Methods for Multiplexing Recombinase Polymerase Amplification

• US 20120015367A1
• Filed: 07/06/2011
• Published: 01/19/2012
• Est. Priority Date: 07/25/2005
• Status: Active Grant

• Current Assignee: Abbott Diagnostics Scarborough, Inc. (Abbott Laboratories Incorporated)
• Original Assignee: Alere San Diego Incorporated (Abbott Laboratories Incorporated)
• Inventors: Piepenburg, Olaf, Williams, Colin H., Armes, Niall A.
• Granted Patent: US 8,580,507 B2
• Time in Patent Office: Days
• US Class Current: 435/6.12
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/507 Recombinase
• CPC Class Codes: C12Q 2525/119 incorporating abasic sites
• CPC Class Codes: C12Q 2525/125 incorporating agents resulting in resistance to degradation
• CPC Class Codes: C12Q 2537/125 Sandwich assay format
• CPC Class Codes: C12Q 2537/1376 Displacement by an enzyme
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/102 Multiple non-interacting labels
• CPC Class Codes: C12Q 2565/625 being a nucleic acid test strip device, e.g. dipsticks, strips, tapes, CD plates
• CPC Class Codes: G01N 2021/7759 Dipstick; Test strip
• CPC Class Codes: G01N 2021/7763 Sample through flow
• CPC Class Codes: G01N 2021/7793 Sensor comprising plural indicators
• CPC Class Codes: G01N 21/78 : producing a change of colour

Abstract

This disclosure provides for methods and reagents for rapid multiplex RPA reactions and improved methods for detection of multiplex RPA reaction products. In addition, the disclosure provides new methods for eliminating carryover contamination between RPA processes.

Claims

1. A process comprising:

(a) contacting a recombinase agent with a first and a second nucleic acid primer and a third extension blocked primer which comprises one or more noncomplementary or modified internal residues to form a first, second and third nucleoprotein primer;

(b) contacting the first and second nucleoprotein primers to a double stranded target nucleic acid comprising a first and second strand, thereby forming a first double stranded structure between said first nucleoprotein primer and said first strand at a first portion of said first strand and a second double stranded structure between said second nucleoprotein primer and said second strand at a second portion of said second strand such that the 3′

ends of said first nucleoprotein primer and said second nucleoprotein primer are oriented toward each other on the same target nucleic acid with a third portion of target nucleic acid between said 3′

ends;

(c) extending the 3′

end of said first nucleoprotein primer and second nucleoprotein primer with one or more polymerases and dNTPs to generate an amplified target nucleic acid with an internal region comprising the third portion of nucleic acid; and

(d) contacting said amplified target nucleic acid to said third nucleoprotein primer to form a third double stranded structure at the third portion of said amplified target nucleic acid in the presences of a nuclease;

wherein said nuclease specifically cleaves said noncomplementary internal residue only after the formation of said third double stranded structure to form a third 5′

primer and a third 3′

extension blocked primer.

2. The process of claim 1 wherein the first double stranded structure is part of a first D-loop and wherein said second double stranded structure is part of a second D-loop.

3. The process of claim 1 wherein said nuclease is a DNA glycosylase or AP endonuclease.

4. The process of claim 1 wherein said modified internal residue is a uracil or inosine residue.

5. The process of claim 4 wherein the nuclease recognizes the uracil or inosine residue and cleaves said third extension blocked primer at the uracil or inosine residue.

6. The process of claim 1 wherein the nuclease recognizes a base mismatch between the noncomplementary base of said third extension blocked primer and said target nucleic acid and cleaves said third extension blocked primer at said noncomplementary base.

7. The process of claim 1 wherein said nuclease is selected from the group consisting of fpg, Nth, MutY, MutS, MutM, E. coli MUG, human MUG, human Ogg1, vertebrate Nei-like (Neil) glycosylases, uracil glycosylase, hypoxanthine-DNA glycosylase, and functional analogs thereof.

8. The process of claim 1 wherein said nuclease is E. coli Nfo or E. coli exonuclease III and wherein the modified residue is a tetrahydrofuran residue or carbon linker.

9. The process of claim 1 wherein the modified internal base is selected from the group consisting of 8-oxoguanine, thymine glycol, and an abasic site mimic.

10. The process of claim 9 wherein the abasic site mimic is a tetrahydrofuran residue or D-spacer.

11. The process of claim 1 wherein said third extension blocked primer comprises a blocked 3′

residue which is resistant to extension by DNA polymerase.

20. The process of claim 1 wherein the third extension blocked primer further comprises one or more detectable labels.

21. The process of claim 20 wherein said process further comprises detecting said detectable label on the third extension blocked primer.

22. The process of claim 20 wherein said detectable label is selected from the group consisting of a fluorophore, an enzyme, a quencher, an enzyme inhibitor, a radioactive label, one member of a binding pair and a combination thereof.

23. The process of claim 22 in which the fluorophore is attached to the third extension blocked primer by a fluorophore-dT residue.

24. The process of claim 22 in which the quencher is attached to the third extension blocked primer by a quencher-dT residue.

