Recombinase polymerase amplification

• US 7,399,590 B2
• Filed: 09/01/2004
• Issued: 07/15/2008
• Est. Priority Date: 02/21/2002
• Status: Active Grant

• Current Assignee: Abbott Diagnostics Scarborough, Inc. (Abbott Laboratories Incorporated)
• Original Assignee: ASM Scientific, Inc.
• Inventors: Stemple, Derek L., Armes, Niall A., Piepenburg, Olaf, Williams, Colin H.
• Primary Examiner(s): Horlick; Kenneth R.
• Application Number:
  US10/931,916
• Publication Number:
  US 20050112631A1
• Time in Patent Office: 1,413 Days
• Field of Search: 435/6, 435/91.1, 435/91.2
• US Class Current: 435/6.14
• 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 describe three 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. Further, the improved processivity of the disclosed methods may allow amplification of DNA up to hundreds of megabases in length.

Claims

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

(a) contacting a recombinase agent selected from the group consisting of uvsX and recA, 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′

end;

(b) contacting the first and the second nucleoprotein primer to said double stranded target nucleic acid molecule thereby forming a first double-stranded structure at a first portion of said first strand and forming a second 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 one another on the same double-stranded template nucleic acid molecule;

(c) extending the 3′

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

(d) continuing the reaction through repetition of (b) and (c) until a desired degree of amplification is reached, wherein said process at steps (b) and (c) is performed in the presence of a crowding agent such that the crowding agent stimulates amplification.

2. The process of claim 1, wherein in step (c), the first and second displaced strands of nucleic acid are converted to double-stranded DNA by the action of a concurrent recombinase polymerase amplification reaction established from the first or second nucleoprotein primer targeting the displaced strands.

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 second nucleoprotein primer to said displaced strand; and

(b) extending said first or second primer with a polymerase and dNTPs.

4. 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 second nucleic acid primer to said first or second displaced strand; and

(b) extending said first or second nucleic acid primer with a polymerase and dNTPs.

5. The process of claim 1, wherein the first or second displaced strand of nucleic acid is converted to double-stranded DNA by:

(a) contacting a first or second nucleoprotein primer to said displaced strand of nucleic acid to form a DNA duplex hybrid; and

(b) extending said first or second nucleoprotein primer with a polymerase and dNTPs.

6. 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 nucleic acid molecule that is double stranded or partially double stranded.

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

8. 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.

9. 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.

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

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

12. The process of claim 1 wherein the recombinase agent is present at a concentration of between about 0.2 μ

M and about 1 μ

M.

13. The process of claim 1 wherein the recombinase agent is present at a concentration of between about 1 μ

M and about 4 μ

M.

14. The process of claim 1 wherein the recombinase agent is present at a concentration of between about 4 μ

M and about 6 μ

M.

15. The process of claim 1 wherein the recombinase agent is present at a concentration of between about 6 μ

M and about 12 μ

M.

16. The process of claim 1 performed in the presence of one or more accessory agents.

17. The process of claim 16 wherein said accessory agent is selected from the group consisting of single-strand binding protein, helicase, resolvase and a combination thereof.

18. The process of claim 16 wherein the accessory agent is selected from the group consisting of single stranded binding protein, RuvA, RuvB, RuvC, RecG, PriA, PriB, PriC, DnaT, DnaB, DnaC, DnaG, DnaX clamp loader, polymerase core complex, DNA ligase, a sliding clamp, and a combination thereof.

19. The process of claim 18 wherein said sliding clamp is the E. coli β

-dimer sliding clamp, the eukaryotic PCNA sliding clamp, or the T4 sliding clamp (gp45).

20. The process of claim 1 wherein said process is performed in the presence of a RecA/ssDNA nucleoprotein filament stabilizing agent.

21. The process of claim 20 wherein said stabilizing agent is selected from the group consisting of RecR, RecO, RecF and a combination thereof.

22. The process of claim 1 wherein said process is performed in the presence of a single strand stabilizing agent selected from the group consisting of gp32, E. coli SSB protein, T4 gp32 protein, and derivatives thereof.

23. The process of claim 22 wherein the derivative of the T4 gp32 protein is selected from the group consisting of T4 gp32 with additional amino acids at the N terminus, T4 gp32 with additional amino acids at the C terminus, T4 gp32 with the substitution of lysine 3 with a non lysine amino acid, and T4 gp32 with the substitution of arginine 4 with a non arginine amino acid and a combination thereof.

