Methods for detection of a target nucleic acid sequence

• US 6,548,250 B1
• Filed: 08/30/2000
• Issued: 04/15/2003
• Est. Priority Date: 10/29/1999
• Status: Expired due to Term

• Current Assignee: Hologic Incorporated
• Original Assignee: Stratagene California
• Inventors: Sorge, Joseph A.
• Primary Examiner(s): FREDMAN, JEFFREY NORMAN
• Assistant Examiner(s): Taylor, Janell E.
• Application Number:
  US09/650,888
• Time in Patent Office: 958 Days
• Field of Search: 435/6, 435/4, 435/18, 435/91.53, 435/183, 435/194, 435/195, 435/196, 435/810, 435/822, 436/94, 530/350
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2561/109 Invader technology
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 435/822 using bacteria or actinomycetales
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The invention relates to a method of generating a signal indicative of the presence of a target nucleic acid sequence in a sample, where the method includes forming a cleavage structure by incubating a sample containing a target nucleic acid sequence with a nucleic acid polymerase and cleaving the cleavage structure with a FEN nuclease to generate a cleaved nucleic acid fragment. The invention also relates to methods of detecting or measuring a target nucleic acid sequence, where the method includes forming a cleavage structure by incubating a target nucleic acid sequence with a nucleic acid polymerase, cleaving the cleavage structure with a FEN nuclease and detecting or measuring the release of a fragment.

Claims

1. A method of generating a signal indicative of the presence of a target nucleic acid sequence in a sample, comprising forming a cleavage structure comprising duplex and single-stranded nucleic acid, wherein said single stranded nucleic acid comprises a flap, by incubating a sample comprising a target nucleic acid sequence with a nucleic acid polymerase and cleaving said cleavage structure with a FEN nuclease to generate a signal, wherein generation of said signal is indicative of the presence of a target nucleic acid sequence in said sample.

2. The method of claim 1 wherein said nucleic acid polymerase substantially lacks 5′

to 3′

exonuclease activity.

5. The method of claim 1 or 3 wherein the nucleic acid polymerase is a DNA polymerase.

6. The method of claim 1 or 3 wherein the nucleic acid polymerase is thermostable.

7. The method of claim 1 or 3 wherein the FEN nuclease is a flap-specific nuclease.

8. The method of claim 1 or 3 wherein the FEN nuclease is thermostable.

9. The method of claim 1 or 3 wherein a cleavage structure is formed comprising at least one labeled moiety capable of providing a signal.

10. The method of claim 1 or 3 wherein a cleavage structure is formed comprising a pair of interactive signal generating labeled moieties effectively positioned to quench the generation of a detectable signal, said labeled moieties being separated by a site susceptible to FEN nuclease cleavage, thereby allowing the nuclease activity of the FEN nuclease to separate the first interactive signal generating labeled moiety from the second interactive signal generating labeled moiety by cleaving at said site susceptible to FEN nuclease, thereby generating a detectable signal.

11. The method of claim 10 wherein said pair of interactive signal generating moieties comprises a quencher moiety and a fluorescent moiety.

12. The method of claim 1 wherein a cleavage structure comprises at least one oligonucleotide primer.

3. A method of detecting or measuring a target nucleic acid sequence comprising forming a cleavage structure comprising duplex and single-stranded nucleic acid, wherein said single stranded nucleic acid comprises a flap, by incubating a sample comprising a target nucleic acid sequence with a nucleic acid polymerase, cleaving said cleavage structure with a FEN nuclease to release a nucleic acid fragment and detecting and/or measuring the release of said fragment as an indication of the presence of the target sequence in the sample.

4. The method of claim 3 wherein said nucleic acid polymerase substantially lacks 5′

to 3′

exonuclease activity.

13. A polymerase chain reaction process for detecting a target nucleic acid sequence in a sample comprising providing a cleavage structure comprising duplex and single-stranded nucleic acid, wherein said single stranded nucleic acid comprises a flap, providing a set of oligonucleotide primers wherein a first primer contains a sequence complementary to a region in one strand of the target nucleic acid sequence and primes the synthesis of a complementary DNA strand, and a second primer contains a sequence complementary to a region in a second strand of the target nucleic acid sequence and primes the synthesis of a complementary DNA strand and amplifying the target nucleic acid sequence employing a nucleic acid polymerase as a template-dependent polymerizing agent under conditions which are permissive for PCR cycling steps of (i) annealing of primers required for amplification to a template nucleic acid sequence contained within the target nucleic acid sequence, (ii) extending the primers wherein said nucleic acid polymerase synthesizes a primer extension product, and (iii) cleaving said cleavage structure employing a FEN nuclease as a cleavage agent for release of labeled fragments from said cleavage structure thereby creating detectable labeled fragments and detecting and/or measuring the release of labeled fragments as an indication of the presence of the target sequence in the sample.

