Isothermal SNP Detection Method

• US 20080118917A1
• Filed: 11/21/2006
• Published: 05/22/2008
• Est. Priority Date: 11/21/2006
• Status: Abandoned Application

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Hardy, Christine D., Martin, Patrick K.
• Application Number:
  US11/562,345
• Publication Number:
  US 20080118917A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6865 Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
• CPC Class Codes: C12Q 1/6867 Replicase-based amplification, e.g. using Q-beta replicase
• CPC Class Codes: C12Q 2521/507 Recombinase
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension

Abstract

In some embodiments, the present teachings provide a method for detecting a nucleotide of interest comprising; forming an amplification reaction mixture comprising a mismatched primer, a target polynucleotide, a strand-displacing polymerase lacking 3′ to 5′ exonuclease activity, a recombinase, and a single-stranded DNA binding protein; hybridizing a mismatched primer to the target polynucleotide to form a primer-target complex; and, detecting the nucleotide of interest by the absence of a primer extension product. In some embodiments, control reactions are performed in which a control polynucleotide is exponentially amplified. Additional methods, as well as reaction mixtures and kits, are also provided.

Claims

1. A method for detecting a nucleotide of interest by the absence of a primer extension product comprising;

forming an amplification reaction mixture comprising a mismatched primer, a target polynucleotide, a strand-displacing polymerase lacking 3′

to 5′

exonuclease activity, a recombinase, and a single-stranded DNA binding protein;

hybridizing a mismatched primer to the target polynucleotide to form a primer-target complex; and

,

detecting the nucleotide of interest by the absence of a primer extension product.

5. The method of any of claims 1-4 wherein the recombinase is selected from the group consisting of bacteriophage T4 UvsX, E. coli recA, Archaebacteria RadA, Archaebacteria RadB, eurkaryotic Rad51, eukaryotic DMC1, and eukaryotic Rad55/57.

6. The method of any of claims 1-4 wherein the single-stranded DNA binding protein is selected from the group consisting of bacteriophage T4 gp32, bacteriophage T7 gene 2.5, Phi 29 gene 5, E. coli ssb, eukaryotic RPA, Aeropyrum pernix ssb, and Pyrococcus furiosus RPA.

7. The method of any of claims 1-4 wherein the strand-displacing polymerase lacking 3′

to 5′

exonuclease activity is selected from the group consisting of Klenow exo⁻

, B. stearothermophilus exo-, Thermus aquaticus polymerase, Aquifex aeolicus polymerase, bacteriophage T5 polymerase, bacteriophage T7 polymerase, Phi29 exo-, Vent pol exo-, and Thermococcus spp. 9°

N_m exo-.

8. The method of any of claims 1-4 further comprising a crowding agent.

9. The method of claim 8 wherein the crowding agent is selected from the group consisting of carbowax20M, a PEG compound, T4 UvsY, Rb 69 UvsY, and E. coli Rec OR.

2. A method for detecting a nucleotide of interest in a first target polynucleotide comprising;

forming an amplification reaction mixture comprising a mismatched primer, a matched primer, a first target polynucleotide, a second target polynucleotide, a strand-displacing polymerase lacking 3′

to 5′

exonuclease activity, a recombinase, and a single-stranded DNA binding protein;

hybridizing the mismatched primer to the first target polynucleotide to form a first primer-target complex and hybridizing the matched primer to the second target polynucleotide to form a second primer-target complex; and

,

measuring the absence of a first amplification product from the first target polynucleotide and the presence of a second amplification product from the second target polynucleotide to detect the nucleotide of interest in the first target polynucleotide.

3. A method for detecting a nucleotide of interest in a first target polynucleotide comprising;

forming a first amplification reaction mixture comprising a mismatched primer, a first target polynucleotide, a strand-displacing polymerase lacking 3′

to 5′

exonuclease activity, a recombinase, and a single-stranded DNA binding protein;

forming a second amplification reaction mixture comprising a matched primer, a second target polynucleotide, a strand-displacing polymerase lacking 3′

to 5′

exonuclease activity, a recombinase, and a single-stranded DNA binding protein;

hybridizing the mismatched primer to the first target polynucleotide to form a first primer-target complex and hybridizing the matched primer to the second target polynucleotide to form a second primer-target complex; and

,

measuring the absence of a first amplification product from the first target polynucleotide in the first amplification reaction mixture and the presence of a second amplification product from the second target polynucleotide in the second amplification reaction mixture to detect the nucleotide of interest in the first target polynucleotide.

