Recombinase Polymerase Amplification

• US 20080293045A1
• Filed: 04/11/2005
• Published: 11/27/2008
• Est. Priority Date: 02/21/2002
• Status: Active Grant

• Current Assignee: Abbott Diagnostics Scarborough, Inc. (Abbott Laboratories Incorporated)
• Original Assignee: Alere San Diego Incorporated (Abbott Laboratories Incorporated)
• Inventors: Armes, Niall A., Piepenburg, Olaf, Williams, Colin H., Stemple, Derek L.
• Granted Patent: US 7,666,598 B2
• Time in Patent Office: Days
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/70 involving virus or bacteriophage immunoassay for viruses G01N33/56983
• CPC Class Codes: C12Q 2521/507 Recombinase
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins
• CPC Class Codes: C12Q 2527/101 Temperature
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]

Abstract

This disclosure describes related novel methods for Recombinase-Polymerase Amplification (RPA) of a target DNA that exploit the properties of recombinase and related proteins, to invade double-stranded DNA with single stranded homologous DNA permitting sequence specific priming of DNA polymerase reactions. The disclosed methods have the advantage of not requiring thermocycling or thermophilic enzymes, thus offering easy and affordable implementation and portability relative to other amplification methods. Further disclosed are conditions to enable real-time monitoring of RPA reactions, methods to regulate RPA reactions using light and otherwise, methods to determine the nature of amplified species without a need for gel electrophoresis, methods to improve and optimize signal to noise ratios in RPA reactions, methods to optimize oligonucleotide primer function, methods to control carryover contamination, and methods to employ sequence-specific third ‘specificity’ probes. Further described are novel properties and approaches for use of probes monitored by light in dynamic recombination environments.

Claims

1. A recombinase polymerase amplification process of DNA amplification of a double stranded target nucleic acid molecule comprising a first and second strand of DNA, comprising the steps of

(a) contacting in a solution a uvsX recombinase agent with a first and a second nucleic acid primer to form a first and a second nucleoprotein primer, wherein said nucleic acid primers comprise a single stranded region at its 3′

end;

(b) contacting the first and the second nucleoprotein primers to said double stranded target nucleic acid molecule thereby forming;

(1) a first double-stranded structure at a first portion of said first strand and

(2) a second double stranded structure 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 one another on the same double-stranded template nucleic acid molecule;

(c) extending the 3′

end of said first and second nucleoprotein primer with dNTPs and one or more DNA polymerases with strand-displacing properties to generate a first and second double-stranded nucleic acid product and a first and second displaced strands of nucleic acid product; and

(d) repeating (b) and (c) until a desired degree of amplification is reached;

wherein the nucleic acid product can serve as the double stranded target sequence in step (d);

wherein said solution further comprises a gp32 single-stranded DNA binding protein at a concentration sufficient to saturate the first and second primers at step (a);

wherein said solution further comprises a recombinase loading factor uvsY at a concentration of between 0.2 and 8 micromolar;

wherein said solution further comprises a crowding agent at a concentration of between 1% and 12% such that the crowding agent stimulates amplification;

wherein said one or more DNA polymerase lack 5′

to 3′

exonuclease activity and lack FLAP endonuclease activity;

wherein said solution further comprises a hydrolysable nucleoside triphosphate sufficient to support uvsX-loaded DNA filament disassembly and assembly; and

wherein the reaction is performed at a temperature of between 20°

C. and 50°

C.

2. The process of claim 1, wherein said first and second displaced strands of nucleic acid product are converted to double-stranded DNA by a concurrent recombinase polymerase reaction with the first or second nucleoprotein primer.

3. The process of claim 1 wherein the first and second displaced strands of nucleic acid are converted to double-stranded DNA by:

(a) contacting the first or the second primer to said displaced strand; and

(b) extending said first or second primer with said one or more polymerases and dNTPs.

5. The process of claim 1 wherein said first and second displaced strands are at least partially complementary to each other and hybridize to form a double stranded or partially double stranded nucleic acid.

6. The process of claim 5 wherein the partially double stranded nucleic acid is converted to a fully double stranded nucleic acid by a polymerase and dNTPs.

7. The process of claim 1 wherein the first and second displaced strands of nucleic acid comprise two regions of nucleic acid at its 3′

end which can form a self-priming hairpin structure.

8. The process of claim 1 wherein said nucleic acid primers are selected from the group consisting of DNA, RNA, PNA, LNA, morpholino backbone nucleic acid, phosphorothiorate backbone nucleic acid, and a combination thereof.

9. The process of claim 1 wherein the uvsX recombinase agent is a fusion protein comprising a short tag sequence at the N or C terminus.

10. The process of claim 1 wherein the uvsX recombinase agent comprises a C terminal deletion of acidic residues.

11. The process of claim 1 wherein the uvsX recombinase agent is present at a concentration selected from the group consisting of between about 0.2 μ

M and about 1 μ

M, between about 1 μ

M and about 4 μ

M, between about 4 μ

M and about 6 μ

M, and between about 6 μ

M and about 12 μ

M.

12. The process of claim 1 wherein said solution further comprises one or more accessory agents selected from the group consisting of single-strand binding protein, helicase, resolvase, RuvA, RuvB, RuvC, RecG, PriA, PriB, PriC, DnaT, DnaB, DnaC, DnaG, DnaX clamp loader, polymerase core complex, DNA ligase, a sliding clamp, E. coli β

-dimer sliding clamp, eukaryotic PCNA sliding clamp, T4 sliding clamp gp45, E. coli SSB protein, gp32 protein, gp32 with additional amino acids at the N terminus, gp32 with additional amino acids at the C terminus, gp32 with the substitution of lysine 3 with a non lysine amino acid, and gp32 with the substitution of arginine 4 with a non arginine amino acid, and derivatives and combinations thereof.

13. The process of claim 12 wherein the additional amino acids comprise a poly His tag.

14. The process of claim 12 wherein said derivative is selected from the group consisting of gp3 2(N), gp3 2(C), gp32(C)K3A, gp3 2(C)R4Q, gp3 2(C)R4T, gp3 2K3A, gp32R4Q, gp32R4T and a combination thereof.

15. The process of claim 12 wherein said single stranded stabilizing agent is present at a concentration between about 1 μ

M and 20 μ

M.

16. The process of claim 1 wherein said T4 gp32 is at a concentration of between 1 μ

M and 30 μ

M.

