said initiation site, and a 5'-terminal phosphate group; and

(viii) a single-stranded RNA comprising said specific nucleic acid sequence:

(B) maintaining conditions such that

(i) said second oligonucleotide primer hybridizes to said single-stranded RNA;

(ii) said DNA polymerase uses said single-stranded 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 polymerase uses said complementary DNA as template to synthesize a DNA segment which terminates at said second oligonucleotide primer by extension of said first oligonucleotide primer;

(v) said DNA ligase joins said DNA segment to said second oligonucleotide primer and thereby forms a DNA second template; and

(vi) said RNA polymerase recognizes said promoter and transcribes said DNA second template,

thereby providing copies of an RNA first template which comprises a sequence complementary to said specific nucleic acid sequence, minus-sense sequences of said promoter and said initiation site, and a 5'-terminal sequence that is self-complementary to said minus-sense sequence of said initiation site, and

(C) maintaining conditions such that a cycle ensues wherein:

(i) said first oligonucleotide primer hybridizes to said RNA first template;

(ii) said DNA polymerase uses said RNA first template to synthesize a DNA second template by extension of said first oligonucleotide primer and thereby forms as RNA-DNA hybrid, said DNA second template comprising plus-sense sequences of said promoter and said initiation site, and a 3'-terminal priming sequence that is self-complementary to said plus-sense sequence of said initiation site;

(iii) said ribonuclease hydrolyses RNA which comprises said RNA-DNA hybrid;

(iv) said 3'-terminal priming sequence of said DNA second template hybridizes to said plus-sense sequence of said initiation site;

(v) said DNA polymerase uses said DNA second template as template to synthesize said promoter by extension of said DNA second template; and

(vi) said RNA polymerase recognizes said promoter and transcribes said DNA second template, thereby providing copies of said RNA first template; and thereafter,

(D) maintaining conditions such that a cycle ensues for a time sufficient to achieve a desired amplification of said specific nucleic acid sequence.

2. 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) an RNA polymerase;

(iii) a DNA polymerase;

(iv) a ribonuclease that hydrolyses RNA of an RNA-DNA hybrid without hydrolysing single- or double-stranded RNA or DNA;

(v) ribonucleoside and deoxyribonucleoside triphosphates;

(vi) a DNA ligase;

(vii) a second oligonucleotide primer comprising a 3'-terminal priming sequence that is complementary to the specific nucleic acid sequence, minus-sense sequences of a promoter and an initiation site that are recognized by said RNA polymerase, a 5'-terminal sequence that is self-complementary to said minus-sense sequence of said initiation site, and a 5'-terminal phosphate group; and

(x) a single-stranded DNA comprising said specific nucleic acid sequence;

(B) maintaining conditions such that

(i) said second oligonucleotide primer hybridizes to said single-stranded DNA;

(ii) said DNA polymerase uses said single-stranded DNA as a template to synthesize a complementary DNA by extension of said second oligonucleotide primer and thereby forms a DNA-DNA hybrid;

(iii) said DNA-DNA hybrid is denatured;

(vi) said first oligonucleotide primer hybridizes to said complementary DNA;

(v) said DNA polymerase uses said complementary DNA as template to synthesize a DNA segment which terminates at said second oligonucleotide primer by extension of said first oligonucleotide primer;

(vi) said DNA ligase joins said DNA segment to said second oligonucleotide primer and thereby forms a DNA second template; and

(vii) said RNA polymerase recognizes said promoter and transcribes said DNA second template,

thereby providing copies of an RNA first template which comprises a sequence complementary to said specific nucleic acid sequence, minus-sense sequences of said promoter and said initiation site, and a 5'-terminal sequence that is self-complementary to at least said minus-sense sequence of said initiation site;

(C) maintaining conditions such that a cycle ensues wherein:

(i) said first oligonucleotide primer hybridizes to said RNA first template;

(ii) said DNA polymerase uses said RNA first template to synthesize a DNA second template by extension of said first oligonucleotide primer and thereby forms as RNA-DNA hybrid, said DNA second template comprising plus-sense sequences of said promoter and said initiation site, and a 3'-terminal priming sequence that is self-complementary to said plus-sense sequence of said initiation site;

