Detecting single nucleotide polymorphism using overlapping hydrolysis probes

Patent metadata

Field	Value
Publication number	US-9689026-B2
Application number	US-201615232070-A
Country	US
Kind code	B2
Filing date	Aug 9, 2016
Priority date	Nov 26, 2014
Publication date	Jun 27, 2017
Grant date	Jun 27, 2017

Abstract

Methods for the rapid detection of the presence or absence of a SNP in a target nucleic acid in a sample are described. The methods can include performing an amplifying step, a hybridizing step utilizing a double stranded probe with two overlapping SNP specific hydrolysis probe sequences where one of the probe sequences can include a hairpin structure toward the 3′ end, and a detecting step. Furthermore, the double stranded SNP specific hydrolysis probes along with kits are provided that are designed for the detection of a SNP in a target nucleic acid.

First claim

What is claimed:

1. A kit for detecting a single nucleotide polymorphism (SNP) that generates a mutant version of a target nucleic acid in a sample, comprising:
a first primer comprising a first nucleic acid sequence and a second primer comprising a second nucleic acid sequence specific to produce an amplification product comprising a sense strand and an anti-sense strand of the target nucleic acid; and
a double stranded probe comprising:
a first mutant specific hydrolysis probe comprising a third nucleic acid sequence complementary to a region of the sense strand that contains the mutant generating SNP, the first mutant specific hydrolysis probe comprising a first interactive label and a second interactive label, a first 5′ end and a first 3′ end; and
a second mutant specific hydrolysis probe comprising a fourth nucleic acid sequence complementary to a region of the anti-sense strand that contains the mutant generating SNP, the second mutant specific hydrolysis probe comprising a third interactive label and a fourth interactive label, a second 5′ end and a second 3′ end;

wherein the target nucleic acid is the rpoB gene in Mycobacterium tuberculosis.

2. The kit of claim 1 , wherein the second mutant specific hydrolysis probe comprises a hairpin structure toward the second 3′ end, the hairpin structure comprising a region of non-naturally occurring nucleic acid sequence by changing or adding at least one non-naturally occurring nucleotide into a naturally occurring sequence.

3. The kit of claim 2 , wherein the first interactive label comprises a first donor fluorescent moiety at the first 5′ terminus, and the second interactive label comprises a first corresponding acceptor fluorescent moiety within no more than 8 nucleotides of the first donor fluorescent moiety on the first mutant specific hydrolysis probe, and wherein the third interactive label comprises a second donor fluorescent moiety at the second 5′ terminus, and the fourth interactive label comprises a second corresponding acceptor fluorescent moiety within no more than 8 nucleotides of the second donor fluorescent moiety on the second mutant specific hydrolysis probe.

4. The kit of claim 2 , wherein the first acceptor fluorescent moiety is a first quencher, and wherein the second acceptor fluorescent moiety is a second quencher.

5. The kit of claim 1 , further comprising a polymerase enzyme having 5′ to 3′ nuclease activity.

6. The kit of claim 1 , wherein the first and second nucleic acid sequences of the primers and/or the third and fourth nucleic acid sequences of the hydrolysis probes comprise at least one modified nucleotide.

7. The kit of claim 1 , wherein the first and second nucleic acid sequences of the primers and/or the third and fourth nucleic acid sequences of the hydrolysis probes have 40 or fewer nucleotides.

8. The kit of claim 1 , wherein the double stranded probe comprises a pair of oligonucleotides with nucleotide sequences selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:2; SEQ ID NO:3 and SEQ ID NO:4; and SEQ ID NO:5 and SEQ ID NO:6.

9. A double stranded probe configured to detect a single nucleotide polymorphism (SNP) that generates a mutant version of a target nucleic acid, the double stranded probe comprising:
a first mutant specific hydrolysis probe comprising a first nucleic acid sequence complementary to a mutant generating SNP-containing region of a sense strand of the target nucleic acid, the first mutant specific hydrolysis probe comprising a first interactive label and a second interactive label, a first 5′ end and a first 3′ end; and
a second mutant specific hydrolysis probe comprising a second nucleic acid sequence complementary to a mutant generating containing region of an anti-sense strand of the target nucleic acid, the second SNP mutant specific hydrolysis probe comprising a third interactive label and a fourth interactive label, a second 5′ end and a second 3′ end;
wherein the target nucleic acid is the rpoB gene in Mycobacterium tuberculosis.

10. The double stranded probe of claim 9 , wherein the second mutant specific hydrolysis probe comprises a hairpin structure toward the second 3′ end, the hairpin structure comprising a region of non-naturally occurring nucleic acid sequence by changing or adding at least one non-naturally occurring nucleotide into a naturally occurring sequence.

11. The double stranded probe of claim 10 , wherein the first interactive label comprises a first donor fluorescent moiety at the first 5′ terminus, and the second interactive label comprises a first corresponding acceptor fluorescent moiety within no more than 8 nucleotides of the first donor fluorescent moiety on the first mutant specific hydrolysis probe, and wherein the third interactive label comprises a second donor fluorescent moiety at the second 5′ terminus, and the fourth interactive label comprises a second corresponding acceptor fluorescent moiety within no more than 8 nucleotides of the second donor fluorescent moiety on the second mutant specific hydrolysis probe.

12. The double stranded probe of claim 10 , wherein the first acceptor fluorescent moiety is a first quencher, and wherein the second acceptor fluorescent moiety is a second quencher.

13. The double stranded probe of claim 9 , wherein first and second nucleic acid sequences of the hydrolysis probes comprise at least one modified nucleotide.

14. The double stranded probe of claim 9 , wherein the first and second nucleic acid sequences of the hydrolysis probes have 40 or fewer nucleotides.

15. The double stranded probe of claim 9 comprising a pair of oligonucleotides with nucleotide sequences selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:2; SEQ ID NO:3 and SEQ ID NO:4; and SEQ ID NO:5 and SEQ ID NO:6.

Assignees

• Roche Molecular Systems Inc

Inventors

• Mehta Rochak

Classifications

• C12Q1/6848

  characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction · CPC title

• C12Q1/6827 Primary

  for detection of mutation or polymorphism · CPC title

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title

• C12Q1/6818

  involving interaction of two or more labels, e.g. resonant energy transfer · CPC title

• C12Q1/6851

  Quantitative amplification · CPC title