17. The process of claim 1 wherein said crowding agent is selected from the group consisting of polyethylene glycol, dextran, ficoll, PEG1450, PEG8000, PEG10000, PEG compound with molecular weight between 15,000 to 20,000 daltons and derivatives and a combinations thereof.

18. The process of claim 17 wherein said polyethylene glycol is between 1 to 12% of weight or volume of the reaction.

19. The process of claim 1 wherein the uvsY further comprise a poly-His tag at the N or C terminus.

20. The process of claim 1 wherein said solution further comprises a cofactor for the recombinase agent selected from the group consisting of ATP, ATP analog, ATP-γ

-S and derivatives and combinations thereof.

21. The process of claim 1, wherein said solution further comprises an ATP regeneration system to convert ADP to ATP, a system to regenerate ADP from AMP, pyrophosphatase or a combination thereof.

22. The process of claim 21 wherein said ATP regeneration system comprises phosphocreatine and creatine kinase, ADP-β

-S, and a combination thereof.

23. The process of claim 1 wherein the one or more DNA polymerase are selected from the group consisting of prokaryotic polymerase, eukaryotic polymerase and phage-encoded polymerase.

24. The process of claim 23 wherein the procaryotic polymerase is selected from the group consisting of E. coli DNA polymerase I Klenow fragment, Bacillus stearothermophilus polymerase I large fragment, Phi-29 DNA polymerase, T7 DNA polymerase, Bacillus subtilis Pol I large fragment, E. coli DNA polymerase I, E. coli DNA polymerase II, E. coli DNA polymerase IV, E. coli DNA polymerase V and a combination thereof.

25. The process of claims 24 wherein at least one of said one or more DNA polymerase is a fusion protein comprising a short tag sequence at the N or C terminus.

26. The process of claim 1 wherein at least one nucleic acid primer comprise a non-phosphate linkage between the two bases at its 3′

end and is resistant to 3′

to 5′

nuclease activity or at least one locked nucleic acid at its 3′

ultimate base or 3′

penultimate base or both.

27. The process of claim 1 wherein the target nucleic acid molecule is selected from the group consisting of supercoiled nucleic acid, linear nucleic acid with two ends and wherein both ends are linked to a non-target nucleic acid molecule, linear nucleic acid with two ends and wherein one end is linked to a non-target nucleic acid molecule, linear nucleic acid, single-stranded nucleic acid which is converted to a double stranded nucleic acid by a polymerase, and a double stranded nucleic acid denatured by the action of heat or chemical treatment.

28. The process of claim 1 wherein the first or second nucleic acid primers are at least 20 residues in length or at least 30 residues in length.

29. The process of claim 1 wherein said first nucleic acid primer and said second nucleic acid primer are oriented with their 3′

ends toward one another on the same double-stranded template nucleic acid molecule and wherein their 3′

ends are separated by a distance selected from the group consisting of between 0 and 10 bases after step (b), 10 and 30 bases after step (b), between 30 and 100 bases after step (b), more than 100 bases after step (b), and more than 1,000,000 bases after step (b).

30. The process of claim 1 wherein said process is performed in the presence of short, 3′

-blocked oligonucleotides with a sequence complementary to the 3′

region of the first or the second nucleic acid primers.

31. The process of claim 30 wherein the short oligonucleotides are tethered to the first or the second nucleic acid primers through a covalent linker between the 5′

extreme of the nucleic acid primer and the 3′

or 5′

end of the short oligonucleotide.

32. The process of claim 1 wherein at least one nucleic acid primer contains a short 5′

region not complementary to the target nucleic acid molecule but complementary to a 3′

region of the same primer.

33. The process of claim 1, wherein said process is performed between 20°

C. and 40°

C. or between 20°

C. and 30°

C.

34. The process of claim 1 wherein said process is performed without temperature induced melting of the template nucleic acid.

35. The process of claim 1, wherein the first and the second nucleoprotein primers are each present in a molar concentration that is at least 1000 fold greater than a concentration of the target nucleic acid molecule.

36. The process of claim 1 wherein at least one nucleic acid primer comprises one or more bases that are not complementary to the target nucleic acid molecule at its 5′

end.

37. The process of claim 36 wherein the one or more bases that are not complementary to the target nucleic acid comprises the sequence of a restriction endonuclease recognition site.

38. The process of claim 1 wherein at least one nucleic acid primer is partially single stranded and partially double stranded, wherein said primer is comprised of a longer invading-strand which possesses a specific 3′

primer sequence and a shorter non-invading strand, which is complementary to the 5′

end of the longer invading strand.

39. The process of claim 1 wherein said first nucleic acid primer comprise a first invading strand and a first non invading strand and said second nucleic acid primer comprise a second invading strand and a second non-invading strand and said first and second non-invading strand are complementary to each other.

40. The process of claim 39 wherein said invading strand, said non-invading strand, or both are labeled with a detectable moiety.

41. The process of claim 39 wherein both the invading strand and the non-invading strand are labeled and the separation of the invading strand and non-invading strand is detected.

42. The process of claim 40 wherein said detectable moiety is selected from the group consisting of a fluorochrome, an enzyme, a fluorescence quencher, an enzyme inhibitor, a radioactive label, and a combination thereof.

43. The process of claim 39 wherein both said invading strand and said non-invading strand are labeled and wherein the invading strand is labeled with a first fluorochrome or a first enzyme and wherein the non-invading strand is labeled with a second fluorochrome, a second enzyme, a fluorescence quencher or an enzyme inhibitor.

44. The process of claim 39 wherein both said invading strand and said non-invading strand are labeled and wherein the non-invading strand is labeled with a first fluorochrome or a first enzyme and wherein the invading strand is labeled with a second fluorochrome, a second enzyme, a fluorescence quencher or an enzyme inhibitor.

45. The process of claim 1 wherein one or both nucleic acid primers is a single stranded primer.

46. The process of claim 45, wherein said nucleic acid primer is labeled with a detectable moiety selected from the group consisting of a fluorochrome, an enzyme, a fluorescence quencher, an enzyme inhibitor, a radioactive label and a combination thereof.

47. The process of claim 1 wherein the uvsX recombinase agent is temperature-sensitive, wherein said temperature sensitive uvsX recombinase agent possesses strand-invasion activity at a permissive temperature and no strand-invasion activity at a non-permissive temperature.