(iii) said ribonuclease hydrolyses RNA which comprises said RNA-DNA hybrid;

(iv) said 3'-terminal priming sequence of said DNA second template hybridizes to said plus-sense sequence of said initiation site;

(v) said DNA polymerase uses said DNA second template as template to synthesize said promoter by extension of said DNA second template; and

(vi) said RNA polymerase recognizes said promoter and transcribes said DNA second template, thereby providing copies of said RNA first template;

and thereafter,

(D) maintaining conditions such that a cycle ensues for a time sufficient to achieve a desired amplification of said specific nucleic acid sequence.

3. A process according to claim 2, further comprising prior to step (A), denaturing double-stranded DNA to provide single-stranded DNA.

4. 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) an RNA polymerase;

(iii) a DNA polymerase;

(iv) a ribonuclease that hydrolyses RNA of an RNA-DNA hybrid without hydrolysing single- or double-stranded RNA or DNA;

(v) ribonucleoside and deoxyribonucleoside triphosphates;

(vi) a second oligonucleotide primer comprising a 3'-terminal priming sequence that is complementary to the specific nucleic acid sequence, minus-sense sequences of a promoter and an initiation site that are recognized by said RNA polymerase, a 5'-terminal sequence that is self-complementary to said minus-sense sequence of said initiation site, and further comprising a 5'-terminal oligoribonucleotide segment; and

(x) a single-stranded RNA comprising said specific nucleic acid sequence:

(B) maintaining conditions such that:

(i) said second oligonucleotide primer hybridizes to said single-stranded RNA;

(ii) said DNA polymerase uses said single-stranded 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 polymerase uses said complementary DNA as template to synthesize a DNA strand;

(vi) said ribonuclease hydrolyses RNA of said second primer within said complementary DNA;

(vii) the 3'-end of said DNA strand hybridizes to a complementary sequence of said DNA strand thereby forming a 3'-stem loop structure;

(viii) said DNA polymerase extends said 3'-end of said DNA strand to provide said promoter;

(vi) said RNA polymerase recognizes said promoter and transcribes said DNA second template,

thereby providing copies of an RNA first template which comprises a sequence complementary to said specific nucleic acid sequence, minus-sense sequence of said promoter and said initiation site, and a 5'-terminal sequence that is self-complementary to at least said minus-sense sequence of said initiation site;

(C) maintaining conditions such that a cycle ensues wherein:

(i) said first oligonucleotide primer hybridizes to said RNA first template;

(ii) said DNA polymerase uses said RNA first template to synthesize a DNA second template by extension of said first oligonucleotide primer and thereby forms as RNA-DNA hybrid, said DNA second template comprising plus-sense sequences of said promoter and said initiation site, and a 3'-terminal priming sequence that is self-complementary to said plus-sense sequence of said initiation site;

(iii) said ribonuclease hydrolyses RNA which comprises said RNA-DNA hybrid;

(iv) said 3'-terminal priming sequence of said DNA second template hybridizes to said plus-sense sequence of said initiation site;

(v) said DNA polymerase uses said DNA second template as template to synthesize said promoter by extension of said DNA second template; and

(vi) said RNA polymerase recognizes said promoter and transcribes said DNA second template, thereby providing copies of said RNA first template; and thereafter,

(D) maintaining conditions such that a cycle ensues for a time sufficient to achieve a desired amplification of said specific nucleic acid sequence.

