Continuous amplification reaction

• US 5,981,179 A
• Filed: 09/14/1995
• Issued: 11/09/1999
• Est. Priority Date: 11/14/1991
• Status: Expired due to Term

• Current Assignee: Digene Corporation (Qiagen NV)
• Original Assignee: Digene Diagnostics, Inc.
• Inventors: DeLaRosa, Abel, Lorinez, Attila T.
• Primary Examiner(s): Zitomer, Stephanie W.
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US08/527,864
• Time in Patent Office: 1,517 Days
• Field of Search: 435/91.2, 435/6, 435/183, 435/195, 536/23.1, 536/25.32, 536/24.3, 536/22.1
• US Class Current: 435/6.18
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• 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]
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/143 incorporating a promoter sequence
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2537/125 Sandwich assay format
• CPC Class Codes: C12Q 2537/137 a displacement step

Abstract

Continuous amplification reaction provide a method of amplifying a specific nucleic acid without the need to cycle a reaction. The method produces RNA transcripts which can be detected by a variety of methods. Amplification and detection kits are also provided.

Claims

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

providing a single-stranded nucleic acid containing a target region;

hybridizing the target region of said nucleic acid to a promoter-primer having a central promoter portion and two regions homologous to non-contiguous portions of the target region forming a circular hybrid;

trimming back single-stranded sequence 3'"'"'to the target region generating a flushed 3'"'"' end of said hybrid;

extending 3'"'"' ends of the target region and the promoter-primer forming a double-stranded intermediate; and

transcribing the double-stranded intermediate producing many RNA transcripts from each target region.

2. A method according to claim 1 further comprising:

denaturing a double stranded nucleic acid, thereby providing a single-stranded nucleic acid.

3. A method according to claim 2 wherein denaturing comprises:

treating said nucleic acid with a base; and

neutralizing with a hybridization neutralization buffer.

4. A method according to claim 1 wherein the single stranded nucleic acid is hybridized to a capture probe.

5. A method according to claim 1 wherein the trimming and extending steps are carried out using at least one DNA polymerase having exonuclease activity.

6. A method according to claim 1 wherein the trimming and extending steps are carried out using at least one DNA polymerase and at least one exonuclease.

7. A method according to claim 1 wherein the promoter-primer comprises modified nucleotides or phosphodiester linkages in at least an ultimate 3'"'"' end position.

8. A method of detecting a nucleic acid containing a target region comprising the steps of:

providing a single-stranded nucleic acid comprising said target region;

hybridizing said nucleic acid to a single stranded promoter-primer having a 5'"'"' primer portion and a 3'"'"' promoter portion, said 3'"'"' primer portion comprising a sequence complementary to a 3'"'"' portion of said target region, said 3'"'"' primer portion further comprising at least one modified nucleotide or modified phosphodiester linkage to resist exonuclease activity, to form a hybrid;

trimming back single-stranded sequence 3'"'"' to the target with an exonuclease activity, thereby generating a flushed 3'"'"' end of said hybrid;

extending 3'"'"' ends of said target region and the promoter-primer forming a double-stranded intermediate;

transcribing the double-stranded intermediate producing many RNA transcripts from each target region; and

detecting RNA transcripts.

9. A method according to claim 8 further comprising:

denaturing a double stranded nucleic acid, thereby providing the single-stranded nucleic acid.

10. A method according to claim 9 wherein denaturing comprises:

treating said nucleic acid with a base; and

neutralizing with a hybridization neutralization buffer.

11. A method according to claim 8 wherein the single stranded nucleic acid is hybridized to a capture probe.

12. A method according to claim 8 wherein the trimming and extending steps are carried out using at least one DNA polymerase having exonuclease activity.

13. A method according to claim 8 wherein the trimming and extending steps are carried out using at least one DNA polymerase and at least one exonuclease.

14. The method of claim 8 wherein the RNA transcripts are detected by a method comprising the steps of:

a--hybridizing the RNA transcript to a complementary DNA probe to form a double-stranded hybrid;

b--capturing the hybrid of step (a) onto a solid phase to form a bound hybrid;

c--eliminating non-hybridized nucleic acid; and

d--detecting the bound hybrid.

15. The method of claim 14 wherein the complementary DNA probe is labelled with at least one ligand.

16. The method of claim 14 wherein the DNA probe is biotinylated and the solid phase is coated with streptavidin.

17. The method of claim 14 wherein an anti-hybrid antibody or anti-hybrid fragment has been immobilized to the solid phase, wherein the antibody or antibody fragment specifically binds to a component of the double-stranded hybrid.

18. The method of claim 14 wherein the probe is double-stranded DNA, comprising the additional step of treating the probe with a base to form single-stranded DNA prior to the hybridization step.

19. A kit for amplification of nucleic acid in a biological sample comprising:

a promoter-primer having a central promoter portion and two regions homologous to non-contiguous sequences in the target region;

a trimming back agent;

a nucleic acid polymerase;

an RNA polymerase; and

a sample transport medium for stabilization of the biological sample.

21. A kit for amplification and detection of nucleic acid in a biological sample comprising the components of claims 19 or 20, further comprising the components of:

a DNA detection probe complementary to the target RNA sequence for formation of a double-stranded nucleic acid hybrid;

a neutralizing probe diluent for diluting and neutralizing the treated probe;

a solid phase coated with a coating to which a hybrid formed by hybridization of the probe and the target nucleic acid sequence, will bind; and

means for detecting the hybrid formed by hybridization of the probe and the target nucleic acid sequence.

22. The kit of claim 21 wherein the solid phase is coated with streptavidin and the probe is biotinylated.

23. The kit of claim 21 wherein the solid phase is coated with an anti-hybrid antibody or an anti-hybrid antibody fragment, wherein the antibody or antibody fragment specifically binds to a component of the double-stranded hybrid.

24. The kit of claim 21 further comprising a means for eliminating non-hybridized probe.

25. The kit of claim 21 wherein the detecting means is an anti-hybrid antibody or an anti-hybrid antibody fragment, wherein the antibody or antibody fragment specifically binds to a component of the double-stranded hybrid and is detectable.

26. The kit according to claim 24 wherein the means for eliminating non-hybridized probe is DNase.

20. A kit for amplification of nucleic acid in a biological sample comprising:

a promoter-primer having a 5'"'"' promoter portion and a 3'"'"' primer portion, said primer portion comprising a sequence complementary to a 3'"'"' portion of said target region, said promoter-primer further comprising at least one modified nucleotide or phosphodiester linkage to resist exonuclease activity;

a trimming back agent comprising an exonuclease activity;

a nucleic acid polymerase;

a RNA polymerase; and

a sample transport medium for stabilization of the biological sample.