48. The process of claim 1 wherein said process amplifies said target nucleic acid in an amount selected from the group consisting of at least 10 fold, at least 100 fold, at least 1000 fold, and at least 1,000,000 fold.

49. The process of claim 1 wherein said steps (b) and (c) are repeated at least once or at least five times.

50. The process of claim 1 wherein the target nucleic acid molecule contains at least one specific mutation that is associated with a genetic disease.

51. The process of claim 1 wherein the target nucleic acid molecule is contained in a pathogenic organism, an oncogene, an activated oncogene, or a repressed oncogene.

52. The process of claim 1 wherein at least one primer is complementary to a sequence associated with a genetic disease, a nucleic acid sequence in a pathogenic organism, an oncogene, an activated oncogene, or a repressed oncogene.

53. A process of nested recombinase polymerase amplification comprising the steps of:

(a) amplifying a region of DNA using the recombinase polymerase amplification process of claim 1 using a first and a second primer to produce a first amplified product;

(b) amplifying said amplified product using a third and a fourth primer using the recombinase polymerase amplification process of claim 1 to produce a second amplified product, wherein said second amplified product is a smaller sequence contained within said first amplified product.

54. The process of claim 53 wherein said first primer, said second primer, said third primer and said fourth primer are each different.

55. The process of claim 53 wherein said one of said first and said second primer is identical to one of said third and fourth primer.

4. The process of claim 4 wherein said contacting step produces a DNA duplex hybrid.

56. A recombinase polymerase amplification process of DNA amplification of a target nucleic acid molecule, said target nucleic acid molecule comprising a first and second strand of DNA, comprising the steps of

(a) contacting in a solution a uvsX recombinase protein with a nucleic acid primer to form a nucleoprotein primer which comprises a single stranded region at its 3′

end;

(b) contacting the nucleoprotein primer to said target nucleic acid molecule thereby forming a double-stranded structure at a portion of said target nucleic acid molecule;

(c) extending a 3′

end of said nucleoprotein primer with dNTPs and one or more polymerases with strand-displacing properties to generate a double-stranded nucleic acid product and a displaced strand of nucleic acid product; and

(d) repeating (b) and (c) until a desired degree of amplification is reached;

wherein the nucleic acid product can serve as the double stranded target sequence in step (d);

wherein said solution further comprises a gp32 single-stranded DNA binding protein at a concentration sufficient to saturate the nucleoprotein primers in step (a);

wherein said solution further comprises a recombinase loading factor uvsY at a concentration between 0.2 and 8 micromolar;

wherein said solution further comprises a crowding agent at a concentration between 1% and 12% such that the crowding agent stimulates amplification;

wherein said one or more polymerases lacks 5′

to 3′

exonuclease activity and lacks FLAP endonuclease activity;

wherein said solution further comprises a hydrolysable nucleoside triphosphate sufficient to support uvsX-loaded DNA filament disassembly and assembly; and

wherein a fraction of said dNTPs comprises chain-terminating dNTPs;

wherein at least a fraction of said dNTPs is labeled with a detectable marker.

57. A process of detecting the presence or absence of a recombinase polymerase amplification amplified nucleic acids product in a sample, comprising the steps of:

(a) contacting in solution a T4 recombinase agent with a first and second nucleic acid primer to form a first and second nucleoprotein primer, wherein said nucleic acid primer comprises a single stranded region at its 3′

, wherein said first nucleic acid primer is labeled with a first member of a first binding pair, and wherein said second nucleic acid primer is labeled with a first member of a second binding pair;

(b) contacting the first and the second nucleoprotein primer to a sample to form a complex of the first and second nucleoprotein primer and amplification product;

(c) contacting said complex with a first mobile solid support coated with a second member of said first binding pair and a second mobile solid support coated with a second member of said second binding pair;

(d) determining if said first mobile solid support is co-localized with said second mobile solid support to determine the presence of said amplification amplified nucleic acid product;

wherein the recombinase loading factor T4 uvsY is included in the reaction at a concentration of between 0.2 and 8 micromolar;

wherein a crowding agent is included at a concentration between 1% and 12% such that the crowding agent stimulates amplification;

wherein a hydrolysable nucleoside triphosphate is employed to support recombinase activity such that uvsX-loaded DNA filaments are capable of efficient disassembly as well as assembly.

58. The process of claim 57 wherein the first nucleic acid primer has the same nucleic acid sequence as a primer used to produce the amplification product, or wherein the second nucleic acid primer has the same nucleic acid sequence as a primer used to produce the amplification product or both.

59. The process of claim 57 wherein the first or second primer is associated with the target DNA by formation of a triple helix mediated by a recombinase after amplification of the target by a recombinase polymerase amplification reaction.

60. A recombinase polymerase amplification process of DNA amplification of a double stranded target molecule comprising the steps of:

(a) contacting in solution a recombinase agent selected from the group consisting of uvsX and recA with a first and a second nucleic acid primer to form a first and a second nucleoprotein primers;

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

ends of said first nucleic acid primer and said second nucleic acid primer are oriented toward each other on the template nucleic acid molecule;

(c) contacting said target nucleic acid molecule to primosome assembly proteins and a clamp loader/polymerase holoenzyme complex to form a replication fork at said first portion of the target nucleic acid molecule and a second replication fork at said second portion of the target nucleic acid molecule;

(d) contacting said target molecule to a DNA polymerase III core, a DNA polymerase I, a primase, a helicase, and a ligase to allow coupled leading and lagging strand DNA synthesis and migration of each replication fork towards each other; and

(e) repeating (b), (c) and (d) until a desired degree of amplification is reached;

wherein a gp32 single-stranded DNA binding protein is included at a concentration at least sufficient to saturate the first and second nucleic acid primers in solution at step (a);

wherein the recombinase loading factor uvsY or the E. coli recO and recR gene products are included in the reaction at a concentration of between 0.2 and 8 micromolar;

wherein a crowding agent is included at a concentration of at least 1% to 12% such that the crowding agent stimulates amplification;

wherein a hydrolysable nucleoside triphosphate is employed to support recombinase activity such that recombinase-loaded DNA filaments are capable of efficient disassembly as well as assembly.

61. The process of claim 60 which is performed in the presence of dNTP, rNTP, single stranded DNA binding protein, or a combination thereof.

62. The process of claim 60 wherein said primosome assembly proteins are selected from the group consisting of PriA, PriB, PriC, DnaT, DnaC, DnaB, DnaG, Pol III holoenzyme, and a combination thereof.