5. 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) an RNA polymerase;

(iii) a DNA polymerase;

(iv) a ribonuclease that hydrolyses RNA of an RNA-DNA hybrid without hydrolysing single- or double-stranded RNA or DNA;

(v) ribonucleoside and deoxyribonucleoside triphosphates;

(vi) a DNA ligase; and

(vii) a single-stranded DNA which comprises a sequence complementary to said specific nucleic acid sequence, minus-sense sequences of a promoter and an initiation site that are recognized by said RNA polymerase, a 5'-terminal sequence that is self-complementary to said minus-sense sequence of said initiation site, and a 5'-terminal phosphate group

(B) maintaining conditions such that:

(i) said first oligonucleotide primer hybridizes to said single-stranded DNA;

(ii) said DNA polymerase uses said single-stranded DNA as a template to synthesize a DNA segment which terminates at said 5'-terminal sequence by extension of said first oligonucleotide primer;

(iii) said DNA ligase joins said DNA segment to said single-stranded DNA and thereby forms a DNA second template; and

(iv) said RNA polymerase recognizes said promoter and transcribes said DNA second template,

thereby providing copies of an RNA first template which comprises a sequence complementary to said specific nucleic acid sequence, minus-sense sequence of said promoter and said initiation site, and a 5'-terminal sequence that is self-complementary to at least said minus-sense sequence of said initiation site:

(C) maintaining conditions such that a cycle ensues wherein:

(i) said first oligonucleotide primer hybridizes to said RNA first template;

(ii) said DNA polymerase uses said RNA first template to synthesize a DNA second template by extension of said first oligonucleotide primer and thereby forms as RNA-DNA hybrid, said DNA second template comprising plus-sense sequences of said promoter and said initiation site, and a 3'-terminal priming sequence that is self-complementary to said plus-sense sequence of said initiation site;

(iii) said ribonuclease hydrolyses RNA which comprises said RNA-DNA hybrid;

(iv) said 3'-terminal priming sequence of said DNA second template hybridizes to said plus-sense sequence of said initiation site:

(v) said DNA polymerase uses said DNA second template as template to synthesize said promoter by extension of said DNA second template; and

(vi) said RNA polymerase recognizes said promoter and transcribes said DNA second template, thereby providing copies of said RNA first template;

and thereafter,

(D) maintaining conditions such that a cycle ensues for a time sufficient to achieve a desired amplification of said specific nucleic acid sequence.

6. A process according to claim 5, wherein step (B) further comprises adding to said reaction medium an RNA-DNA hybrid comprising said single-stranded DNA, such that said ribonuclease hydrolyses RNA which comprises said RNA-DNA hybrid.

7. 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) an RNA polymerase;

(ii) a DNA polymerase;

(iii) a ribonuclease that hydrolyses RNA of an RNA-DNA hybrid without hydrolysing single- or double-stranded RNA or DNA;

(iv) ribonucleoside and deoxyribonucleoside triphosphates;

(v) a DNA ligase;

(vi) a first oligonucleotide primer comprising a 5' complementary sequence;

(vii) a second oligonucleotide primer comprising a 3'-terminal priming sequence that is complementary to the specific nucleic acid sequence, minus-sense sequences of a promoter and an initiation site that are recognized by said RNA polymerase, a 5'-terminal sequence that is self-complementary to said minus-sense sequence of said initiation site, and a 5'-terminal phosphate group; and

(viii) a single-stranded RNA comprising said specific nucleic acid sequence:

(B) maintaining conditions such that

(i) said second oligonucleotide primer hybridizes to said single-stranded RNA;

(ii) said DNA polymerase uses said single-stranded RNA as a template to synthesize a complementary DNA by extension of said second oligonucleotide primer and thereby forms an KNA-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 polymerase uses said complementary DNA as template to synthesize a DNA segment which terminates at said second oligonucleotide primer by extension of said first oligonucleotide primer;

(vi) said DNA ligase joins said DNA segment to said second oligonucleotide primer and thereby forms a DNA second template; and

(vii) said RNA polymerase recognizes said promoter and transcribes said DNA second template,

thereby providing copies of an RNA first template which comprises a sequence complementary to said specific nucleic acid sequence, minus-sense sequences of said promoter and said initiation site, and a 5'-terminal sequence that is self-complementary to said minus-sense sequence of said initiation site and a 3'-terminal priming sequence that hybridizes to a complementary sequence of the RNA first template thereby forming an RNA:RNA stem loop, and

(C) maintaining conditions such that a cycle ensues wherein:

