Format of probes to detect nucleic acid differences

• US 8,409,802 B2
• Filed: 08/09/2010
• Issued: 04/02/2013
• Est. Priority Date: 08/14/2009
• Status: Active Grant

• Current Assignee: Roche Molecular Systems (Roche Holding AG)
• Original Assignee: Roche Molecular Systems (Roche Holding AG)
• Inventors: Will, Stephen G.
• Primary Examiner(s): Lu, Frank W
• Application Number:
  US12/852,936
• Publication Number:
  US 20110039264A1
• Time in Patent Office: 967 Days
• Field of Search: 435/6.1, 435/6.11, 435/6.12, 435/91.1, 435/91.2, 435/183, 436/94, 436/501, 536/23.1, 536/24.3, 536/24.33, 536/25.3
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2535/131 Allele specific probes

Abstract

The invention provides, inter alia, novel probes, methods, reaction mixtures, and kits for detecting the presence or absence of a target nucleic acid sequence.

Claims

1. A method of detecting the presence or absence of a target nucleic acid in a biological sample, the method comprising:

a) contacting a detectably-labeled probe comprising an anchor nucleic acid domain and a reporter nucleic acid domain with the sample; and

b) detecting the presence or absence of binding of the probe to the target nucleic acid,

wherein the anchor and reporter domains are linked by a non-nucleoside linker, and neither the anchor nor the reporter domain forms a stem loop in the absence of the target nucleic acid; and

wherein the probe is not extendible by a polymerase; and

the linker is linked to the anchor domain within 2 nucleotides of the 3′

end of the anchor domain and the linker is linked to the reporter domain within 2 nucleotides of the 5′

end of the reporter domain, wherein the anchor domain is not linked to a detectable label; and

each of the anchor domain and the reporter domain comprises a contiguous sequence of at least 6 nucleotides complementary to the same strand of the target nucleic acid.

2. The method of claim 1, wherein each of the anchor domain and the reporter domain comprises a contiguous sequence of at least 10 nucleotides complementary to the same strand of the target nucleic acid.

3. The method of claim 1 further comprising measuring the melting temperature of a complex formed between the reporter domain and the target nucleic acid when the target nucleic acid is detected in the biological sample.

4. The method of claim 1, wherein the length of the reporter domain is between 4 to 20 nucleotides.

5. The method of claim 1, wherein the anchor region is between 6-40 nucleotides.

6. The method of claim 1, wherein the detectably-labeled probe comprises a fluorescent label and further comprises contacting the probe and sample with a soluble intercalating quencher such that the quencher alters fluorescence from the label when the reporter domain forms a complex with the target nucleic acid and the target nucleic acid is in the biological sample.

7. The method of claim 1, wherein the target nucleic acid comprises a single nucleotide polymorphism (SNP) and the reporter domain is 100% complementary to the allele of the SNP.

8. The method of claim 1, wherein the reporter domain of the probe comprises at least one non-natural nucleotide and the non-natural nucleotide increases the melting temperature of the reporter domain.

9. The method of claim 1, wherein the linker is polyethylene glycol.

10. The method of claim 9, wherein the linker is hexaethylene glycol.