63. The process of claim 60 wherein the clamp loader is a DnaX clamp loader complex.

64. The process of claim 60 wherein the DNA polymerase III holoenzyme is E. coli polymerase III.

65. The process of claim 60 wherein the DNA polymerase I is E. coli polymerase I.

66. The process of claim 60 wherein the primase is E. coli DnaG primase.

67. The process of claim 60 wherein the helicase is E. coli DnaB helicase.

68. A recombinase polymerase amplification process of DNA amplification comprising the steps of:

(a) combining the following reagents in a solution

(1) at least one recombinase;

(2) at least one single stranded DNA binding protein;

(3) at least one DNA polymerase;

(4) dNTPs or a mixture of dNTPs and ddNTPs;

(5) a crowding agent such that the crowding agent stimulates amplification;

(6) a buffer;

(7) a reducing agent;

(8) ATP or hydrolysable ATP analog;

(9) at least one recombinase loading protein;

(10) a first primer and optionally a second primer; and

(11) a target nucleic acid molecule; and

(b) incubating said solution until a desired degree of amplification is achieved.

69. The process of claim 68 wherein said recombinase is selected from the group consisting of uvsX, recA and derivatives and combinations thereof.

70. The process of claim 68 wherein said recombinase is a fusion protein comprising a short tag sequence at a N or C terminus.

71. The process of claim 68 wherein the recombinase comprises a C terminal deletion of acidic residues.

72. The process of claim 68 wherein the recombinase is present at a concentration selected from the group consisting of between about 0.2 μ

M and about 1 μ

M, between about 1 μ

M and about 4 μ

M, between about 4 μ

M and about 6 μ

M, between about 6 μ

M and about 12 μ

M, and between about 0.2 μ

M and about 12 μ

M.

73. The process of claim 68 wherein said single stranded DNA binding protein is selected from the group consisting of gp32, and derivatives and combinations thereof.

74. The process of claim 73 wherein said gp32 derivative is selected from the group consisting of gp32(N), gp32(C), gp32(C)K3A, gp32(C)R4Q, gp32(C)R4T, gp32K3A, gp32R4Q, gp32R4T and a combination thereof.

75. The process of claim 68 wherein said single stranded DNA binding protein is present at a concentration of between 1 μ

M and 30 μ

M.

76. The process of claim 68 wherein said DNA polymerase is a eukaryotic polymerase or a prokaryotic polymerase.

77. The process of claim 76 wherein said eukaryotic polymerase is selected from the group consisting of pol-α

, pol-β

, pol-δ

, pol-ε and

derivatives and combinations thereof.

78. The process of claim 76 wherein said prokaryotic polymerase is selected from the group consisting of E. coli DNA polymerase I Klenow fragment, bacteriophage T4 gp43 DNA polymerase, Bacillus stearothermophilus polymerase I large fragment, Phi-29 DNA polymerase, T7 DNA polymerase, Bsu Pol I, E. coli DNA polymerase I, E. coli DNA polymerase II, E. coli DNA polymerase III, E. coli DNA polymerase IV, E. coli DNA polymerase V and derivatives and combinations thereof.

79. The process of claim 68 wherein said DNA polymerase is a fusion protein comprising a short tag sequence at the N or C terminus.

80. The process of claim 79 wherein said DNA polymerase is at a concentration of between 10,000 units/ml to 16 units/ml or between 5000 units/ml to 500 units/ml.

81. The process of claim 68 wherein the one or more DNA polymerase includes at least one DNA polymerase which lacks 3′

-5′

exonuclease activity.

82. The process of claim 68 wherein said one or more DNA polymerase comprises a DNA polymerase with strand displacing properties.

83. The process of claim 68 wherein said dNTP is at a concentration of 1 mM to 40 μ

M.

84. The process of claim 68 wherein said crowding agent is polyethylene glycol, dextran, ficoll or a combination thereof.

85. The process of claim 84 wherein said polyethylene glycol is between 1 to 12% of a weight or a volume of the reaction.

86. The process of claim 85 wherein said polyethylene glycol is selected from the group consisting of PEG1450, PEG8000, PEG10000, PEG mixture with molecular weight between 15,000 to 20,000 daltons, and a combination thereof.

87. The process of claim 68 wherein said buffer is a Tris-HCl buffer, a Tris-Acetate buffer, or a combination thereof.

88. The process of claim 87 wherein said buffer is at a concentration of between 10 to 60 mM and at a pH of between 6.5 and 9.0.

89. The process of claim 68 wherein said reducing agent is DTT.

90. The process of claim 68 wherein said reducing agent is at a concentration of 1 to 10 mM.

91. The process of claim 68 wherein said ATP or ATP analog is selected from the group consisting of ATP, ATP-γ

-S, ATP-β

-S and ddATP.

92. The process of claim 91 wherein said ATP or ATP analog is at a concentration of 1 to 10 mM.

93. The process of claim 68 wherein said recombinase loading protein is selected from the group consisting of T4uvsY, E. coli recO, E. coli recR and derivatives and combinations thereof.

94. The process of claim 68 wherein said recombinase loading protein further comprise a poly-His tag at an N terminus, a C terminus, or at both an N and a C terminus.

95. The process of claim 68 wherein said recombinase loading protein is at a concentration between 0.2 and 8 μ

M.

96. The process of claim 68 wherein the first or second primer comprises DNA, RNA, PNA, LNA, morpholino backbone nucleic acid, phosphorothiorate backbone nucleic acid or a combination thereof.

97. The process of claim 68 wherein said first or second primer is at a concentration between 100 nM and 1000 nM

98. The process of claim 68 wherein the first or second primer comprises a non-phosphate linkage between the two bases at its 3′

end and is resistant to 3′

to 5′

nuclease activity.

99. The process of claim 68 wherein the first or second primer comprises at least one locked nucleic acid at its 3′

ultimate base or 3′

penultimate base.

100. The process of claim 68 wherein the first or second primer is at least 20 bases in length.

101. The process of claim 68 wherein the first or second primer is at least 30 bases in length.

102. The process of claim 68 wherein the first or second primer further comprises one or more bases at its 5′

end that are not complementary to the target nucleic acid.

103. The process of claim 102 wherein said one or more bases comprise the sequence of a restriction endonuclease recognition site.