(i) said DNA polymerase uses said RNA first template to synthesize a DNA second template by extension of said 3'-terminal priming sequence and thereby forms an RNA-DNA hybrid, said DNA second template comprising plus-sense sequences of said promoter and said initiation site, and a 3'-terminal priming sequence that is self-complementary to said plus-sense sequence of said initiation site;

(ii) said ribonuclease hydrolyses RNA which comprises said RNA-DNA hybrid but not RNA of said RNA-RNA stem loop;

(iii) said 3'-terminal priming sequence of said DNA second template hybridizes to said plus-sense sequence of said initiation site;

(iv) said DNA polymerase uses said DNA second template as template to synthesize said promoter by extension of said DNA second template; and

(v) said RNA polymerase recognizes said promoter and transcribes said DNA second template, thereby providing copies of said RNA first template;

and thereafter,

(D) maintaining said conditions for a time sufficient to achieve a desired amplification of said specific nucleic acid sequence.

8. 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) an RNA polymerase;

(ii) a DNA polymerase;

(iii) a ribonuclease that hydrolyses RNA of an RNA-DNA hybrid without hydrolysing single- or double-stranded RNA or DNA;

(iv) ribonucleoside and deoxyribonucleoside triphosphates;

(v) a DNA ligase;

(vi) a first oligonucleotide primer comprising a 5' complementary sequence;

(vii) a second oligonucleotide primer comprising a 3'-terminal priming sequence that is complementary to the specific nucleic acid sequence, minus-sense sequences of a promoter and an initiation site that are recognized by said RNA polymerase, a 5'-terminal sequence that is self-complementary to said minus-sense sequence of said initiation site, and a 5'-terminal phosphate group; and

(x) a single-stranded DNA comprising said specific nucleic acid sequence;

(B) under conditions such that

(i) said second oligonucleotide primer hybridizes to said single-stranded DNA;

(ii) said DNA polymerase uses said single-stranded DNA as a template to synthesize a complementary DNA by extension of said second oligonucleotide primer and thereby forms a DNA-DNA hybrid;

(iii) said DNA-DNA hybrid is denatured;

(vi) said first oligonucleotide primer hybridizes to said complementary DNA;

(v) said DNA polymerase uses said complementary DNA as template to synthesize a DNA segment which terminates at said second oligonucleotide primer by extension of said first oligonucleotide primer;

(vi) said DNA ligase joins said DNA segment to said second oligonucleotide primer and thereby forms a DNA second template; and

(vii) said RNA polymerase recognizes said promoter and transcribes said DNA second template,

thereby providing copies of an RNA first template which comprises a sequence complementary to said specific nucleic acid sequence, minus-sense sequences of said promoter and said initiation site, and a 5'-terminal sequence that is self-complementary to at least said minus-sense sequence of said initiation site and a 3'-terminal priming sequence that hybridizes to a complementary sequence of the RNA first template thereby forming an RNA:RNA stem loop;

(C) maintaining conditions such that a cycle ensues wherein:

(i) said DNA polymerase uses said RNA first template to synthesize a DNA second template by extension of said 3'-terminal priming sequence and thereby forms an RNA-DNA hybrid, said DNA second template comprising plus-sense sequences of said promoter and said initiation site, and a 3'-terminal priming sequence that is self-complementary to said plus-sense sequence of said initiation site;

(ii) said ribonuclease hydrolyses RNA which comprises said RNA-DNA hybrid but not RNA of said RNA-RNA stem loop;

(iii) said 3'-terminal priming sequence of said DNA second template hybridizes to said plus-sense sequence of said initiation site;

(iv) said DNA polymerase uses said DNA second template as template to synthesize said promoter by extension of said DNA second template; and

(v) said RNA polymerase recognizes said promoter and transcribes said DNA second template, thereby providing copies of said RNA first template;

and thereafter,

(D) maintaining said conditions for a time sufficient to achieve a desired amplification of said specific nucleic acid sequence.

