Methods, compositions, and kits for rare allele detection

• US 8,815,515 B1
• Filed: 09/24/2010
• Issued: 08/26/2014
• Est. Priority Date: 09/24/2009
• Status: Active Grant

• Current Assignee: University of Utah Research Foundation (State of Utah)
• Original Assignee: University of Utah Research Foundation (State of Utah)
• Inventors: Zhou, Luming
• Primary Examiner(s): Kim, Young J
• Application Number:
  US12/890,041
• Time in Patent Office: 1,432 Days
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2535/137 Amplification Refractory Mutation System [ARMS]

Abstract

Methods are provided for nucleic acid analysis. In an illustrative method, Snapback-ARMS primers are used to amplify preferentially a target nucleic acid that is present in a low allele fraction. In another embodiment, tailed primers are used to identify the preferentially amplified allele.

Claims

1. A method for detecting a minor allele in a sample comprising,

amplifying DNA comprising the minor allele and a major allele, wherein the minor allele is present in a lower concentration than the major allele, and the minor allele differs from the major allele by a nucleotide, wherein the amplifying step is performed using

a pair of primers comprising an ARMS primer, wherein the ARMS primer is a Snapback ARMS primer, and a second primer, wherein a 3′

terminal nucleotide of the ARMS primer is complementary to the nucleotide of the minor allele and is not complementary to the nucleotide of the major allele, and

a probe, wherein the probe is a probe element located as a 5′

end of the ARMS primer, wherein the probe has a nucleotide that is complementary to the nucleotide of the major allele and is not complementary to the minor allele, and

detecting the minor allele using the probe under annealing or melting conditions.

2. The method of claim 1, wherein

the amplifying step is performed using an additional Snapback ARMS primer, wherein the additional Snapback ARMS primer comprises a 3′

nucleotide that matches a third allele and a probe element located at the 5′

end that is different from the probe element of the Snapback ARMS primer, and

the detecting step comprises generating a melt curve and determining whether the melt curve matches the 5′

element of the Snapback ARMS primer or the 5′

element of the additional Snapback ARMS primer.

3. The method of claim 1, wherein the ARMS primer is provided at a concentration of less than 0.5 μ

M.

4. The method of claim 3 wherein the ARMS primer is provided at a concentration of about 0.05 μ

M.

5. The method of claim 3, wherein the ARMS primer is provided at a concentration lower than the second primer.

6. The method of claim 5, wherein the second primer is provided at a concentration about 10 times of the ARMS primer.

7. The method of claim 1 wherein the amplifying step includes using an annealing temperature that is between a Tm of the minor allele and the probe and a Tm of the major allele and the probe.