104. The process of claim 68 wherein said first or second primer is partially double stranded with a single stranded region at its 3′

end.

105. The process of claim 68 wherein at least one primer is labeled with a detectable label.

106. The process of claim 105 wherein said detectable label is selected from the group consisting of a fluorochrome, an enzyme, a fluorescence quencher, an enzyme inhibitor, a radioactive label and a combination thereof.

107. The process of claim 68 wherein the target nucleic acid molecule is selected from the group consisting of supercoiled nucleic acid, linear nucleic acid with two ends and wherein both ends are linked to a non-target nucleic acid molecule, linear nucleic acid with two ends and wherein one end is linked to a non-target nucleic acid molecule, linear nucleic acid, and a single-stranded nucleic acid molecule which is converted to a double stranded nucleic acid by a polymerase or a double stranded nucleic acid denatured by the action of heat or chemical treatment.

108. The process of claim 68 wherein the target nucleic acid is at a concentration of less than 1000 copies per ml or between 1 and 10 copies per reaction.

109. The process of claim 68 wherein said incubation is between 5 minutes and 16 hours.

110. The process of claim 68 wherein said incubation is between 15 minutes and 3 hours.

111. The process of claim 68 wherein said incubation is between 30 minutes and 2 hours.

112. The process of claim 68 wherein said incubation is performed until a desired degree of amplification is achieved.

113. The process of claim 112 wherein said desired degree of amplification is selected from the group consisting of at least 10 fold, at least 100 fold, at least 1000 fold, at least 10,000 fold, at least 100,000 fold and at least 1000000 fold.

114. The process of claim 68 wherein said incubation is performed between 20°

C. and 50°

C., between 20°

C. and 40°

C., or between 20°

C. and 30°

C.

115. The process of claim 68 which is performed without temperature induced melting of the template nucleic acid.

116. The process of claim 68 wherein step (a) further comprises an accessory agent.

117. The process of claim 116 wherein said accessory agent is selected from the group consisting of helicase, resolvase and a combination thereof.

118. The process of claim 116 wherein the accessory agent are selected from the group consisting of RuvA, RuvB, RuvC, RecG, PriA, PriB, PriC, DnaT, DnaB, DnaC, DnaG, DnaX clamp loader, polymerase core complex, DNA ligase, a sliding clamp, DNA Polymerase III holoenzyme complex consisting of β

-Clamp, DnaX Clamp Loader, and the Polymerase Core Complex.

119. The process of claim 118 wherein said sliding clamp is the E. coli β

-dimer sliding clamp, the eukaryotic PCNA sliding clamp, or the T4 sliding clamp gp45 and a combination thereof.

120. The process of claim 68 wherein step (a) further comprises a RecA/ssDNA nucleoprotein filaments stabilizing agent.

121. The process of claim 120 wherein said stabilizing agent is selected from the group consisting of RecR, RecO, RecF and a combination thereof.

122. The process of claim 120 wherein said stabilizing agent is at a concentration of between 0.01 μ

M to 20 μ

M.

123. The process of claim 68 wherein said solution further comprises an ATP regeneration system to convert ADP to ATP, a system to regenerate ADP from AMP, a system to convert pyrophosphate to phosphate, or a combination thereof.

124. The process of claim 123 wherein said ATP regeneration system comprises phosphocreatine and creatine kinase.

125. The process of claim 68, wherein one or more reagents are freeze dried before said step (a), said reagents selected from the group consisting of the recombinase, the single stranded DNA binding protein, the DNA polymerase, the dNTPs or the mixture of dNTPs and ddNTPs, the reducing agent, the ATP or ATP analog, the recombinase loading protein, crowding agent, buffer, salts, DTT, and the first primer and optionally a second primer or a combination thereof.

126. A recombinase polymerase amplification process of DNA amplification comprising the steps of:

(a) combining the following reagents in a reaction

(1) a uvsX recombinase at a concentration of between 0.2 to 12 μ

M;

(2) a gp32 single stranded DNA binding protein at a concentration between 1 to 30 μ

M;

(3) Bsu polymerase at a concentration between 50 to 5000 units per ml;

(4) dNTPs or a mixture of dNTPs and ddNTPs at a concentration of between 1-500 μ

M;

(5) polyethylene glycol at a concentration of between 1% to 12% by weight or by volume such that the polyethylene glycol stimulates amplification;

(6) Tris-acetate buffer at a concentration of between 25 mM to 60 mM;

(7) DTT at a concentration of between 1 mM-10 mM;

(8) ATP at a concentration of between 1.5 mM-5 mM;

(9) uvsY at a concentration of between 0.2 μ

M-8 μ

M;

(10) a first primer and optionally a second primer, wherein said primers are at a concentration of between 10 nM to 1 μ

M; and

(11) a target nucleic acid molecule of at least one copy;

(b) incubating said reaction until a desired degree of amplification is achieved.

127. A recombinase polymerase amplification process of DNA amplification comprising the steps of:

(a) combining the following reagents in a reaction;

(1) 100-200 ng/μ

l uvsX recombinase;

(2) 600 ng/μ

l gp32;

(3) 20 ng/μ

l Bsu polymerase;

(4) 200 μ

M dNTPs;

(5) 1 mM DTT

(6) 3 mM ATP or a hydrolysable ATP analog;

(7) 16 ng/μ

l to 60 ng/μ

l uvsY

(8) 300 nM of a first primer and 300 nM of a second primer;

(9) 100 mM Potassium acetate

(10) 8-16 mM Magnesium acetate

(11) 25 mM Phosphocreatine

(12) 100 ng/μ

l Creatine kinase

(b) freeze drying the reagents from said step (a) to form freeze dried reagents;

(c) reconstituting said freeze dried reagents with

(1) Tris-acetate buffer at a concentration of between 1 mM to 60 mM;

(2) polyethylene glycol at a concentration of between 1% to 12% by weight or by volume such that the polyethylene glycol stimulates amplification;

(3) a target nucleic acid;

(d) incubating said reaction until a desired degree of amplification is achieved.

130. The process of claims 127, 128 or 129 wherein trehalose is added to the reagent before freeze drying.

131. The process according to claim 130 wherein trehalose is added to the reagent to a concentration of between 1 0 mM to 200 mM.