24. The process of claim 23 wherein the additional amino acids comprise a poly His tag.

25. The process of claim 22 wherein said T4 gp32 derivative is selected from the group consisting of T4 gp32(N), T4 gp32(C), T4 gp32(C)K3A, T4 gp32(C)R4Q, T4 gp32(C)R4T, T4 gp32K3A, T4 gp32R4Q, T4 gp32 R4T and a combination thereof.

26. The process of claim 22 wherein said single stranded DNA stabilizing agent is present at a concentration of between 1 μ

M and 20 μ

M.

27. The process of claims 22 wherein the T4 gp32 is at a concentration sufficient to saturate all of the primers in the reaction.

28. The process of claim 22 wherein said T4 gp32 is between 1 μ

M and 20 μ

M.

29. The process of claim 1 wherein said crowding agent is selected from the group consisting of polyethylene glycols, dextran, ficoll and a combination thereof.

30. The process of claim 29 wherein said polyethylene glycol selected from the group consisting of PEG1450, PEG8000, PEG10000, PEG compound (molecular weight 15,000 to 20,000 and also known as Carbowax 20M), and a combination thereof.

31. The process of claim 29 wherein said polyethylene glycol is between 1 to 12% of a weight or a volume of the reaction.

32. The process of claim 1 wherein the crowding agent has a concentration of between 1 and 12% weight/volume of the reaction.

33. The process of claim 1 wherein said process is performed in the presence of a recombinase loading protein.

34. The process of claim 33 wherein the recombinase loading protein is selected from the group consisting of T4 uvsY, E. coli recO, E. coli recR and a combination thereof.

35. The process of claim 34 wherein the T4 uvsY further comprise a poly-His tag at the N or C terminus.

36. The process of claim 33 wherein the recombinase loading agent is at a concentration of between 0.2 μ

M and 8 μ

M.

37. The process of claim 1 wherein said recombinase polymerase amplification process further comprises a cofactor for the recombinase agent selected from the group consisting of ATP, ATP analog, and a combination thereof.

38. The process of claim 37 wherein said ATP analog is ATP-γ

-S or ddATP.

39. The process of claim 1, wherein said recombinase polymerase amplification process further comprises an ATP regeneration system to convert ADP to ATP.

40. The process of claim 39 wherein said ATP regeneration system comprises phosphocreatine and creatine kinase.

41. The process of claim 1 wherein said recombinase polymerase amplification process further comprises ADP-β

-S as a recombinase agent modulator.

42. The process of claim 1, wherein said recombinase polymerase amplification process further comprises a system to regenerate ADP from AMP.

43. The process of claim 1, wherein said recombinase polymerase amplification process further comprises pyrophosphatase to convert pyrophosphate to phosphate.

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

45. The process of claim 44 wherein said eukaryotic polymerase is selected from the group consisting of pol-α

, pol-β

, pol-δ

, pol-δ and

a combination thereof.

46. The process of claim 44 wherein the procaryotic 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, Bacillus subtilis Pol I large fragment (Bsu polymerase), 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 a combination thereof.

47. The process of claim 44 wherein said one or more DNA polymerase is a fusion protein comprising a short tag sequence at the N or C terminus.

48. The process of claim 1 wherein the one or more DNA polymerase includes at least one DNA polymerase which lacks 3′

-5′

exonuclease activity.

49. The process of claim 1 wherein said one or more DNA polymerase comprises a DNA polymerase with strand displacing properties.

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

end and is resistant to 3′

to 5′

nuclease activity.

51. The process of claim 1 wherein at least one primer comprise at least one locked nucleic acid at its 3′

ultimate base or 3′

penultimate base.

52. The process of claim 1 wherein target nucleic acid molecule is supercoiled.

53. The process of claim 1 wherein the target nucleic acid molecule is linear with two ends and wherein both ends are linked to a non-target nucleic acid molecule.

54. The process of claim 1 wherein the target nucleic acid molecule is linear with two ends and wherein one end is linked to a non-target nucleic acid molecule.

55. The process of claim 1 wherein the target nucleic acid molecule is a linear nucleic acid molecule.

56. The process of claim 1 wherein the target nucleic acid molecule is a single-stranded nucleic acid 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.

57. The process of claim 53, 54, 55 or 56 wherein the nucleic acid primers possess additional 5′

bases that are not complementary to the target nucleic acid molecule.

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

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

60. 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 are separated by between 0 and 10 bases after step (b).

61. 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 are separated by between 10 and 30 bases after step (b).

62. 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 are separated by between 30 and 100 bases after step (b).

63. 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 are separated by more than 100 bases after step (b).

64. 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 are separated by more than 1,000,000 bases after step (b).

