Methods for multiplexing recombinase polymerase amplification

• US 7,435,561 B2
• Filed: 07/25/2006
• Issued: 10/14/2008
• Est. Priority Date: 07/25/2005
• Status: Active Grant

• Current Assignee: Abbott Diagnostics Scarborough, Inc. (Abbott Laboratories Incorporated)
• Original Assignee: ASM Scientific, Inc.
• Inventors: Armes, Niall A., Piepenburg, Olaf, Williams, Colin H.
• Primary Examiner(s): Horlick; Kenneth R.
• Assistant Examiner(s): WILDER, CYNTHIA B
• Application Number:
  US11/493,677
• Publication Number:
  US 20070054296A1
• Time in Patent Office: 812 Days
• Field of Search: 435/91.2, 435/91.1
• US Class Current: 435/91.2
• 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 RPA process of DNA amplification of a target nucleic acid molecule comprising a first and a second strand of DNA, comprising the steps of:

(a) contacting a recombinase agent with a first and a second nucleic acid primer and a third extension blocked primer which extension blocked primer 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 second 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 a first 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 presence of a nuclease;

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

primer double stranded structure and a third 3′

extension blocked primer double stranded structure;

(e) extending the 3′

end of said third 5′

primer with one or more polymerase and dNTP to generate a second double stranded amplified nucleic acid which comprises said first nucleic acid primer and said third 5′

primer;

(f) continuing the reaction through repetition of (b) through (e) until a desired degree of the second double stranded amplified nucleic acid is reached.

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 Ogg, 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 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.

12. The process of claim 11 wherein the blocked 3′

residue comprises a blocking moiety which prevents the extension of the primer by polymerase.

13. The process of claim 12 wherein the blocking moiety is attached to the 3′

or 2′

site of the 3′

residue sugar.

14. The process of claim 12 wherein the blocking moiety is a detectable label.

15. The process of claim 14 wherein said detectable label is selected from the group consisting of a fluorophore, an enzyme, a quencher, an enzyme inhibitor, a radioactive label, a member of a binding pair, and a combination thereof.

16. The process of claim 11 wherein the blocked 3′

residue is a dideoxy nucleotide.

17. The process of claim 1 wherein said first nucleic acid primer comprises a first detectable label and said third extension blocked primer comprises a second detectable label.

18. The process of claim 17 wherein said first and second detectable label are different and the production of said second double stranded amplified nucleic acid is monitored by detecting the presence of the first and second detectable label on a single double stranded DNA molecule.

19. The process of claim 18 wherein the production of said second double stranded amplified nucleic acid is detected by a sandwich assay wherein a first antibody binds said first detectable label and a second antibody binds said second detectable label.

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 the step of monitoring the progress of the RPA reaction by 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 amidite residue.

24. The process of claim 22 in which the quencher is attached to the third extension blocked primer by a quencher-dT amidite 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 wherein said dark quencher is a non-fluorescent chromophore.

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 12 to 40 residues in length.

37. The process of claim 1 wherein the first primer, second primer or third extension blocked primer 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 folds.

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

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

46. The process of claim 1, wherein said dNTP comprises dUTP and wherein said RPA process is performed in the presence of uracil glycosylase for a first period of less than 20 minutes and wherein said process is performed in the presence of uracil glycosylase inhibitor after said first period.

47. The process of claim 46 wherein said process is performed without temperature based inactivation of said uracil glycosylase.

48. The process of claim 46 wherein said uracil glycosylase inhibitor is Bacillus subtilis phages PBS1 uracil glycosylase inhibitor or Bacillus subtilis phages PBS2 uracil glycosylase inhibitor.

49. The process of claim 46 wherein said dNTP consists of dATP, dUTP, dCTP and dGTP.

50. The process of claim 46 wherein said dNTP does not contain dTTP.

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 process comprise the following steps:

(a) contacting a recombinase agent with a first and a second nucleic acid primer and a third extension blocked primer which extension blocked primer 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 second 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 a first 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 presence of a nuclease;

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

primer double stranded structure and a third 3′

extension blocked primer double stranded structure;

(e) extending the 3′

end of said third 5′

primer with one or more polymerase and dNTP to generate a second double stranded amplified nucleic acid which comprises said first nucleic acid primer and said third 5′

primer;

(f) continuing the reaction through repetition of (b) through (e) until a desired degree of the second double stranded amplified nucleic acid is reached;

wherein each RPA process is performed with a different combination of said first and second nucleic acid primer and wherein each process is performed with the same third 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.

58. The process of claim 51 further comprising the step of detecting the formation of said second double stranded amplified nucleic acid to determine the cumulative amplification of any of said more than one RPA process.

59. The process of claim 51 wherein the first nucleic acid primer of each RPA process is labeled with the same first detectable label, wherein said third extension blocked primer is labeled with a second detectable label, and wherein said detecting step comprises detecting a double stranded nucleic acid comprising both said first detectable label and said second detectable label.

60. The process of claim 59 wherein the production of said second double stranded amplified nucleic acid is detected by a sandwich assay wherein a first antibody binds said first detectable label and a second antibody binds said second detectable label.

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, or 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 51 wherein said third extension blocked primer comprises a blocked 3′

residue which is resistant to extension by DNA polymerase.

68. The process of claim 67 wherein the blocked 3′

residue comprises a blocking moiety which prevents the extension of the primer by polymerase.

69. The process of claim 68 wherein the blocking moiety is attached to the 3′

or 2′

site of the 3′

residue sugar.

70. The process of claim 69 wherein the blocked 3′

residue is a dideoxy nucleotide.

71. The process of claim 51 wherein said dNTP comprises dUTP and wherein said RPA process is performed in the presence of uracil glycosylase for a first period of less than 20 minutes and wherein said process is performed in the presence of uracil glycosylase inhibitor after said first period.

72. The process of claim 51 wherein said process is performed without temperature based inactivation of said uracil glycosylase.

73. The process of claim 51 wherein said uracil glycosylase inhibitor is Bacillus subtilis phages PBS1 uracil glycosylase inhibitor or Bacillus subtilis phages PBS2 uracil glycosylase inhibitor.

74. The process of claim 51 wherein said dNTP consists of dATP, dUTP, dCTP and dGTP.

75. The process of claim 51 wherein said dNTP does not contain dTTP.