128. A recombinase polymerase amplification process of DNA amplification comprising the steps of:

(a) combining the following reagents in a reaction;

(1) 100-200 ng/μ

l uvsX recombinase;

(2) 300-1000 ng/μ

l gp32;

(3) 10-50 ng/μ

l Bsu polymerase or T4 polymerase;

(4) 50-500 μ

M dNTPs;

(5) 0.1 to 10 mM DTT

(6) 3 mM ATP or a hydrolysable ATP analog;

(7) 16 ng/μ

l to 60 ng/μ

l uvsY

(8) 50-1000 nM of a first primer and 50-1000 nM of a second primer;

(9) 40-160 mM Potassium acetate

(10) 5-20 mM Magnesium acetate

(11) 10-40 mM Phosphocreatine

(12) 50-200 ng/μ

l Creatine kinase

(b) freeze drying the reagents from said step (a) to form freeze dried reagents;

(c) reconstituting said freeze dried reagents with

(1) Tris-acetate buffer at a concentration of between 1 mM to 60 mM;

(2) polyethylene glycol at a concentration of between 1% to 12% by weight or by volume such that the polyethylene glycol stimulates amplification;

(3) a target nucleic acid;

(d) incubating said reaction until a desired degree of amplification is achieved.

129. A recombinase polymerase amplification process of DNA amplification comprising the steps of:

(a) combining the following reagents in a reaction;

(1) 100-200 ng/μ

l uvsX recombinase;

(2) 300-1000 ng/μ

l gp32;

(3) 10-50 ng/μ

l Bsu polymerase or T4 polymerase;

(4) 50-500 μ

M dNTPs;

(5) 0.1 to 10 mM DTT

(6) 3 mM ATP or an ATP analog;

(7) 16 ng/μ

l to 60 ng/μ

l uvsY

(8) 50-1000 nM of a first primer and 50-1000 nM of a second primer;

(9) 40-160 mM Potassium acetate

(10) 5-20 mM Magnesium acetate

(11) 10-40 mM Phosphocreatine

(12) 50-200 ng/μ

l Creatine kinase

(13) polyethylene glycol at a concentration of between 1% to 12% by weight or by volume such that the polyethylene glycol stimulates amplification;

(b) freeze drying the reagents from said step (a) to form freeze dried reagents;

(c) reconstituting said freeze dried reagents with

(1) Tris-acetate buffer at a concentration of between 1 mM to 60 mM;

(2) a target nucleic acid;

(d) incubating said reaction until a desired degree of amplification is achieved.

132. An RPA process of DNA amplification of a double stranded target sequence, said target sequence comprising a first and a second strand of DNA, said process comprising the steps of:

(a) contacting a recombinase agent with a first and a second nucleic acid primer to form a first and a second nucleoprotein primer;

(b) contacting the first and second nucleoprotein primers to said double stranded target sequence thereby forming a first double stranded structure at a first portion of said first strand and form a double stranded structure at a second portion of said second strand such that the 3′

ends of said first nucleic acid primer and said second nucleic acid primer are oriented toward each other on the same template nucleic acid molecule;

(c) extending the 3′

end of said first and second nucleoprotein primer with one or more polymerases and dNTPs to generate a first and second double stranded nucleic acid and a first and second displaced strand of nucleic acid;

(d) continuing the reaction through repetition of (b) and (c) until a desired degree of amplification is reached

wherein the generated double stranded nucleic acid can serve as the double stranded target sequence.

133. The process of claim 132 wherein said first or second nucleic acid primers have a length that is below the length required for optimal strand exchange to decrease nonspecific amplification reactions.

134. The process of claim 132 wherein said first or second nucleic acid primers is between 25 and 29 residues in length or between 16 and 24 residues in length.

135. The process of claim 132 further comprising the step of ligating additional nucleic acid sequence to at least one end of said target nucleic acid before step (b) to reduce foldback priming or to modulate nucleoprotein recombination/elongation rate.

136. The process of claim 132 wherein said first or second nucleic acid primers comprises one or more modified sugar residues.

137. The process of claim 136 wherein said modified sugar residues is selected from the group consisting of locked nucleic acid, ribose, 2′

-O-methyl ribose, acyclic sugar residues and a combination thereof.

138. The process of claim 136 wherein said modified sugar residue is at the 3′

most position of the primer.

139. The process of claim 132 further comprising the step of monitoring total DNA concentration with a sensor during said process.

140. The process of claim 139 wherein said sensor detects DNA concentration by fluorescence measurements, detects a fluorescence from SYBR gold, or SYBR green, detects DNA concentration in a sequence specific manner or a combination thereof.

141. The process of claim 139 wherein the sensor functions by hybridization to single-stranded targets in the reaction.

142. The process of claim 141 wherein said hybridization is aided by melting/annealing agents such as single-stranded DNA binding proteins or recombinase.

143. The process of claim 139 wherein the sensor functions by recombinase-mediated targeting to double-stranded DNA targets.

144. The process of claim 139 wherein the sensor comprises two or more, fluorescently labeled sequence-specific oligonucleotides which undergo specific fluorescence resonance energy transfer only when hybridized to adjacent target sites in the target DNA.

145. The process of claim 139 wherein a fluorophore present in the sensor is separated from a quencher also attached to the sensor by the action of a nuclease which functions only when the sensor is bound to its target site.

146. The process of claim 145 wherein the nuclease is a double stranded nuclease.

147. The process of claim 146 wherein said double stranded nuclease is a restriction endonuclease.

148. The process of claim 132 wherein said RPA process is started or controlled by a light-driven photodeprotection of one or more caged substance.

149. The process of claim 148 wherein said caged substance is caged ATP or caged oligonucleotides.

150. The process of claim 148 wherein one or more of said primers, ATP or nucleotides is a caged molecule and wherein steps (a), (b) or (c) is controlled or initiated by light mediated photo-deprotection of said caged molecule to produce uncaged molecules.

151. The process of claim 148 wherein the concentration of uncaged molecules are monitored.

152. The process of claim 148 wherein said uncaged molecule is consumed in one or more cycles of RPA thereby stopping said RPA reaction until additional uncaged molecules are produced.

153. The process of claim 148 wherein said caged molecule is selected from the group consisting of caged ATP, caged dATP, and caged UTP.

154. The process of claim 132 wherein one or more of said primers, ATP or NTPs are at a concentration which can only support one cycle of steps (a), (b) or (c), and wherein additional steps of (a) (b) and (c) are initiated by the addition of a new amount of one or more of said primers, ATP or NTPs.

