Primers with modified phosphate and base in allele-specific PCR

• US 9,382,581 B2
• Filed: 11/27/2013
• Issued: 07/05/2016
• Est. Priority Date: 12/13/2012
• Status: Active Grant

• Current Assignee: Roche Molecular Systems Inc (Roche Holding AG)
• Original Assignee: Roche Molecular Systems Inc (Roche Holding AG)
• Inventors: Froehner, Stefanie, Heindl, Dieter, Kessler, Dirk, Schoenbrunner, Nancy, Tsan, Alison
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US14/092,133
• Publication Number:
  US 20140170650A1
• Time in Patent Office: 951 Days
• Field of Search: 435/6.1, 435/91.1
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification

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 both a nucleotide with a base covalently modified at the exocyclic amino group and a modified phosphate.

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 oligonucleotide and a second oligonucleotide 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 complementary to only one variant of the target sequence;

wherein said second oligonucleotide comprises a modified phosphate having a structure;

3. The method of claim 1, wherein said nucleotide with said base covalently modified at the exocyclic amino group occurs at positions -5, -4, -3, -2 or -1 relative to the 3′

-terminal nucleotide.

4. The method of claim 3, wherein said base covalently modified at the exocyclic amino group is selected from a group consisting of N6-benzyl-adenine, N6-para-tert-butyl-benzyl adenine, N4-para-tert-butyl-benzyl cytosine, N4-ethyl-cytosine, and N4-benzyl-cytosine.

5. The method of claim 1, wherein said variant of the target sequence is a mutation of the human EGFR gene.

6. The method of claim 5 wherein said second oligonucleotide is selected from a group consisting of SEQ ID NO:

7, 13, 14, 22.

2. A method of detecting a variant of a target sequence, the target existing in the form of several variant sequences, the method comprising:

(a) hybridizing a first oligonucleotide and a second oligonucleotide 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 complementary to only one variant of the target sequence;

wherein said second oligonucleotide comprises a modified phosphate having a structure;