9. 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) an RNA polymerase;

(ii) a DNA polymerase;

(iii) a ribonuclease that hydrolyses RNA of an RNA-DNA hybrid without hydrolysing single- or double-stranded RNA or DNA;

(iv) ribonucleoside and deoxyribonucleoside triphosphates;

(v) a first oligonucleotide primer comprising a 5' complementary sequence;

(vi) a second oligonucleotide primer comprising a 3'-terminal priming sequence that is complementary to the specific nucleic acid sequence, minus-sense sequences of a promoter and an initiation site that are recognized by said RNA polymerase, a 5'-terminal sequence that is self-complementary to said minus-sense sequence of said initiation site, and further comprising a 5'-terminal oligoribonucleotide segment; and

(x) a single-stranded RNA comprising said specific nucleic acid sequence:

(B) under conditions such that:

(i) said second oligonucleotide primer hybridizes to said single-stranded RNA;

(ii) said DNA polymerase uses said single-stranded 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 polymerase uses said complementary DNA as template to synthesize a DNA strand;

(vi) said ribonuclease hydrolyses RNA of said second primer within said complementary DNA;

(vii) the 3'-end of said DNA strand hybridizes to a complementary sequence of said DNA strand thereby forming a 3'-stem loop structure;

(viii) said DNA polymerase extends said 3'-end of said DNA strand to provide said promoter;

(vi) said RNA polymerase recognizes said promoter and transcribes said DNA second template,

thereby providing copies of an RNA first template which comprises a sequence complementary to said specific nucleic acid sequence, minus-sense sequence of said promoter and said initiation site, and a 5'-terminal sequence that is self-complementary to at least said minus-sense sequence of said initiation site and a 3'-terminal priming sequence that hybridizes to a complementary sequence of the RNA first template thereby forming an RNA:RNA stem loop;

(C) maintaining conditions such that a cycle ensues wherein:

(i) said DNA polymerase uses said RNA first template to synthesize a DNA second template by extension of said 3'-terminal priming sequence and thereby forms an RNA-DNA hybrid, said DNA second template comprising plus-sense sequences of said promoter and said initiation site, and a 3'-terminal priming sequence that is self-complementary to said plus-sense sequence of said initiation site;

(ii) said ribonuclease hydrolyses RNA which comprises said RNA-DNA hybrid but not RNA of said RNA-RNA stem loop;

(iii) said 3'-terminal priming sequence of said DNA second template hybridizes to said plus-sense sequence of said initiation site;

(iv) said DNA polymerase uses said DNA second template as template to synthesize said promoter by extension of said DNA second template; and

(v) said RNA polymerase recognizes said promoter and transcribes said DNA second template, thereby providing copies of said RNA first template;

and thereafter,

(D) maintaining said conditions for a time sufficient to achieve a desired amplification of said specific nucleic acid sequence.

10. A process according to claim 1, wherein said RNA polymerase is bateriophase T7 RNA polymerase and wherein said minus-sense sequence of said initiation site and said minus-sense sequence of said promoter together comprise the nucleotide sequence complementary to 5'-AATTCTAATACGACTCACTATAGGGAGA-3'(nucleotides 1-28 of SEQ ID NO: 5).

11. A process according to claim 1, wherein said process further comprises, after step (D), a step (E) of monitoring said reaction medium for consumption of any of said reagents or for accumulation of any product of said cycle.

12. A process according to claim 1 wherein said ribonuclease comprises calf thymus ribonuclease H.

13. A process according to claim 1, wherein said RNA polymerase is a bacteriophage RNA polymerase.

14. A process according to claim 13, wherein said bacteriophage RNA polymerase is selected from the group consisting of bacteriophage T7 RNA polymerase, bacteriophage T3 polymerase, bacteriophage .phi.II polymerase, Salmonella bacteriophage sp6 polymerase, and Pseudomonas bacteriophage gh-1 polymerase.

15. A process according to claim 1, wherein said DNA polymerase is a retrovirus reverse transcriptase.

16. A process according to claim 15, wherein said retrovirus reverse transcriptase is selected from the group consisting of avian myeloblastosis virus polymerase, and a Moloney murine leukaemia virus polymerase.

