Detecting single nucleotide polymorphism using hydrolysis probes with 3′ hairpin structure

• US 9,297,033 B2
• Filed: 12/13/2013
• Issued: 03/29/2016
• Est. Priority Date: 12/13/2013
• Status: Active Grant

• Current Assignee: Roche Molecular Systems (Roche Holding AG)
• Original Assignee: Roche Molecular Systems (Roche Holding AG)
• Inventors: Mehta, Rochak
• Primary Examiner(s): Lu, Frank
• Application Number:
  US14/106,456
• Publication Number:
  US 20150167059A1
• Time in Patent Office: 837 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: 1/1
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• 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 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2561/101 Taqman
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

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 SNP specific hydrolysis probe including a hairpin structure toward the 3′ end, and a detecting step. Furthermore, SNP specific hydrolysis probe including a hairpin structure toward the 3′ end, along with kits are provided that are designed for the detection of a SNP in a target nucleic acid.

Claims

1. A method for detecting a single nucleotide polymorphism (SNP) in a target nucleic acid in a sample, the method comprising:

performing an amplifying step comprising contacting the sample with a primer comprising a first nucleic acid sequence to produce an amplification product comprising a region containing the SNP if the target nucleic acid is present in the sample;

adding a SNP specific hydrolysis probe comprising a second nucleic acid sequence complementary to the region containing the SNP of the amplification product into the sample and performing a hybridizing step comprising contacting the amplification product with the SNP specific hydrolysis probe if the target nucleic acid is present in the sample, the SNP specific hydrolysis probe comprising a donor fluorescent moiety and an acceptor moiety of the donor fluorescent moiety, a 5′

end and a 3′

end, and a hairpin structure located on the 3′

end, the hairpin structure comprising a region of non-naturally occurring nucleic acid sequence which is located on the 3′

end of the hairpin structure and comprises one or more non-naturally occurring nucleotides, wherein the acceptor moiety is in an internal position of the SNP specific hydrolysis probe; and

detecting the presence or absence of the amplification product, wherein the presence of the amplification products is indicative of the presence of the SNP in the target nucleic acid.

2. The method of claim 1, wherein the donor fluorescent moiety is at the 5′

end, and the acceptor moiety is within no more than 5 nucleotides from the donor fluorescent moiety on the hydrolysis probe.

3. The method of claim 1, wherein the acceptor moiety is a quencher.

4. The method of claim 1, wherein the amplifying step employs a polymerase enzyme having 5′

to 3′

exonuclease activity.

5. The method of claim 1, wherein the first nucleic acid sequence of the primer and/or the second nucleic acid sequence of the hydrolysis probe comprises/comprise at least one modified nucleotide.

6. The method of claim 1, wherein the first nucleic acid sequence of the primer and/or the second nucleic acid sequence of the hydrolysis probe has/have 40 or fewer nucleotides.