Method for amplifying a polynucleotide

• US 5,914,229 A
• Filed: 06/14/1996
• Issued: 06/22/1999
• Est. Priority Date: 06/14/1996
• Status: Expired due to Term

• Current Assignee: Sarnoff Corporation (SRI International, Inc.)
• Original Assignee: Sarnoff Corporation (SRI International, Inc.)
• Inventors: Loewy, Zvi
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US08/663,688
• Time in Patent Office: 1,103 Days
• Field of Search: 435/91.2, 435/91.52, 435/91.1, 435/6, 536/25.32, 536/25.5
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6865 Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]

Abstract

In one aspect, the present invention relates to methods of amplifying a target nucleic acid, the methods involving the production of a double-stranded promoter to provide for transcription of the target sequence, and the provision of a poly-A tail in the transcription product. In another aspect, the present invention relates to methods of detecting a target nucleic acid in a sample.

Claims

1. A method of amplifying a target nucleic acid in a sample comprising the steps of:

(a) providing the target nucleic acid of the sample as single-stranded nucleic acid;

(b) combining with the sample at least one oligonucleotide, wherein the oligonucleotide or oligonucleotides include a double-stranded promoter, a single-stranded segment of nucleic acid complementary to a segment of the target nucleic acid, and a homopolynucleotide;

(c) annealing oligonucleotide(s) comprising the double-stranded promoter, complementary segment and homopolynucleotide to the target nucleic acid; and

(d) adding an RNA polymerase and ribonucleoside triphosphates or analogs thereof, thereby generating transcripts.

2. The method of claim 1, further comprising repeating steps (a) through (d) at least twice.

3. The method of claim 1, further comprising detection of the presence or absence of the transcripts resulting from step (d).

4. The method of claim 3, wherein the ribonucleoside triphosphates or analogs thereof include a reporter molecule, and the method further comprises detection of the presence or absence of the reporter molecule, wherein the reporter molecule comprises biotin, poly G, a fluorescent dye, or a radioisotope.

5. The method of claim 3, further comprising detection of the presence or absence of the homopolynucleotide or a complement thereof.

7. The method of claim 1, wherein at least one of the oligonucleotides of step (b) has a reporter molecule attached thereto which is annealed to the target nucleic acid.

8. The method of claim 4, wherein the reporter molecule comprises the fluorescent dye or the radioisotope.

9. The method of claim 1, wherein the double-stranded promoter comprises two oligonucleotides, each of which is single-stranded and each of which includes a promoter sequence complementary to the other'"'"'s promoter sequence.

10. The method of claim 1, wherein the double-stranded promoter comprises a single polynucleotide with a promoter in a double-stranded region of the polynucleotide.

11. The method of claim 10, wherein the double-stranded region consists of a hairpin structure.

14. The method of claim 1 wherein the oligonucleotides in step (b) include at least two oligonucleotides having a sequence complementary to a sequence in the target nucleic acid, the sequences of the two oligonucleotides being contiguous in the target nucleic acid.

15. The method of claim 14, wherein the amplification of the target nucleic acid includes detection of transcripts, said detection being used to determine presence or absence of at least one allele or a point mutation in the target nucleic acid.

16. The method of claim 14 wherein the oligonucleotides having complementary contiguous sequences are ligated together by a joining agent.

17. The method of claim 16 wherein ligation occurs prior to step (c).

18. The method of claim 16 wherein ligation occurs after step (c).

19. The method of claim 16, wherein the joining agent is ligase, cyanogen bromide, or carbodiimide.

20. The method of claim 1 wherein the RNA polymerase does not cause transcription from a substrate that is a single stranded polynucleotide.

21. The method of claim 20 wherein the RNA polymerase is an E. coli polymerase.

22. The method of claim 1 further comprising the addition of an agent that destroys single-stranded polynucleotide prior to step (d).

23. The method of claim 1 wherein the promoter is a lac promoter.

24. The method of claim 1 wherein the oligonucleotides of step (b) comprise:

(i) a first oligonucleotide that includes a promoter, a segment of nucleic acid complementary to a segment of the target nucleic acid, and the homopolynucleotide; and

(ii) a second oligonucleotide that comprises a segment of nucleic acid complementary to the promoter.

