Pyrophosphorolysis activated polymerization (PAP): application to allele-specific amplification and nucleic acid sequence determination

• US 20030092051A1
• Filed: 10/15/2002
• Published: 05/15/2003
• Est. Priority Date: 02/23/2000
• Status: Active Grant

• Current Assignee: Arroyo Optics, Inc. (NVIDIA Corporation)
• Original Assignee: Arroyo Optics, Inc. (NVIDIA Corporation)
• Inventors: Sommer, Steve S., Liu, Qiang
• Granted Patent: US 7,105,298 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)

Abstract

A novel method of pyrophosphorolysis activated polymerization (PAP) has been developed. In PAP, pyrophosphorolysis and polymerization by DNA polymerase are coupled serially for each amplification by using an activatable oligonucleotide P* that has a non-extendable 3′-deoxynucleotide at its 3′ terminus. PAP can be applied for exponential amplification or for linear amplification. PAP can be applied to amplification of a rare allele in admixture with one or more wild type alleles by using an activatable oligonucleotide P* that is an exact match at its 3′ end for the rare allele but has a mismatch at or near its 3′ terminus for the wild type allele. PAP is inhibited by a mismatch in the 3′ specific subsequence as far as 16 nucleotides away from the 3′ terminus. PAP can greatly increase the specificity of detection of an extremely rare mutant allele in the presence of the wild type allele. Specificity results from both pyrophosphorolysis and polymerization since significant nonspecific amplification requires the combination of mismatch pyrophosphorolysis and misincorporation by the DNA polymerase, an extremely rare event. Using genetically engineered DNA polymerases greatly improves the efficiency of PAP.

Claims

1. A process which comprises serial coupling of two reactions, the second reaction being amplification of a nucleic acid by extension of an oligonucleotide on a nucleic acid template in the presence of four nucleoside triphosphates and a nucleic acid polymerase, the first reaction being activation of the oligonucleotide by removal of a 3′

end block which, if not removed, would prevent the oligonucleotide from being extended on the template.

2. Process of claim 1 where the oligonucleotide is at least partially hybridized to the template before and during the first reaction.

3. Process of claim 2 where the 3′

end block on the oligonucleotide is a 3′

chemical cap or 3′

mismatch of the template.

4. Process of claim 3 where the 3′

block is removed by 3′

base repair, dephosphorylation or restriction endonuclease cleavage.

5. Process of claim 4 where the oligonucleotide contains a methylated endonuclease recognition sequence and is annealed to the target with the unmethylated restriction endonuclease sequence, and the oligonucleotide is activated by restriction endonuclease cleavage of the methylated site.

6. Process of claim 5 where the methylated endonuclease recognition sequence is G^mATC

7. Process of claim 6 where the restriction endonuclease is DpnI.