14. The polymerase chain reaction process of claim 13 wherein said nucleic acid polymerase substantially lacks 5′

to 3′

exonuclease activity.

15. The method of claim 13 wherein said oligonucleotide primers of step b are oriented such that the forward primer is located upstream of said cleavage structure and the reverse primer is located downstream of said cleavage structure.

16. The polymerase chain reaction process of claim 13 wherein the nucleic acid polymerase is a DNA polymerase.

17. The polymerase chain reaction process of claim 13 wherein the nucleic acid polymerase is thermostable.

18. The polymerase chain reaction process of claim 13 wherein the nucleic acid polymerase is selected from the group consisting of 5′

to 3′

exonuclease deficient Taq polymerase and Pfu polymerase.

19. The polymerase chain reaction process of claim 13 wherein the FEN nuclease is thermostable.

20. The polymerase chain reaction process of claim 13 wherein the FEN nuclease is a flap-specific nuclease.

21. The polymerase chain reaction process of claim 13 wherein the FEN nuclease is selected from the group consisting of FEN nuclease enzyme derived from Archaeglobus fulgidus, Methanococcus jannaschii, Pyrococcus furiosus, Taq, Tfl and Bca.

22. The polymerase chain reaction process of claim 13 wherein the labeled cleavage structure is formed by the addition of at least one labeled moiety capable of providing a signal.

23. A polymerase chain reaction process for simultaneously forming a cleavage structure comprising duplex and single-stranded nucleic acid, wherein said single stranded nucleic acid comprises a flap, amplifying a target nucleic acid sequence in a sample and cleaving said cleavage structure comprising:

(a) providing an upstream oligonucleotide primer complementary to a region in one strand of the target nucleic acid sequence and a downstream labeled probe complementary to a region in the same strand of the target nucleic acid sequence, wherein the upstream primer contains a sequence complementary to a region in one strand of the target nucleic acid sequence and primes the synthesis of a complementary DNA strand, and the downstream probe contains a sequence complementary to a region in a second strand of the target nucleic acid sequence and primes the synthesis of a complementary DNA strand; and

(b) detecting a nucleic acid which is produced in a reaction comprising amplification of said target nucleic acid sequence and cleavage thereof wherein a nucleic acid polymerase is a template-dependent polymerizing agent under conditions which are permissive for PCR cycling steps of (i) annealing of primers to a target nucleic acid sequence, (ii) extending the primers of step (a) wherein said nucleic acid polymerase synthesizes primer extension products, and wherein the primer extension product of the primer of step (a) partially displaces the downstream probe of step (a) to form a cleavage structure; and

(iii) cleaving said cleavage structure employing a FEN nuclease as a cleavage agent for release of labeled fragments from said cleavage structure thereby creating detectable labeled fragments.

24. The polymerase chain reaction process of claim 23 wherein said nucleic acid polymerase substantially lacks 5′

to 3′

exonuclease activity.

25. A method of forming a cleavage structure comprising duplex and single-stranded nucleic acid, wherein said single stranded nucleic acid comprises a flap, comprising providing a target nucleic acid sequence, providing an upstream primer complementary to said target nucleic acid sequence, providing a downstream probe complementary to said target nucleic acid sequence, extending the 3′

end of the upstream primer with a nucleic acid polymerase that substantially lacks 5′

to 3′

exonuclease activity; and

displacing the 5′

end of the downstream probe.

26. A composition comprising a nucleic acid polymerase wherein said nucleic acid polymerase substantially lacks 5′

to 3′

exonuclease activity, and a FEN nuclease.

27. A kit for generating a signal indicative of the presence of a target nucleic acid sequence in a sample, comprising, a FEN nuclease, a labeled nucleic acid complementary to said target nucleic acid sequence, a suitable buffer and a nucleic acid polymerase substantially lacking 5′

to 3′

exonuclease activity, wherein said FEN nuclease, said labeled nucleic acid complementary to said target nucleic acid sequence, said suitable buffer and said nucleic acid polymerase are in the same composition.

28. The kit of claim 27 wherein said nucleic acid polymerase is thermostable.

29. The kit of claim 27 wherein said FEN nuclease is thermostable.