Pyrophosphorolysis activated polymerization (PAP)

• US 7,238,480 B2
• Filed: 03/12/2004
• Issued: 07/03/2007
• Est. Priority Date: 02/23/2000
• Status: Expired due to Fees

• Current Assignee: City of Hope
• Original Assignee: City of Hope
• Inventors: Sommer, Steve S., Liu, Qiang, Riggs, Arthur D.
• Primary Examiner(s): Horlick; Kenneth R.
• Application Number:
  US10/798,844
• Publication Number:
  US 20050095608A1
• Time in Patent Office: 1,208 Days
• Field of Search: 435/6
• US Class Current: 435/6.11
• 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. A method for determining the presence or absence of a nucleic acid hybrid in a sample comprising the following steps:

(a) providing a reaction mixture comprising (i) a sample that may contain a nucleic acid hybrid wherein one strand of the hybrid comprises an activatable oligonucleotide having a non-extendible 3′

-terminus, (ii) pyrophosphate, (iii) an enzyme that catalyzes the release of a nucleotide from a nucleic acid hybrid, by pyrophosphorolysis of the non-extendible 3′

-terminus of a strand of the nucleic acid hybrid in the presence of pyrophosphate, and (iv) a suitable nucleotide that can be incorporated in the place of said released nucleotide;

(b) maintaining said reaction mixture for a time period and under conditions that permit (i) pyrophosphorolysis of the non-extendible 3′

-terminus of a strand of a nucleic acid hybrid to produce a released nucleotide and a modified 3′

-terminus as well as (ii) the incorporation of said suitable nucleotide into the modified 3′

-terminus of the nucleic acid hybrid to produce an incorporated modified 3′

-terminus, thereby forming a treated sample; and

(c) assaying the treated sample to determine whether incorporation of said suitable nucleotide into the hybrid occurred and thereby determining the presence or absence of a nucleic acid hybrid in said sample.

2. The method of detecting the presence or absence of a nucleic acid hybrid according to claim 1 wherein the nucleotide suitable for incorporation into the modified 3′

-terminus of the nucleic acid hybrid includes a label.

3. The method of detecting the presence or absence of a nucleic acid hybrid according to claim 1 wherein the method of assaying whether incorporation of a suitable nucleotide occurred comprises determining whether the label is associated with the nucleic acid hybrid.

4. The method of detecting the presence or absence of a nucleic acid hybrid according to claim 2 wherein the label is a fluorescent label.

5. The method of detecting the presence or absence of a nucleic acid hybrid according to claim 1 wherein said nucleic acid hybrid is formed between a target nucleic acid strand and a probe nucleic acid strand.

6. The method of detecting the presence or absence of a nucleic acid hybrid according to claim 1 wherein said nucleic acid hybrid comprises a label.

7. The method of detecting the presence or absence of a nucleic acid hybrid according to claim 2 wherein said nucleotide suitable for incorporation into the modified 3′

-terminus of the nucleic acid hybrid is a chain terminating or other polymerization-blocking nucleotide.

8. The method of detecting the presence or absence of a nucleic acid hybrid according to claim 5 wherein assaying to determine whether incorporation of said suitable nucleotide occurred is carried out by detecting an increase in the length of said probe.

20. The method for determining the presence or absence of a nucleic acid hybrid in a sample according to claim 1 wherein said nucleic acid hybrid is affixed to a solid support.

21. The method for determining the presence or absence of a nucleic acid hybrid in a sample according to claim 20 wherein said nucleic acid hybrid is affixed to a solid support through attachment of a strand of the nucleic acid hybrid to said solid support.

9. A method for determining the presence or absence of a nucleic acid target in a sample comprising the following steps:

(a) providing a reaction mixture comprising (i) a sample that may contain a nucleic acid target, (ii) a nucleic acid probe corresponding to said nucleic acid target, wherein the nucleic acid probe comprises an activatable oligonucleotide having a non-extendible 3′

terminus, (iii) pyrophosphate, (iv) an enzyme that catalyzes the release of a nucleotide from a nucleic acid hybrid, wherein one strand of the hybrid comprises an activatable oligonucleotide having a non-extendible 3′

-terminus, by pyrophosphorolysis of the non-extendible 3′

-terminus of a strand of the nucleic acid hybrid in the presence of pyrophosphate, and (v) a suitable nucleotide that can be incorporated in the place of said released nucleotide;

(b) maintaining said reaction mixture for a time period and under conditions that permit (i) hybridization of the nucleic acid target with the nucleic acid probe to form a nucleic acid hybrid that comprises a non-extendible 3′

-terminus, (ii) pyrophosphorolysis of the non-extendible 3′

-terminus of a strand of a nucleic acid hybrid to produce a released nucleotide and a modified 3′

-terminus as well as (iii) the incorporation of said suitable nucleotide into the modified 3′

-terminus of the nucleic acid hybrid to produce an incorporated modified 3′

-terminus, thereby forming a treated sample; and

(c) assaying the treated sample to determine whether incorporation of said suitable nucleotide occurred and thereby determining the presence or absence of the nucleic acid target in said sample.

10. The method for determining the presence or absence of a nucleic acid target in a sample according to claim 9 wherein said reaction mixture may comprise a plurality of nucleic acid targets and their corresponding nucleic acid probes.