25. The method of claim 1, wherein the target nucleic acid or an oligonucleotide is converted from a double-stranded form to a single-stranded form by thermal means, wherein the melting temperature used in the thermal means is from about 90°

C. to about 100°

C. in the absence of formamide, or less than about 90°

C. in the presence of formamide.

26. The method of claim 1, wherein the target nucleic acid or an oligonucleotide is converted from a double-stranded form to a single-stranded form by non-thermal means.

27. The method of claim 26, wherein the non-thermal means includes contacting the nucleic acid or the oligonucleotide with base or acid.

28. The method of claim 26, wherein the non-thermal means includes use of electrostatics.

6. A method of amplifying a target nucleic acid in a sample comprising the steps of:

(a) providing the target nucleic acid of the sample as single-stranded nucleic acid;

(b) combining with the sample at least one oligonucleotide, wherein the oligonucleotide or oligonucleotides include a double-stranded promoter, a single-stranded segment of nucleic acid complementary to a segment of the target nucleic acid, and a homopolynucleotide;

(c) annealing oligonucleotide(s) comprising the double-stranded promoter, complementary segment and homopolynucleotide the target nucleic acid;

(d) adding an RNA polymerase and ribonucleoside triphosphates or analogs thereof, thereby generating the complement of the complementary segment and homopolynucleotide, and

(e) detecting the complementary polynucleotide which is produced as a transcript in step (d), and wherein detection comprises;

(i) providing the homopolynucleotide attached to a solid substrate;

(ii) combining the substrate-attached homopolynucleotide and the polynucleotide to be detected under conditions that promote hybridization therebetween; and

(iii) detecting the polynucleotide to be detected associated with the solid substrate.

29. The method of claim 6, wherein the solid substrate is a paramagnetic bead.

12. A method of amplifying a target nucleic acid in a sample comprising the steps of:

(a) providing the target nucleic acid of the sample as single-stranded nucleic acid;

(b) combining with the sample at least one oligonucleotide, wherein the oligonucleotide or oligonucleotides include a double-stranded promoter, a single-stranded segment of nucleic acid complementary to a segment of the target nucleic acid, and a homopolynucleotide;

(c) annealing oligonucleotide(s) comprising the double-stranded promoter, complementary segment and homopolynucleotide to the target nucleic acid; and

(d) adding an RNA polymerase and ribonucleoside triphosphates or analogs thereof, thereby generating the complement of the complementary segment and homopolynucleotide,

wherein the target nucleic acid has a first and a second sequence on the same strand, said first and second sequences being contiguous; and

wherein the oligonucleotides in step (b) comprises at least three oligonucleotides, the three oligonucleotides comprising;

(i) a first oligonucleotide including a promoter and a first segment of nucleic acid complementary to the first sequence on the target nucleic acid;

(ii) a second oligonucleotide including a segment of nucleic acid complementary to the promoter of the first oligonucleotide;

(iii) and a third oligonucleotide including a segment of nucleic acid complementary to the second sequence on the target nucleic acid and the homopolynucleotide.

13. The method of claim 12, wherein the promoter is a bacteriophage promoter.

30. A method of detecting a target nucleic acid in a sample comprising the steps of:

(a) providing the nucleic acid of the sample as single-stranded nucleic acid;

(b) combining with the sample (i) at least one first oligonucleotide, wherein the at least one first oligonucleotide can form a double-stranded promoter which is joined to a first single-stranded segment of nucleic acid complementary to a first segment of the target nucleic acid, and (ii) a second oligonucleotide that includes a second single-stranded segment of nucleic acid complementary to a second segment of the target nucleic acid which is adjacent to the first segment of the target nucleic acid, wherein the second oligonucleotide comprises a homopolynucleotide label;

(c) contacting the oligonucleotides comprising the first and second single-stranded segments with the sample nucleic acid and, if the target nucleic acid is present, ligating the first and second single-stranded segments; and

(d) forming the double-stranded promoter and, if the ligation of step (c) has occurred, catalyzing with an RNA polymerase the formation of a nucleic acid segment complementary to the first and second single-stranded segments and the homopolynucleotide label.