Use of G-clamp for improved allele-specific PCR

• US 9,476,091 B2
• Filed: 09/21/2012
• Issued: 10/25/2016
• Est. Priority Date: 09/23/2011
• Status: Active Grant

• Current Assignee: Roche Molecular Systems Inc (Roche Holding AG)
• Original Assignee: Roche Molecular Systems Inc (Roche Holding AG)
• Inventors: Bodepudi, Veeraiah, Schoenbrunner, Nancy, Tsan, Alison
• Primary Examiner(s): Kim, Young J
• Application Number:
  US13/624,077
• Publication Number:
  US 20130078630A1
• Time in Patent Office: 1,495 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

The present invention includes a method of allele-specific amplification, utilizing an allele-specific oligonucleotide, at least partially complementary to more than one variant of the target sequence, but having at least one selective nucleotide complementary to only one variant of the target sequence and incorporating at least one “G-clamp” nucleotide.

Claims

1. A method of allele-specific amplification of a variant of a target sequence, the target existing in the form of several variant sequences, the method comprising:

(a) hybridizing a first and a second oligonucleotides to at least one variant of the target sequence;

wherein the first oligonucleotide is at least partially complementary to one or more variants of the target sequence, and the second oligonucleotide is at least partially complementary to one or more variants of the target sequence, and has at least one selective nucleotide at the 3′

terminal nucleotide that is complementary to only one variant of the target sequence;

wherein said second oligonucleotide incorporates at least one G-clamp nucleotide at a position between 1 and 5 nucleotide upstream of the 3′

terminal nucleotide;

(b) extending the second oligonucleotide with a nucleic acid polymerase, wherein said polymerase is capable of extending said second oligonucleotide efficiently when said second oligonucleotide is hybridized to a variant of the target sequence which is complementary to the one selective nucleotide at the 3′

terminus, and substantially less efficiently when said second oligonucleotide is hybridized to a variant of the target sequence which is not complementary to the one selective nucleotide at the 3′

terminus.

2. The method of claim 1, further comprising a step (c) of detecting the product of primer extension of step (b).

3. The method of claim 1, wherein said nucleic acid polymerase is selected from a group consisting of Taq DNA polymerase, Z05 DNA polymerase, Δ

Z05 DNA polymerase and Δ

Z05-Gold DNA polymerase.

4. The method of claim 1 wherein said nucleic acid polymerase possesses 3′

-5′

nuclease activity.

5. The method of claim 4, wherein said nucleic acid polymerase is selected from a group consisting of Pfu DNA polymerase and Thermatoga Maritima.

6. The method of claim 1, wherein said variant of the sequence in step (a) is a mutation of the human PIK3CA or EGFR gene.

7. The method of claim 1, wherein said second oligonucleotide is selected from a group consisting of SEQ ID NO:

6, 7, and 12.

8. An oligonucleotide for performing an allele-specific amplification of a target sequence, said target existing in the form of several variant sequences, the oligonucleotide comprising

(a) a sequence at least partially complementary to a portion of one or more variants of said target sequence;

(b) one selective nucleotide at the 3′

terminal nucleotide that is complementary to only one variant of the target sequence; and

(c) at least one G-clamp nucleotide at a position between 1 and 5 nucleotides upstream of the 3′

terminal nucleotide.

9. The oligonucleotide of claim 8 with a sequence selected from a group consisting of SEQ ID NO:

6, 7 and 12.