11. The method for determining the presence or absence of a nucleic acid target in a sample according to claim 10 wherein said nucleic acid probes are distinguishable from one another on the basis of length, thereby permitting the determination of the presence or absence of a plurality of nucleic acid targets.

12. The method for determining the presence or absence of a nucleic acid target in a sample according to claim 10 wherein the suitable nucleotides incorporated to form the incorporated modified 3′

-terminus permit distinction between the probes, thereby permitting the determination of the presence or absence of a plurality of nucleic acid targets.

13. The method for determining the presence or absence of a nucleic acid target in a sample according to claim 12 wherein the nucleic acid probes having incorporated modified 3′

-termini are distinguishable from one another on the basis of the suitable nucleotide incorporated or on the basis of length, thereby permitting the determination of the presence or absence of a plurality of nucleic acid targets.

14. The method for determining the presence or absence of a nucleic acid target in a sample according to claim 9 wherein said reaction mixture may comprise a plurality of nucleic acid targets that differ from one another by a single base.

15. The method for determining the presence or absence of a nucleic acid target in a sample according to claim 14 wherein said plurality of nucleic acid targets differ from one another by a single base at an interrogation position.

16. The method for determining the presence or absence of a nucleic acid target in a sample according to claim 15 wherein the penultimate 3′

-terminal residue of the corresponding nucleic acid probe base pairs with the interrogation position of the nucleic acid target.

17. A method for determining the presence or absence of a specific nucleic acid base at an interrogation position of a nucleic acid target in a sample comprising the following steps:

(a) providing a reaction mixture comprising (i) a sample that may contain a nucleic acid target having a nucleic acid residue at an interrogation position, (ii) a nucleic acid probe comprising a nucleic acid residue in its 3′

-terminus that base pairs with the interrogation position of the nucleic acid target when the nucleic acid target and the nucleic acid probe are hybridized to form a nucleic acid hybrid, wherein the nucleic acid probe comprises an activatable oligonucleotide having a non-extendible 3′

terminus, (ii) pyrophosphate, (iii) an enzyme that catalyzes the release of a nucleotide from a nucleic acid hybrid, a wherein one strand of the hybrid comprises an activatable oligonucleotide having a non-extendible 3′

-terminus, by pyrophosphorolysis of the non-extendible 3′

-terminus of a strand of the nucleic acid hybrid in the presence of pyrophosphate, and (iv) a suitable nucleotide that can be incorporated in the place of said released nucleotide;

(b) maintaining said reaction mixture for a time period and under conditions that permit (i) formation of a nucleic acid hybrid between the nucleic acid probe and the nucleic acid target, (ii) pyrophosphorolysis of the non-extendible 3′

-terminus of a strand of a nucleic acid hybrid to produce a released nucleotide and a modified 3′

-terminus and (iii) the incorporation of said suitable nucleotide into the modified 3′

-terminus of the nucleic acid hybrid to produce an incorporated modified 3′

-terminus, thereby forming a treated sample; and

(c) assaying the treated sample to determine whether incorporation of said suitable nucleotide occurred and thereby determining the presence or absence of a specific nucleic acid base at an interrogation position of a nucleic acid target in said sample.

18. The method for determining the presence or absence of a specific nucleic acid base at an interrogation position of a nucleic acid target in a sample according to claim 17 wherein the nucleic acid residue of the nucleic acid probe that corresponds with the interrogation position of the nucleic acid target is the 3′

-terminal residue.

19. The method for determining the presence or absence of a specific nucleic acid base at an interrogation position of a nucleic acid target in a sample according to claim 17 wherein the nucleic acid residue of the nucleic acid probe that corresponds with the interrogation position of the nucleic acid target is the penultimate 3′

-terminal residue.

22. A method of determining a nucleotide sequence of a nucleic acid hybrid that comprises the following steps:

(a) providing a reaction mixture comprising (i) a sample that contains a nucleic acid hybrid wherein one strand of the hybrid comprises an activatable oligonucleotide having a non-extendible 3′

-terminus, (ii) pyrophosphate, (iii) an enzyme that catalyzes the release of a nucleotide from a nucleic acid hybrid, by pyrophosphorolysis of the non-extendible 3′

-terminus of a strand of the nucleic acid hybrid in the presence of pyrophosphate, and (iv) a suitable nucleotide that can be incorporated in the place of said released nucleotide;

(b) maintaining said reaction mixture for a time period and under conditions that permit (i) pyrophosphorolysis of the non-extendible 3′

-terminus of a strand of a nucleic acid hybrid to produce a released nucleotide and a modified 3′

-terminus as well as (ii) the incorporation of said suitable nucleotide into the modified 3′

-terminus of the nucleic acid hybrid to produce an incorporated modified 3′

-terminus, thereby forming a treated sample; and

(c) assaying the treated sample to determine where incorporation of said suitable nucleotide into the hybrid occurred and thereby determining nucleotide sequence of the nucleic acid hybrid.

23. The method of determining a nucleotide sequence of a nucleic acid hybrid according to claim 22 wherein said nucleic acid hybrid is formed by a combination of a nucleic acid probe with a nucleic acid target.