Methods of detection of a target nucleic acid sequence

• US 7,736,854 B2
• Filed: 11/29/2006
• Issued: 06/15/2010
• Est. Priority Date: 10/29/1999
• Status: Expired due to Fees

• Current Assignee: Hologic Incorporated
• Original Assignee: Hologic Incorporated
• Inventors: Sorge, Joseph A.
• Primary Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US11/606,518
• Publication Number:
  US 20070292863A1
• Time in Patent Office: 1,294 Days
• Field of Search: 435/6, 435/91.2, 536/23.1, 536/24.3
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/307 Single strand endonuclease
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2561/101 Taqman
• CPC Class Codes: C12Q 2561/109 Invader technology
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide

Abstract

The present invention is directed to methods of generating a signal indicative of the presence of a target nucleic acid in a sample by forming a cleavage structure comprising duplex and single-stranded nucleic acid by incubating a sample comprising a target nucleic acid with a thermostable nucleic acid polymerase substantially lacking 5′ to 3′ exonuclease activity and cleaving said cleavage structure with a thermostable Fen nuclease lacking 5′ to 3′ synthetic activity to generate a signal.

Claims

1. A method of generating a signal indicative of the presence of a target nucleic acid in a sample, comprising:

in the same reaction vessel, forming a cleavage structure comprising duplex and single-stranded nucleic acid by incubating a sample comprising a target nucleic acid with a thermostable nucleic acid polymerase substantially lacking 5′

to 3′

exonuclease activity and cleaving said cleavage structure with a thermostable Fen nuclease lacking 5′

to 3′

synthetic activity to generate a signal, wherein generation of said signal is indicative of the presence of a target nucleic acid in said sample.

3. The method of claim 1 or 2, wherein said method comprises, in said reaction vessel, a pair of amplification primers complementary with said target nucleic acid to permit amplification of a sequence of said target nucleic acid between said primers.

4. The method of claim 3, wherein said method comprises, in said reaction vessel, one or more dNTPs.

5. The method of claim 3, wherein said method comprises, in said reaction vessel, an oligonucleotide that specifically hybridizes to a region of the target nucleic acid between said amplification primers.

6. The method of claims 1 or 2, wherein said method comprises, in said reaction vessel, said thermostable Fen nuclease selected from the group consisting of Fen nuclease enzyme of Archaeglobus fulgidus, Methanococcus jannaschii, Pyrococcus furiosus, and Pyrococcus horikoshii.

7. The method of claims 1 or 2, wherein said method comprises, in said reaction vessel, a thermostable polymerase selected from the group consisting of Pfu, Pfu-exo, Taq, Taq-exo, Deep vent and Deep vent exo.

8. The method of claims 1 or 2, wherein a cleavage structure is formed prior to the addition of said Fen nuclease, said cleavage structure comprising a 5′

flap, and said Fen nuclease cleaves said 5′

flap of said cleavage structure.

9. The method of claim 8, wherein said 5′

flap comprises a label comprising a first member of a pair of interactive labels effectively positioned to quench the generation of a detectable signal, the first and second members of said pair being separable 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 signal.

10. The method of claim 9, wherein said pair of interactive labels comprises a quencher moiety and a fluorescent moiety.

11. The method of claim 3, wherein said amplification primers are between about 10 to 100 nucleotides in length.

12. The method of claim 3, wherein said amplification primers are between about 17-50 nucleotides in length.

13. The method of claim 3, wherein said amplification primers are between about 17-45 nucleotides in length.

14. The method of claim 1, wherein said Fen nuclease is a Fen-1 nuclease.

2. A method of detecting and/or measuring a target nucleic acid in a sample, comprising:

in the same reaction vessel, forming a cleavage structure comprising duplex and single-stranded nucleic acid, wherein said single stranded nucleic acid comprises a 5′

flap, by incubating a sample comprising a target nucleic acid with a thermostable nucleic acid polymerase substantially lacking 5′

to 3′

exonuclease activity and cleaving said cleavage structure with a thermostable Fen nuclease substantially lacking 5′

to 3′

synthetic activity to release a nucleic acid fragment, anti detecting and/or measuring the release of said fragment as an indication of the presence of the target in the sample.