65. The process of claim 1 wherein said recombinase polymerase amplification is performed in the presence of short, 3′

-blocked, oligonucleotides with a sequence complementary to the 3′

region of the nucleic acid primers.

66. The process of claim 65 wherein the short oligonucleotides are tethered to the 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.

67. 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.

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

C. and 50°

C.

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

C. and 40°

C.

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

C. and 30°

C.

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

72. The process of claim 1, wherein the two nucleoprotein primer are each present in a molar concentration that is at least 1000 fold greater than the concentration of the target nucleic acid molecule.

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

end.

74. The process of claim 73 wherein said one or more nucleotides is an oligonucleotide that comprises the sequence of a restriction endonuclease recognition site.

75. The process of claim 1 wherein at least one 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.

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

77. The process of claim 75 wherein said invading strand, said non-invading strand, or both are labeled with a detectable moiety.

78. The process of claim 77 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.

79. The process of claim 77 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.

80. The process of claim 75 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.

81. The process of claim 75 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.

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

83. The process of claim 82, wherein said single stranded 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.

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

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

86. The process of claim 1 wherein said steps (b) and (c) are repeated at least once.

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

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

89. 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.

90. The process of claim 1 wherein at least one primer is complementary to a sequence associated with a genetic disease.

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

92. 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.

93. The process of claim 92 wherein said first primer, said second primer, said third primer and said fourth primer are each different.

94. The process of claim 92 wherein one of said first and said second primer is identical to one of said third and fourth primer.

95. 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 a recombinase agent 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 the 3′

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

(d) continuing the reaction through repetition of (b) and (c) until a desired degree of amplification is reached, wherein said process at steps (b) and (c) is performed in the presence of a crowding agent such that the crowding agent stimulates amplification.

96. The process of claim 95 wherein a fraction of said dNTPs comprises chain-terminating dNTPs.

97. The process of claim 95 wherein at least a fraction of said dNTPs is labeled with a detectable marker.

183. The process of claim 95 wherein said crowding agent is selected from the group consisting of polyethylene glycols, dextran, ficoll and a combination thereof.

184. The process of claim 183 wherein said polyethylene glycol is selected from the group consisting of PEG1450, PEG8000, PEG10000, PEG compound (molecular weight 15,000 to 20,000 and also known as Carbowax 20M), and a combination thereof.

185. The process of claim 183 wherein said polyethylene glycol is between 1 to 12% of a weight or a volume of the reaction.

186. The process of claim 95 wherein the crowding agent has a concentration of between 1 and 12% weight/volume of the reaction.

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

(a) contacting 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 primer;

(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 molecule and a second double stranded structure at a second portion of said target molecule 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) contacting said target nucleic acid molecule to primosome assembly proteins and a clamp loader/polymerase III holoenzyme complex to form a replication fork at said first portion of target molecule and a second replication fork at said second portion of the target 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 the migration of the replication forks towards each other; and

(e) continuing the reaction through repetition of steps (b), (c) and (d) until a desired degree of amplification is reached, wherein said process at steps (b) through (d) is performed in the presence of a crowding agent such that the crowding agent stimulates amplification.

99. The process of claim 98 which is performed in the presence of dNTP, rNTP, single stranded DNA binding protein, or a combination thereof.

100. The process of claim 98 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.

101. The process of claim 98 wherein the clamp loader is a DnaX clamp loader complex.

102. The process of claim 98 wherein the DNA polymerase III holoenzyme is E. coli polymerase III.

103. The process of claim 98 wherein DNA polymerase I is E. coli polymerase I.

104. The process of claim 98 wherein the primase is E. coli DnaG primase.

105. The process of claim 98 wherein the helicase is E. coli DnaB helicase.

187. The process of claim 98 wherein said crowding agent is selected from the group consisting of polyethylene glycols, dextran, ficoll and a combination thereof.

188. The process of claim 187 wherein said polyethylene glycol is selected from the group consisting of PEG1450, PEG8000, PEG10000, PEG compound (molecular weight 15,000 to 20,000 and also known as Carbowax 20M), and a combination thereof.

189. The process of claim 187 wherein said polyethylene glycol is between 1 to 12% of a weight or a volume of the reaction.

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

(a) combining the following reagents in a reaction

(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;

(6) a buffer;

(7) a reducing agent;

(8) ATP or 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;

(b) incubating said reaction until a desired degree of amplification is achieved and wherein the crowding agent stimulates amplification.

107. The process of claim 106 wherein said recombinase is selected from the group consisting of uvsX, recA and derivatives and combinations thereof.

108. The process of claim 106 wherein said recombinase is a fusion protein comprising a short tag sequence at the N or C terminus.