155. The process of claim 154 wherein said new amount of one or more of said primers, ATP or NTPs is supplied at a concentration which can only support one cycle of steps (a), (b) or (c).

156. The process of claim 154 wherein said new amount of one or more of said primers, ATP or NTPs is supplied by the action of an enzymatic system.

157. The process according to claim 132 wherein said RPA process is performed in the presence of an enzyme system selected from the group consisting of:

an enzyme to remove ADP accumulating in the reaction by its direct conversion to a compound that does not regulate recombinase activity;

an enzyme system to remove ADP accumulating in the reaction by its direct conversion to a compound that is not ADP, ATP or an equivalent;

an enzyme system to convert all ADP to ATP to reduce ADP concentration in said reaction; and

an enzyme system to reduce ATP in said process to stop the RPA reaction after steps (b) and (c) and wherein addition steps (b) and (c) are initiated with the addition of ATP.

158. The process of claim 157 wherein said addition of ATP is performed by uncaging a caged ATP.

159. The process of claim 158 wherein said caged ATP is uncaged by light excitation.

160. The process of claim 159 wherein said light excitation leads to an effective concentration increase of between 10 μ

M to 30 μ

M ATP, 30 μ

M to 60 μ

M ATP, 60 μ

M to 90 μ

m ATP, 90 μ

m to 200 μ

M ATP, 200 μ

M to 500 μ

M ATP or 500 μ

M to 3000 μ

m ATP.

161. The process of claim 132 or 160 wherein ATP concentration in the reaction is monitored by a sensor.

162. The process of claim 161 wherein said ATP sensor comprises luciferase and luciferin.

163. The process of claim 132 wherein said primers comprise high cytosine content to improve recombinase interaction.

164. The process of claim 163 wherein said cytosine content is greater than 75% of the bases of the primer.

165. The process of claim 132 which is performed in a gel matrix which restricts the mobility of the template nucleic acid and the RPA product.

166. The process of claim 165 wherein said gel matrix is a polony gel matrix.

167. A process of nested RPA comprising the steps of:

(a) performing an RPA reaction of claim 132 in the presence of a first pair of outer primers and a second pair of inner primers in said step (a);

wherein said first pair of outer primers comprise nucleic acid sequence such that the 3′

ends of said pair of outer primers are oriented toward each other on the same template nucleic acid molecule after step (b);

wherein said second pair of inner primers comprise nucleic acid sequence such that the 3′

ends of said pair of inner primers are oriented toward each other on the same template nucleic acid molecule after step (b),

further wherein said second pair of inner primers hybridize to the target nucleic acid at a location that is within the hybridization location of said first pair of outer primers.

168. The process of claim 167 wherein the first pair of outer primers support more rapid amplification kinetics because of length or composition compared to the inner primer pair.

169. The process of claim 167 wherein the first pair of outer primers are at a concentration that is lower than the concentration of the second pair of inner primers.

170. The process of claim 167 wherein at least one primer is immobilized or immobilizable and at least one other primer is a labeled primer, and the hybridization of the first primer to the second primer is determined after step (d).

171. The process of claim 170 wherein the immobilizable primer and the labeled primer are the only primers in said RPA reaction.

172. The process according to claim 170 in which the immobilizable primer hybridizes to a unique sequence within a DNA generated by 2 or more other primers in the RPA reaction.

173. The process of claim 170 wherein said immobilizable primer is a backfire primer that stable product capture via formation a non-branch migratable duplex.

174. The process of claim 170 wherein the labeled primer is a backfire primer that permits stable product capture via formation of a non-branch migratable duplex.

175. The process of claim 167 wherein said process is performed in the presence of dUTP such that dUTP is incorporated into the RPA product wherein said RPA product is susceptible to a dUTP deglycosylase.

176. The process of claim 167 further comprising the step of monitoring said RPA reaction by monitoring the fluorescence of a primer, wherein one of said primer is a dual-labeled oligonucleotide probe consisting of a fluorophore and a quencher separated by a distance of no more than 10-12 residues such that the quencher is more efficient when the primer is in the unhybridized state than in the hybridized state.

177. The process of claim 167 wherein further comprising the step of monitoring said RPA reaction by monitoring the fluorescence of a primer, wherein one of said primer is a dual-labeled oligonucleotide probe consisting of a fluorophore and a quencher separated by a distance of no more than 10-12 residues and wherein fluorescence is increased as a consequence of nuclease activity acting preferentially on duplex hybrids between the probe and the target leading ultimately to lower association between the quencher and the fluorophore.

178. The process of claim 167 wherein one or more of the primers is initially 3′

-blocked, and contains a tetrahydrofuranyl residue, and wherein an E. coli Nfo protein or functional equivalent is present in said RPA process, wherein the blocked oligonucleotide forms duplex hybrids with the target DNA and is subsequently processed by the Nfo protein or functional homolog; and

wherein a polymerase extends the free 3′

end of the probe generated by the Nfo cleavage.

179. The process of claim 178 wherein said blocked primer contains a tetrahydrofuranyl residue, a fluorophore, and a quenching minor groove binding dye, such that on forming duplex hybrids the quenching is decreased by virtue of a change in absorption properties of the minor groove binding dye such that a rise in fluorescence emission from the fluorophore increases as a consequence of DNA amplification.

180. The process of claim 167 wherein said recombinase is selected from the group consisting of T4 phage UvsX, E. coli RecA, and eukaryotic Rad51.

181. The process of claim 167 wherein said RPA is performed in the presence of a crowding agent such that the crowding agent stimulates amplification.

182. The process of claim 181 wherein the molecular crowding agent is selected from the group consisting of polyethylene glycol, Ficoll, Dextan, and polyvinylpyrollidone.

183. The process of claim 167 wherein said RPA is performed in the presence of a re-loading promoting agent.

184. The process of claim 183 in which the re-loading promoting agent is selected from the group consisting of T4 UvsY, E. coli RecO, E. coli RecR, and combinations and homologs thereof.

185. The process of claim 167 wherein said process is performed in the presence of an enzyme selected from the group consisting of DNA ligase, helicase, RNA polymerase, reverse transcriptase, FLAP endonuclease, 5′

-3′

exonuclease, 3′

-5′

exonuclease, restriction endonuclease, endonuclease and base-specific damaged base-specific nuclease.