4. A method of preventing amplification of a first target polynucleotide while allowing amplification of a second target polynucleotide comprising;

forming an amplification reaction mixture comprising a mismatched primer, a matched primer, a first target polynucleotide, a second target polynucleotide, a strand-displacing polymerase lacking 3′

to 5′

exonuclease activity, a recombinase, and a single-stranded DNA binding protein;

hybridizing the mismatched primer to the first target polynucleotide to form a first primer-target complex and hybridizing the matched primer to the second target polynucleotide to form a second primer-target complex; and

,

preventing amplification of the first target polynucleotide while allowing amplification of the second target polynucleotide, with the proviso that the amplifying is isothermal.

10. A reaction mixture comprising;

a first composition comprising a first target polynucleotide hybridized to a complementary portion of a mismatch primer, wherein the mismatch primer further comprises a one nucleotide non-complementary portion at its 3′

end;

a second composition comprising a second target polynucleotide hybridized to a matched primer; and

,

a strand-displacing polymerase lacking 3′

to 5′

exonuclease activity, a recombinase, and a single-stranded DNA binding protein.

11. The reaction composition according to claim 10 wherein the recombinase is selected from the group consisting of bacteriophage T4 UvsX, E. coli recA, Archaebacteria RadA, Archaebacteria RadB, eurkaryotic Rad51, eukaryotic DMC1, and eukaryotic Rad55/57.

12. The reaction composition according to claim 10 wherein the single-stranded DNA binding protein is selected from the group consisting of bacteriophage T4 gp32, bacteriophage T7 gene 2.5, Phi 29 gene 5, E. coli ssb, eukaryotic RPA, Aeropyrum pernix ssb, and Pyrococcus furiosus RPA.

13. The reaction composition of claim 10 wherein the strand-displacing polymerase lacking 3′

to 5′

exonuclease activity is selected from the group consisting of Klenow exo-, B. stearothermophilus exo-, Thermus aquaticus polymerase, Aquifex aeolicus polymerase, bacteriophage T5 polymerase, bacteriophage T7 polymerase, Phi29 exo-, Vent pol exo-, and Thermococcus spp. 9°

N_m exo-.

14. The reaction composition according to claim 10 further comprising a crowding agent.

15. The reaction composition according to claim 14 wherein the crowding agent is selected from the group consisting of carbowax20M, a PEG compound, T4 UvsY, Rb 69 UvsY, and E. coli Rec OR.

16. A kit comprising;

a matched primer and a mismatched primer, wherein the matched primer and the mismatched primer differ only in the nucleotide at their 3′

ends; and

,

a strand-displacing polymerase lacking 3′

to 5′

exonuclease activity, a recombinase, and a single-stranded DNA binding protein.

17. The kit according to claim 16 wherein the recombinase is selected from the group consisting of bacteriophage T4 UvsX, E. coli recA, Archaebacteria RadA, Archaebacteria RadB, eurkaryotic Rad51, eukaryotic DMC1, and eukaryotic Rad55/57.

18. The kit according to claim 16 wherein the single-stranded DNA binding protein is selected from the group consisting of bacteriophage T4 gp32, bacteriophage T7 gene 2.5, Phi 29 gene 5, E. coli ssb, eukaryotic RPA, Aeropyrum pernix ssb, and Pyrococcus furiosus RPA.

19. The kit according to claim 16 wherein the strand-displacing polymerase lacking 3′

to 5′

exonuclease activity is selected from the group consisting of Klenow exo-, B. stearothermophilus exo-, Thermus aquaticus polymerase, Aquifex aeolicus polymerase, bacteriophage T5 polymerase, bacteriophage T7 polymerase, Phi29 exo-, Vent pol exo-, and Thermococcus spp. 9°

N_m exo-.

20. The kit according to claim 16 further comprising a crowding agent.

21. The reaction composition according to claim 20 wherein the crowding agent is selected from the group consisting of carbowax20M, a PEG compound, T4 UvsY, Rb 69 UvsY, and E. coli Rec OR.