109. The process of claim 106 wherein the recombinase comprises a C terminal deletion of acidic residues.

110. The process of claim 106 wherein the recombinase is present at a concentration of between about 0.2 μ

M and about 1 μ

M.

111. The process of claim 106 wherein the recombinase is present at a concentration of between about 1 μ

M and about 4 μ

M.

112. The process of claim 106 wherein the recombinase is present at a concentration of between about 4 μ

M and about 6 μ

M.

113. The process of claim 106 wherein the recombinase is present at a concentration of between about 6 μ

M and about 12 μ

M.

114. The process of claim 106 wherein the recombinase is present at a concentration of between about 0.2 μ

M and about 12 μ

M.

115. The process of claim 106 wherein said single stranded DNA binding protein is selected from the group consisting of gp32, E. coli SSB, T4 gp32, and derivatives and combinations thereof.

116. The process of claim 115 wherein said T4 gp32 derivative is selected from the group consisting of T4 gp32(N), T4 gp32(C), T4 gp32(C)K3A, T4 gp32(C)R4Q, T4 gp32(C)R4T, T4 gp32K3A, T4 gp32R4Q, T4 gp32R4T and combination thereof.

117. The process of claim 106 wherein said single stranded DNA binding protein is present at a concentration of between 1 μ

M and 30 μ

M.

118. The process of claim 106 wherein said DNA polymerase is a eukaryotic polymerase or a prokaryotic polymerase.

119. The process of claim 118 wherein said eukaryotic polymerase is selected from the group consisting of pol-α

, pol-β

, pol-δ

, pol-ε and

derivatives and combinations thereof.

120. The process of claim 118 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.

121. The process of claim 118 wherein said DNA polymerase is a fusion protein comprising a short tag sequence at the N or C terminus.

122. The process of claim 106 wherein said DNA polymerase is at a concentration of between 10,000 units/ml to 16 units/ml.

123. The process of claim 106 wherein said DNA polymerase is at a concentration of between 5000 units/ml to 500 units/ml.

124. The process of claim 106 wherein the one or more DNA polymerase includes at least one DNA polymerase which lacks 3′

-5′

exonuclease activity.

125. The process of claim 106 wherein said one or more DNA polymerase comprises a DNA polymerase with strand displacing properties.

126. The process of claim 106 wherein said dNTP is at a concentration of 1 mM to 40 μ

M.

127. The process of claim 106 wherein said crowding agent is selected from the group consisting of polyethylene glycols, dextran, ficoll and a combination thereof.

128. The process of claim 127 wherein said polyethylene glycol is between 1 to 12% of a weight or a volume of the reaction.

129. The process of claim 127 wherein said polyethylene glycol is selected from the group consisting of PEG1450, PEG8000, PEG10000, PEG compound (molecular weight 15,000 to 20,000 and also known as Carbowax 20M), and a combination thereof.

130. The process of claim 106 wherein said buffer is a Tris-HCl buffer, a Tris-Acetate buffer, or a combination thereof.

131. The process of claim 130 wherein said buffer is at a concentration of between 10 to 60 mM and at a pH of between 6.5 to 9.0.

132. The process of claim 106 wherein said reducing agent is DTT.

133. The process of claim 132 wherein said reducing agent is at a concentration of 1 to 10 mM.

134. The process of claim 106 wherein said ATP or ATP analog is selected from the group consisting of ATP, ATP-γ

-S, ATP-β

-S and ddATP.

135. The process of claim 134 wherein said ATP or ATP analog is at a concentration of 1 to 10 mM.

136. The process of claim 106 wherein said recombinase loading protein is selected from the group consisting of T4uvsY, E. coli recO, E. coli recR and derivatives and combinations thereof.

137. The process of claim 136 wherein said recombinase loading protein further comprise a poly-His tag at the N terminus, C terminus, or at both the N and C terminus.

138. The process of claim 106 wherein said recombinase loading protein is at a concentration of between 0.2 and 8 μ

M.

139. The process of claim 106 wherein the first or second primer is selected from the group consisting of DNA, RNA, PNA, LNA, morpholino backbone nucleic acid, phosphorothiorate backbone nucleic acid and a combination thereof.

140. The process of claim 106 wherein said primer is at a concentration of 100 nM to 1000 nM.

141. The process of claim 106 wherein the first or second primer comprise a non-phosphate linkage between the two bases at its 3′

end and is resistant to 3′

to 5′

nuclease activity.

142. The process of claim 106 wherein the first or second primer comprise at least one locked nucleic acid at its 3′

ultimate base or 3′

penultimate base.