25. The process of claim 22 wherein said third extension blocked primer comprises a fluorophore and a quencher.

26. The process of claim 25 wherein said fluorophore and quencher are separated by between 0 to 2 bases.

27. The process of claim 25 wherein said fluorophore and quencher are separated by between 0 to 5 bases.

28. The process of claim 25 wherein said fluorophore and quencher are separated by between 0 to 8 bases.

29. The process of claim 25 wherein said fluorophore and quencher are separated by between 0 to 10 bases.

30. The process of claim 25 wherein said fluorophore and quencher are separated by a greater distance when the extension blocked primer is unhybridized than when the extension blocked primer is hybridized to the target nucleic acid.

31. The process of claim 25 wherein the fluorophore or the quencher is attached to the noncomplementary or modified internal residue and wherein the fluorophore and quencher are separated following cleavage of the modified internal base by the nuclease.

32. The process of claim 25 in which the fluorophore is selected from the group of fluorescein, FAM, TAMRA.

33. The process of claim 25 in which the quencher is a dark quencher.

34. The process of claim 33 wherein said dark quencher is selected from the group consisting of Dark Quencher 1, Dark Quencher 2, Black Hole Quencher 1 and Black Hole-Quencher 2.

35. The process of claim 1 wherein the first primer, second primer or third extension blocked primer is 12 to 30 residues in length.

36. The process of claim 1 wherein the first primer, second primer or third extension blocked primer is 12 to 40 residues in length.

37. The process of claim 1 wherein the first primer, second primer or third extension blocked primer is 12 to 60 residues in length.

38. The process of claim 1 wherein said process is performed at a temperature of between 14°

C. and 21°

C.

39. The process of claim 1 wherein said process is performed at a temperature, of between 21°

C. and 25°

C.

40. The process of claim 1 wherein said process is performed at a temperature of between 25°

C. and 30°

C.

41. The process of claim 1 wherein said process is performed at a temperature of between 30°

C. and 37°

C.

42. The process of claim 1 wherein said process is performed at a temperature of between 40°

C. and 43°

C.

43. The process of claim 1 wherein said process amplifies at least the third portion of said target nucleic acid at least 10⁷ fold.

44. The process of claim 1 wherein said process is performed in the presence of 1% to 12% PEG.

45. The process of claim 1 wherein said process is performed in the presence of 6% to 8% PEG.

12-19. -19. (canceled)

46-50. -50. (canceled)

51. A multiplex process of RPA comprising the steps of performing more than one RPA process on one or more double stranded target nucleic acid in one reaction wherein each RPA process comprises the following steps:

(a) contacting a recombinase agent with a first and a second nucleic acid primer and a third extension blocked primer which comprises one or more noncomplementary or modified internal residue to form a first, second and third nucleoprotein primer;

(b) contacting the first and second nucleoprotein primers to said double stranded target nucleic acid thereby forming a first double stranded structure between said first nucleoprotein primer and said first strand of DNA at a first portion of said first strand and a second double stranded structure between said second nucleoprotein primer and said second strand of DNA at a second portion of said second strand such that the 3′

ends of said first nucleoprotein primer and said first nucleoprotein primer are oriented toward each other on the same target nucleic acid molecule with a third portion of target nucleic acid between said 3′

ends;

(c) extending the 3′

end of said first nucleoprotein primer and second nucleoprotein primer with one or more polymerases and dNTPs to generate an amplified target nucleic acid with an internal region comprising the third portion of nucleic acid;

(d) contacting said amplified target nucleic acid to said third nucleoprotein primer to form a third double stranded structure at the third portion of said amplified target nucleic acid in the presences of a nuclease;

wherein said nuclease specifically cleaves said noncomplementary internal residue only after the formation of said third double stranded structure to form a third 5′

primer and a third. 3′

extension blocked primer.

52. The method, of claim 51 wherein said more than one RPA processes comprises at least 2 separate RPA processes.

53. The method of claim 51 wherein said more than one RPA processes comprises at least 4 separate RPA processes.

54. The method of claim 51 wherein said more than one RPA processes comprises at least 5 separate RPA processes.

55. The method of claim 51 wherein said more than one RPA processes comprises at least 7 separate RPA processes.

56. The method of claim 51 wherein said more than one RPA processes comprises at least 10 separate RPA processes.

57. The process of claim 51 wherein said modified internal-residue is a uracil or inosine residue.

61. The process of claim 51 wherein said nuclease is a DNA glycosylase or AP endonuclease.

62. The process of claim 51 wherein the nuclease recognizes a base mismatch between the noncomplementary base of said third extension blocked primer and said target nucleic acid and cleaves said third extension blocked primer at said noncomplementary base.

63. The process of claim 51 wherein said nuclease is selected from the group consisting of fpg, Nth, MutY, MutS, MutM, E. coli MUG, human MUG, human Ogg1, vertebrate Nei-like (Neil) glycosylases, uracil glycosylase, hypoxanthine-DNA glycosylase, and functional analogs thereof.

64. The process of claim 51 wherein said nuclease is E. coli Nfo or E. coli exonuclease III and wherein the modified residue is a tetrahydrofuran residue or carbon linker.

65. The process of claim 51 wherein the modified internal base is selected from the group consisting of 8-oxoguanine, thymine glycol, and an abasic site mimic.

66. The process of claim 65 wherein the abasic site mimic is a tetrahydrofuran residue or D-spacer.

67. The process of claim 66 wherein the abasic site mimic is a tetrahydrofuran residue.

58-60. -60. (canceled)

68-78. -78. (canceled)