186. The process of claim 185 wherein said FLAP endonuclease is human FEN1 or equivalents thereof.

187. The process of claim 185 wherein said endonuclease is a helix-distortion recognizing endonuclease or base-specific damaged base-specific nuclease

188. The process of claim 187 wherein said helix-distortion recognizing endonuclease is an S1 nuclease.

189. The process of claim 187 wherein said base-specific damaged base-specific nuclease is selected from the group consisting of E. coli Fpg protein, E. coli Nth, and E. coli Nfo.

190. The process of claim 167 wherein at least one said primer comprise additional nucleic acid sequences which permit optimal seeding and phasing properties of recombinase on the primer.

191. The process of claim 190 wherein the additional DNA sequences comprises more than 70% pyrimidines or more than 75% of cytosine residues.

192. The process of claim 167

wherein the target DNA sequence is partially or completely double-stranded during the replication reaction such that a polymerase with inherent strand-displacing activity is required;

wherein at least one polymerase has significant strand displacing activity;

wherein a single-stranded DNA binding protein is present;

wherein a crowding agent is present at 1% weight per volume or more in the reaction such that the crowding agent stimulates amplification; and

wherein said process is performed at a temperature of between about 30°

C. to about 39°

C.

193. An RPA process of DNA amplification of a double stranded target sequence comprising a first and a second strand of DNA comprising the steps of:

(a) contacting a recombinase agent with a first nucleic acid primer to form a first nucleoprotein primer;

(b) providing a second nucleic acid primer which does not contain the recombinase agent;

(c) contacting the first nucleoprotein primers to said double stranded target sequence thereby forming a first double stranded structure at a first portion of said first strand;

(d) extending the 3′

end of said first nucleoprotein primer with one or more polymerases and dNTPs to generate a double stranded nucleic acid and a first displaced strand of nucleic acid;

(e) hybridizing said second nucleic acid primer to said first displaced strand of nucleic acid and extending the 3′

end of said second nucleic acid primer with one or more polymerases and dNTPs to generate a double stranded nucleic acid;

(f) continuing the reaction through repetition of (c) through (e) until a desired degree of amplification is reached;

wherein said steps (c) to (e) is performed simultaneously or sequentially and wherein the generated double stranded nucleic acid can serve as the double stranded target sequence.

194. The process according to claim 193 wherein the second primer is too short for recombinase function and for engaging in substantial recombinase-mediated sequence targeting on said target nucleic acid and wherein the second primer is of sufficient length for hybridization and elongation reaction.

195. The process according to claim 193 wherein the second primer is suppressed from engaging in substantial recombinase-mediated sequence targeting.

196. The process of claim 193 wherein said second primer comprises one or more modified nucleotides and is suppressed from engaging in substantial recombinase-mediated sequence targeting.

197. The process of claim 196 wherein the modified nucleotide comprises a locked nucleic acid sugar.

198. A process for analyzing a polymorphic DNA sample comprising the steps of:

(i) hybridizing a sample DNA with one or more probes to form one or more hybrid regions in a reaction that includes a recombinase, wherein said one or more probes corresponds to the possible sequence variants in said sample DNA, wherein either the sample or the probe is double-stranded;

(ii) enhanced destabilization of imperfect hybrid regions;

(iii) detecting perfect hybrids formed between the probe and sample by detecting the presence or absence of a label initially present on either probe or sample nucleic acids.

199. The process of claim 198 wherein said enhancing step is performed by an enzyme;

200. The process of claim 199 wherein the enzyme is a recombinase.

201. The process of claim 200 wherein said recombinase is T4 uvsX protein.

202. The process of claim 198 wherein said process is performed in the presences of an additional component selected from the group consisting of T4 gp32 protein, T4uvsY protein, ATP, and a crowding agent such that the crowding agent stimulates amplification.

203. The process of claim 202 wherein said crowding agent is selected from the group consisting of polyvinylchloride, polyethylene glycol, polyethylene glycol mixture, ficoll, polydextrans, bovine serum albumin, and polyvinylpyrollidone.

204. The process of claim 203 wherein said polyethylene glycol mixture comprises polyethylene glycol with molecular weight of between 15,000 to 20,000 daltons.

205. The process according to claim 203 further comprising an ATP regeneration system.

206. The process according to claim 198 in which a helicase is included to aid destabilization of imperfect hybrids.

207. The process according to claim 206 in which the helicase belongs to the group including E. coli PriA, E. coli DnaB helicase, E. coli RuvAB helicase, T4 phage gp41, T4 phage dda helicase.

208. The process according to claims 206 in which the helicase physically disrupts a stable non-covalent interaction between probe and a surface, such as a biotin-streptavidin interaction.

209. The process according to claim 198 in which a nuclease is included which acts upon DNA templates in which there is a mis-paired region or in which there is a bubble in the template.

210. The process according to claim 209 in which the nuclease belongs to the group including S1 nuclease, P1 nuclease, T7 endonuclease I, BAL31 nuclease, Cell nuclease.

211. The process according to claim 198 in which a DNA polymerase capable of initiating synthesis from a nick or small gap is included in the reaction to product an amplified DNA sample.

212. The process according to claim 211 in which the amplified DNA sample is significantly single-stranded in nature and the probe is principally double-stranded or single stranded in nature at the start of the reaction,

213. The process according to claim 211 in which the amplified sample DNA or the probe nucleic acid contains a label at one end of one or both strands.

214. The process according to claims 213 in which the label is selected from the group consisting of a fluorescent group, a quantum dot, a quencher, a paramagnetic particle, and a catalytic moiety.

215. The process of claim 214 wherein said catalytic moiety is an enzyme or inorganic catalyst.

216. The process according to claim 213 in which the label is an entity capable of forming a high affinity non-covalent interaction with another molecule, in particular including antibodies and known antigens.

217. The process according to claim 198 in which both probe and sample nucleic acids contain a label.

218. The process according to claim 217 in which the labels consist of a fluorophore and/or a quencher.

219. An apparatus for performing an RPA reaction comprising:

(a) a reaction chamber comprising a light detecting sensor; and

(b) a non cyclic temperature control means for maintaining a temperature of between 33-39°

C.

220. The apparatus of claim 219 wherein said light detecting sensor is a fluorescence detecting sensor.

221. The apparatus of claim 219 further comprising one or more light emitting diodes for illuminating said reaction chamber for making a light measurement by said light detecting sensor.

222. The apparatus of claim 219 wherein said sensor comprises a pin diode or an array of pin diodes.