143. The process of claim 106 wherein the first or second primer is at least 20 bases in length.

144. The process of claim 106 wherein the first or second primer is at least 30 bases in length.

145. The process of claim 106 wherein the first or second primer further comprises one or more bases at its 5′

end that is not complementary to the target nucleic acid.

146. The process of claim 145 wherein said one or more bases comprises the sequence of a restriction endonuclease recognition site.

147. The process of claim 106 wherein said first or second primer is partially double stranded with a single stranded region at its 3′

end.

148. The process of claim 106 wherein at least one primer is labeled with a detectable label.

149. The process of claim 148 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.

150. The process of claim 106 wherein the target nucleic acid molecule is supercoiled.

151. The process of claim 106 wherein the target nucleic acid molecule is linear with two ends and wherein both ends are linked to a non-target nucleic acid molecule.

152. The process of claim 106 wherein the target nucleic acid molecule is linear with two ends and wherein one end is linked to a non-target nucleic acid molecule.

153. The process of claim 106 wherein the target nucleic acid molecule is a linear nucleic acid molecule.

154. The process of claim 106 wherein the target nucleic acid molecule is a single-stranded nucleic acid 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.

155. The process of claim 106 wherein the target nucleic acid is at a concentration of less than 1000 copies per μ

l.

156. The process of claim 106 wherein the target nucleic acid is at a concentration of between 1 and 10 copies per μ

l.

157. The process of claim 106 wherein said incubation is between 5 minutes and 16 hours.

158. The process of claim 106 wherein said incubation is between 15 minutes and 3 hours.

159. The process of claim 106 wherein said incubation is between 30 minutes and 2 hours.

160. The process of claim 106 wherein said incubation is performed until a desired degree of amplification is achieved.

161. The process of claim 160 wherein said desired degree of amplification is selected from the group consisting of 10 fold, 100 fold, 1000 fold, 10,000 fold, 100,000 fold and 1000000 fold.

162. The process of claim 106 wherein said incubation is performed between 20°

C. and 50°

C.

163. The process of claim 106 wherein said incubation is performed between 20°

C. and 40°

C.

164. The process of claim 106 wherein said incubation is performed between 20°

C. and 30°

C.

165. The process of claim 106 which is performed without temperature induced melting of the template nucleic acid.

166. The process of claim 106 wherein step (a) further comprises an accessory agent.

167. The process of claim 166 wherein said accessory agent is selected from the group consisting of helicase, resolvase and a combination thereof.

168. The process of claim 166 wherein the accessory agent is 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 and a sliding clamp.

169. The process of claim 168 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.

170. A process of claim 166 wherein that accessory agent is a DNA Polymerase III holoenzyme complex consisting of β

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

171. The process of claim 106 wherein step (a) further comprises a RecA/ssDNA nucleoprotein filaments stabilizing agent.

172. The process of claim 171 wherein said stabilizing agent is selected from the group consisting of RecR, RecO, RecF and a combination thereof.

173. The process of claim 171 wherein said stabilizing agent is at a concentration of between 0.01 μ

M to 20 μ

M.

174. The process of claim 106 wherein step (a) further comprises an ATP regeneration system to convert ADP to ATP.

175. The process of claim 174 wherein said ATP regeneration system comprises phosphocreatine and creatine kinase.

176. The process of claim 106 wherein step (a) further comprises a system to regenerate ADP from AMP.

177. The process of claim 106 wherein step (a) further comprises a system to convert pyrophosphate to phosphate.

178. The process of claim 106, 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 and a combination thereof.

190. The process of any one of claims 98 or 106 wherein the crowding agent has a concentration of between 1 and 12% weight/volume of the reaction.

179. 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) a T4 gp43 DNA polymerase or Bsu polymerase at a concentration between 500 to 5000 units per ml;

(4) dNTPs or a mixture of dNTPs and ddNTPs at a concentration of between 1-300 μ

M;

(5) polyethylene glycol at a concentration of between 1% to 12% by weight or by volume;

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

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

(8) ATP at a concentration of between 1 mM-10 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 50 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;

and wherein said polyethylene glycol stimulates ampliflcation.

180. 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 or T4 polymerase;

(4) 200 μ

M dNTPs;

(5) 1 mM DTT

(6) 3 mM ATP or an 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) 80 mM Potassium acetate

(10) 10 mM Magnesium acetate

(11) 20 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;

(3) a target nucleic acid;

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

and wherein said polyethylene glycol stimulates amplification.

181. 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

(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;

(3) a target nucleic acid;

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

and wherein said polyethylene glycol stimulates amplification.

182. 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

(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;

and wherein said polyethylene glycol stimulates amplification.