17. A process according to claim 1, wherein said DNA polymerase lacks exonuclease activity.

18. A process according to claim 1, wherein all DNA polymerases in said reaction medium lack exonuclease and DNA endonuclease activity.

19. A process according to claim 1, wherein said DNA polymerase is selected from the group consisting of avian myeloblastosis virus polymerase, DNA polymerase .beta., and calf thymus DNA polymerase.

20. A process according to claim 1, wherein step (C) comprises maintaining said conditions for a time between 30 minutes and 4 hours.

21. 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.

22. A process according to claim 21, further comprising the step of expressing a product encoded by said DNA product of said cycle in an expression system.

FIELD OF THE INVENTION

This invention relates to a process for amplifying the number of copies of a specific nucleic acid sequence or its complement by using a template having a terminal sequence complementary to another sequence within the template.

BACKGROUND OF THE INVENTION

The presence of nucleic acids in a sample may indicate that a source from which the sample is taken has a disease, disorder or abnormal physical state. Certain diagnostics determine whether nucleic acids are present in a sample. These diagnostics invariably require amplification of nucleic acids because of the copy number problem. In a virus or cell, for example, there is usually a single copy of a particular gene. Without amplification of specific nucleic acids of the gene, it is often difficult to detect the presence of the nucleic acids.

One approach is to increase the copy number of the specific sequence, in preference to other sequences present in the sample, using an in vitro amplification method. The "polymerase chain reaction" (PCR) is one such technique (Mullis, K. et al., Cold Spring Harbor Symp. Quant. Biol. 52:263-273 (1986), Mullis, K., et al., U.S. Pat. No. 4,683,202) to selectively increase the copy number of a DNA segment having a particular sequence. In general, PCR involves treating the sample suspected of containing a target DNA sequence with oligonucleotide primers such that a primer extension product is synthesized by a DNA-dependent DNA polymerase. The primer extension product DNA strand is separated from the template strand in the preferred embodiment using heat denaturation. Both the original template and the primer extension product then serve as templates in the next and subsequent cycles of extension, resulting approximately in the doubling of the number of target DNA sequences in the sample at the end of each cycle. Consequently, multiple cycles result in the quasi-exponential amplification of the target nucleic acid sequence. Optimal practice of the PCR requires the use of a thermocycler capable of rapid changes of temperature and of a DNA polymerase, such as Taq polymerase (Saiki et al., Science 239:487-491 (1988) and Saiki, R. et al., U.S. Pat. No. 4,683,195) that resists the denaturation caused by repeated exposure to temperatures above 90.degree. C. required to separate the DNA strands.

Another in vitro amplification method referred to as the T7RT method (Burg et al., U.S. Ser. No. 080,479, abandoned) uses an RNA polymerase in addition to a DNA polymerase (a reverse transcriptase) to increase the yield of products per cycle of amplification. The method involves the use of two primers, one of which contains a promoter for the synthesis of a double-strand DNA intermediate from an RNA product by a series of primer hybridization, primer extension and product denaturation steps. The double-stranded DNA intermediate containing a promoter derived from one of the primers which directs the synthesis of multiple copies of RNA which can be used for the synthesis of additional copies of the double-stranded DNA intermediate. Multiple cycles result in an exponential amplification. The yield of products per amplification cycle exceeds that of PCR by at least an order of magnitude, thus requiring fewer cycles to obtain the same overall level of amplification. The major drawback to the T7RT method is the inherent heat denaturation step which is necessary to separate the eDNA intermediate from the RNA product but inactivates both of the thermolabile enzymes used in the process. Consequently, fresh enzymes must be added to the reaction mixture at each cycle following the heat denaturation step.

