Oligonucleotide probe/primer methods for polynucleotide detection

• US 20080081335A1
• Filed: 06/28/2007
• Published: 04/03/2008
• Est. Priority Date: 05/02/2005
• Status: Abandoned Application

• Current Assignee: Stratagene California (Agilent Technologies, Inc.)
• Original Assignee: Stratagene California (Agilent Technologies, Inc.)
• Inventors: Sorge, Joseph
• Application Number:
  US11/823,630
• Publication Number:
  US 20080081335A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 2521/325 Single stranded exonuclease
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2531/113 PCR

Abstract

The invention is related to a labeled oligonucleotide pair for detecting a target nucleic acid and methods, kits and compositions containing the labeled oligonucleotide pair. The labeled oligonucleotide pair forms a complex comprising a nucleic acid primer and a nucleic acid probe.

Claims

1. A method of detecting a target nucleic acid in a sample, said method comprising:

(a) contacting a target nucleic acid with a PCR reaction mixture comprising a labeled oligonucleotide pair having a nucleic acid primer, a nucleic acid probe and a pair of interactive labels, wherein a first member of said pair of interactive labels is coupled to said nucleic acid primer and a second member of said interactive pair of labels is coupled to said nucleic acid probe, and wherein when said primer and said probe form a hybrid said labels interact, and when said primer and probe are dissociated said labels do not interact; and

b. permitting the cleavage of said nucleic acid probe when said nucleic acid primer is hybridized to said target nucleic acid so as to generate a detectable signal, wherein said signal is indicative of the presence of said target nucleic acid in the nucleic acid sample.

3. The method of claim 1 or 2, further comprising providing a nucleic acid polymerase

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

to 3′

nuclease activity.

5. The method of claim 4, further comprising a nuclease.

6. The method of claim 5, wherein said nuclease is an exonuclease.

7. The method of claim 5, wherein said nuclease is a FEN nuclease.

8. The method of claim 1 or 2, wherein said primer comprises a first portion and a second portion, wherein said first portion is complementary to said target nucleic acid and said second portion is complementary to said nucleic acid probe and is not complementary to said target nucleic acid.

9. The method of claim 8, wherein said probe comprises a portion complementary to said second portion of said nucleic acid primer, but not comprising a portion complementary to said first portion of said nucleic acid primer.

2. A method of detecting a target nucleic acid in a sample, said method comprising:

a. performing a PCR amplification reaction with a PCR amplification reaction mixture comprising a target nucleic acid, and a labeled oligonucleotide pair comprising a nucleic acid primer, a nucleic acid probe which is complementary to said nucleic acid primer, and a pair of interactive labels;

b. performing a nuclease cleavage reaction; and

c. detecting a signal generated by a member of said pair of interactive labels, wherein said signal is indicative of the presence of the target nucleic acid.