Nucleic acid amplification with allele-specific suppression of sequence variants

• US 8,206,929 B2
• Filed: 01/06/2010
• Issued: 06/26/2012
• Est. Priority Date: 01/07/2009
• Status: Active Grant

• Current Assignee: Roche Molecular Systems (Roche Holding AG)
• Original Assignee: Roche Molecular Systems (Roche Holding AG)
• Inventors: Grow, Michael, Brophy, Victoria
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US12/683,111
• Publication Number:
  US 20100173311A1
• Time in Patent Office: 902 Days
• Field of Search: 435/6.12, 435/91.2
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2535/131 Allele specific probes
• CPC Class Codes: C12Q 2537/159 Reduction of complexity, e.g. amplification of subsets, removing duplicated genomic regions
• CPC Class Codes: C12Q 2549/101 Hot start

Abstract

The present invention is an improved method of selective amplification of certain variants of the target sequence, enhanced by allele-specific suppression of amplification of one or more of the other variants of the target sequence. The improvement is accomplished by providing an oligonucleotide, capable of hybridizing to the desired variant of the target sequence with the lesser affinity than to the undesired variants of the target sequence and optionally, by providing chemically modified primers and hot-start conditions.

Claims

1. A method of selective amplification of a desired variant of a target sequence, which target sequence exists in the form of more than one variant, the method comprising the steps of:

a) providing a sample possibly comprising at least one variant of the target sequence in a reaction mixture;

b) providing a first oligonucleotide, capable of hybridizing to more than one variant of the target sequence;

c) providing a second oligonucleotide, capable of hybridizing to more than one variant of the target sequence, wherein at least a fraction of said second oligonucleotide contains a modified base in one or more nucleotides at or near the 3′

-terminus;

d) providing a third oligonucleotide, capable of hybridizing to the desired variant of the target sequence with lesser affinity than to an undesired variant of the target sequence and designed to hybridize to the same strand and between 0 and 60 nucleotides downstream of said second oligonucleotide;

e) providing a nucleic acid polymerase substantially lacking 5′

-3′

nuclease activity;

f) subjecting said reaction mixture to polymerase chain reaction, wherein said third oligonucleotide substantially inhibits extension of said second oligonucleotide by said nucleic acid polymerase when said third oligonucleotide is hybridized to the undesired variant of the target sequence, but does not substantially inhibit extension of said second oligonucleotide by said nucleic acid polymerase when said third oligonucleotide is hybridized to the desired variant of the target sequence.

2. The method of claim 1, wherein the amounts of said first and second oligonucleotides are unequal, such that the first oligonucleotide is present in excess.

3. The method of claim 1, wherein said modified base in said second oligonucleotide is modified at an exocyclic amino group.

4. The method of claim 1, wherein at least one of said first, second and third oligonucleotides is labeled.

5. The method of claim 1 further comprising detection of the amplified nucleic acid sequence.

6. The method of claim 1, wherein the first oligonucleotide is SEQ ID NO:

2.

7. The method of claim 1, wherein the second oligonucleotide is SEQ ID NO:

3.

8. The method of claim 1, wherein the third oligonucleotide is SEQ ID NO:

5.

9. The method of claim 1, wherein said nucleic acid polymerase possesses a hot-start capability.