U.S. Ser. No. 07/211,384, U.S. Pat. No. 5,409,818, of Cangene Corporation describes another amplification process known as NASBA.TM., which involves the use of two primers, one of which has a promoter, and three enzymes; an RNA polymerase, a DNA polymerase (a reverse transcriptase) and a ribonuclease (RNase H) that specifically degrades the RNA strand of an RNA-DNA hybrid. The cyclic process takes place at a relatively constant temperature throughout and without serial addition of reagents, wherein the first primer hybridizes to the RNA product, reverse transcriptase uses the RNA product as template to synthesize a DNA product by extension of the first primer, RNase H degrades the RNA of the resulting RNA-DNA hybrid, the second primer with the promoter hybridizes to the DNA product, reverse transcriptase uses the second primer as template to synthesize a double-stranded promoter by extension of the DNA product, an RNA polymerase uses the promoter and DNA product to transcribe multiple copies of the same RNA product. The unique addition of RNase H distinguishes NASBA.TM. from the T7RT process by eliminating the need for heat denaturation to separate the DNA product from its RNA template.

U.S. Pat. No. 5,130,238 of Cangene Corporation describes an enhanced nucleic acid amplification process known as enhanced NASBA.TM.. The process is similar to that described in U.S. Ser. No. 07/211,384, and U.S. Pat. No. 5,409,818, and is enhanced by addition to the reaction mixture of an alkylated sulfoxide (for example, dimethyl sulfoxide) and BSA.

U.S. Ser. No. 08/275,250 of Cangene Corporation describes a further improvement of NASBA.TM.. To overcome thermal denaturation during entry into the amplification cycle from DNA, this process of amplification uses RNA polymerase, specifically, E. coli RNA polymerase to eliminate the heating steps involved in entering the amplification cycle.

Notwithstanding these amplification processes, a need exists for improvements to the amplification process. It would be preferable if the amplification process required fewer steps and fewer manipulations by a user.

One step which would be helpful to eliminate is the addition of a promoter sequence to derived DNA to allow subsequent transcription. For example, it is known that DNA synthesis from a DNA or an RNA template requires a DNA or an RNA primer with a 3'-OH group. It is also known that normal synthesis of an RNA using T7 RNA polymerase requires a double-stranded DNA promoter immediately upstream of a template from which the RNA is transcribed. Such transcribed RNA would not contain a promoter sequence; thus one would need to add such promoter sequence to derived DNA to allow subsequent transcription. In NASBA.TM. and enhanced NASBA.TM. amplification reactions, these basic requirements are met by providing two primers, a first primer which hybridizes to the RNA product, and a second primer which has a plus-sense sequence of a T7 promoter and hybridizes to the DNA product. The first primer is extended using the RNA product as template to form the DNA product which serves as the template for transcription of the RNA product. The DNA product is extended using the second primer as template to form a double-stranded promoter for the transcription of the RNA product.

This invention improves upon NASBA.TM. and enhanced NASBA.TM. amplification reactions by reducing the number of primers required in the amplification cycle through the use of an RNA with an inverted repeat sequence at its 5'-end adjacent to a minus-sense sequence of the promoter recognized by an RNA polymerase. A eDNA copy of this RNA has an inverted repeat sequence at its 3'-end adjacent to a plus-sense sequence of a promoter. Upon removal of an RNA strand, the cDNA is capable of self-prig to form a partially double-stranded DNA stem-loop structure containing a double-stranded promoter oriented toward the apex of the stem-loop. Transcription of this DNA results in multiple copies of the same RNA with an inverted repeat sequence at its 5'-end adjacent to a minus-sense sequence of the promoter. Thus, the RNA product of the transcription encodes the minus-sense of the promoter sequence recognized by an RNA polymerase, and consequently the DNA copy of this RNA is fully functional as a template for transcription without the need for the addition of a promoter-bearing primer.

Other researchers have described the use of primers with inverted repeats or "hairpins" capable of transcription in nucleic acid amplification processes, namely, Dattagupta, N., EP 0 427 073 A2, and EP 0 427 074 A2, both published May 15, 1991. However, these hairpin primers encode plus-sense promoters that direct transcription of the target sequence without incorporating the sequence of the promoter itself into the product. Thus the processes described in these patents merely mimic the transcription phase of NASBA.TM. and enhanced NASBA.TM.. Furthermore,