PYROPHOSPHOROLYSIS ACTIVATED POLYMERIZATION (PAP)

• US 20090239283A1
• Filed: 03/11/2009
• Published: 09/24/2009
• Est. Priority Date: 02/23/2000
• Status: Active Grant

• Current Assignee: City of Hope
• Original Assignee: City of Hope
• Inventors: SOMMER, Steve S., LIU, Qiang
• Granted Patent: US 7,914,995 B2
• Time in Patent Office: Days
• US Class Current: 435/183
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2521/319 Exonuclease
• 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 2535/131 Allele specific probes
• 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-extendible 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 sequence 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. An isolated oligonucleotide primer having a non-extendible 3′

terminus, wherein said oligonucleotide primer with the non-extendible 3′

terminus is incapable of being extended by a DNA polymerase and wherein said non-extendible 3′

terminus is removable by pyrophosphorolysis.

2. The isolated oligonucleotide primer of claim 1 which consists of 18-60 nucleotides.

3. The isolated oligonucleotide primer of claim 1, wherein said non-extendible 3′

terminus is selected from the group consisting of a 3′

deoxynucleotide, a 2′

,3′

-dideoxynucleotide, an acyclonucleotide, 3′

-deoxyadenosine (cordycepin), 3′

-azido-3′

-deoxythymidine (AZT), 2′

,3′

-dideoxyinosine (ddI), 2′

,3′

-dideoxy-3′

-thiacytidine (3TC) and 2′

,3′

-didehydro-2′

,3′

-dideoxythymidine (d4T).

4. The isolated oligonucleotide primer of claim 1 which has at least one nucleotide at or near its 3′

terminus that mismatches the corresponding nucleotide of an alleleic strand of a target nucleic acid template.

5. A kit for pyrophosphorolysis activated polymerization (PAP) comprising an oligonucleotide primer having a non-extendible 3′

terminus and a DNA polymerase, wherein said oligonucleotide primer with the non-extendible 3′

terminus is incapable of being extended by said DNA polymerase and wherein said non-extendible 3′

terminus is removable by pyrophosphorolysis.

6. The kit of claim 5, wherein said DNA polymerase has pyrophosphorolysis activity.

7. The kit of claim 5 which further comprises and a reaction buffer that promotes pyrophosphorolysis

8. The kit of claim 5, wherein said oligonucleotide primer having a non-extendible 3′

terminus consists of 18-60 nucleotides.

9. The kit of claim 5, wherein said non-extendible 3′

terminus is selected from the group consisting of a 3′

deoxynucleotide, a 2′

,3′

-dideoxynucleotide, an acyclonucleotide, 3′

-deoxyadenosine (cordycepin), 3′

-azido-3′

-deoxythymidine (AZT), 2′

,3′

-dideoxyinosine (ddI), 2′

,3′

-dideoxy-3′

-thiacytidine (3TC) and 2′

,3′

-didehydro-2′

,3′

-dideoxythymidine (d4T).

10. The kit of claim 5 for PAP-based allele-specific amplification, wherein said oligonucleotide primer having a non-extendible 3′

terminus has at least one nucleotide at or near its 3′

terminus that mismatches the corresponding nucleotide of an alleleic strand of a target nucleic acid template.