Nucleic acid amplification

• US 20030143587A1
• Filed: 10/15/2002
• Published: 07/31/2003
• Est. Priority Date: 10/15/2001
• Status: Active Grant

• Current Assignee: Qiagen GmbH (Qiagen NV)
• Original Assignee: Molecular Staging Inc. (Qiagen NV)
• Inventors: Dean, Frank B., Lasken, Roger S., Faruqi, A. Fawad, Hosono, Seiyu, Wisniewski, Michele, Fang, Linhua, Song, Wanmin, Driscoll, Mark D., Alsmadi, Osama A.
• Granted Patent: US 7,074,600 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2525/125 incorporating agents resulting in resistance to degradation
• CPC Class Codes: C12Q 2527/119 pH
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis

Abstract

Disclosed are compositions and a method for amplification of nucleic acid sequences of interest. The disclosed method generally involves replication of a target sequence such that, during replication, the replicated strands are displaced from the target sequence by strand displacement replication of another replicated strand. In one form of the disclosed method, the target sample is not subjected to denaturing conditions. It was discovered that the target nucleic acids, genomic DNA, for example, need not be denatured for efficient multiple displacement amplification. The primers used can be hexamer primers. The primers can also each contain at least one modified nucleotide such that the primers are nuclease resistant. The primers can also each contain at least one modified nucleotide such that the melting temperature of the primer is altered relative to a primer of the same sequence without the modified nucleotide(s). The DNA polymerase can be φ29 DNA polymerase.

Claims

1. A method of amplifying a whole genome, the method comprising,

exposing cells to alkaline conditions to form a cell lysate, wherein the cell lysate comprises a whole genome,

reducing the pH of the cell lysate to form a stabilized cell lysate, and

incubating the stabilized cell lysate under conditions that promote replication of the genome,

wherein replication of the genome results in replicated strands, wherein during replication at least one of the replicated strands is displaced from the genome by strand displacement replication of another replicated strand.

2. The method of claim 1 wherein the cells are exposed to alkaline conditions by mixing the cells with a lysis solution.

3. The method of claim 2 wherein the lysis solution comprises a base.

4. The method of claim 3 wherein the base is an aqueous base.

5. The method of claim 3 wherein the base is potassium hydroxide, sodium hydroxide, potassium acetate, sodium acetate, ammonium hydroxide, lithium hydroxide, calcium hydroxide, magnesium hydroxide, sodium carbonate, sodium bicarbonate, calcium carbonate, ammonia, aniline, benzylamine, n-butylamine, diethylamine, dimethylamine, diphenylamine, ethylamine, ethylenediamine, methylamine, N-methylaniline, morpholine, pyridine, triethylamine, trimethylamine, aluminum hydroxide, rubidium hydroxide, cesium hydroxide, strontium hydroxide, barium hydroxide, or DBU (1,8-diazobicyclo[5,4,0]undec-7-ene).

6. The method of claim 5 wherein the base is potassium hydroxide.

7. The method of claim 6 wherein the lysis solution comprises 400 mM KOH.

8. The method of claim 7 wherein the lysis solution comprises 400 mM KOH, 100 mM dithiothreitol, and 10 mM EDTA.

9. The method of claim 8 wherein the lysis solution consists of 400 mM KOH, 100 mM dithiothreitol, and 10 mM EDTA.

10. The method of claim 3 wherein the lysis solution comprises a plurality of basic agents.

11. The method of claim 2 wherein the cells are mixed with an equal volume of the lysis solution.

12. The method of claim 11 wherein the lysis solution comprises 400 mM KOH, 100 mM dithiothreitol, and 10 mM EDTA.

13. The method of claim 2 wherein the lysis solution comprises a buffer.

14. The method of claim 13 wherein the buffer is a phosphate buffer, Good buffer, BES, BICINE, CAPS, EPPS, HEPES, MES, MOPS, PIPES, TAPS, TES, TRICINE, sodium cacodylate, sodium citrate, triethylammonium acetate, triethylammonium bicarbonate, Tris, Bis-tris, or Bis-tris propane.

15. The method of claim 13 wherein the lysis solution comprises a plurality of buffering agents.

16. The method of claim 1 wherein the pH of the cell lysate is reduced to the range of about pH 7.0 to about pH 6.8.

17. The method of claim 1 wherein the pH of the cell lysate is reduced by mixing the cell lysate with a stabilization solution.

18. The method of claim 17 wherein the stabilization solution comprises a buffer.

19. The method of claim 18 wherein the buffer is phosphate buffer, Good buffer, BES, BICINE, CAPS, EPPS, HEPES, MES, MOPS, PIPES, TAPS, TES, TRICINE, sodium cacodylate, sodium citrate, triethylammonium acetate, triethylammonium bicarbonate, Tris, Bis-tris, or Bis-tris propane.

20. The method of claim 19 wherein the buffer is Tris-HCl at pH 4.1.

21. The method of claim 20 wherein the stabilization solution comprises 800 mM Tris-HCl, pH 4.1.

22. The method of claim 21 wherein the stabilization solution consists of 800 mM Tris-HCl, pH 4.1.

23. The method of claim 18 wherein the stabilization solution comprises a plurality of buffering agents.

24. The method of claim 17 wherein the cell lysate is mixed with an equal volume of the stabilization solution.

25. The method claim 24 wherein the stabilization solution comprises 800 mM Tris-HCl, pH 4.1.

26. The method of claim 17 wherein the stabilization solution comprises an acid.

27. The method of claim 26 wherein the acid is hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, acetylsalicylic acid, ascorbic acid, carbonic acid, citric acid, formic acid, nitric acid, perchloric acid, HF, HBr, HI, H₂S, HCN, HSCN, HClO, monochloroacetic acid, dichloroacetic acid, trichloroacetic acid, or a carboxylic acid.

28. The method of claim 27 wherein the carboxylic acid is ethanoic, propanoic, or butanoic.

29. The method of claim 17 wherein the stabilization solution comprises a plurality of acidic agents.

30. The method of claim 1 wherein the pH of the cell lysate is reduced to about pH 9.0 or below.

31. The method of claim 1 wherein the pH of the cell lysate is reduced to about pH 8.5 or below.

32. The method of claim 1 wherein the pH of the cell lysate is reduced to about pH 8.0 or below.

33. The method of claim 1 wherein the pH of the cell lysate is reduced to about pH 7.5 or below.

34. The method of claim 1 wherein the pH of the cell lysate is reduced to the range of about pH 9.0 to about pH 6.0.

35. The method of claim 1 wherein the pH of the cell lysate is reduced to the range of about pH 9.0 to about pH 7.0.

36. The method of claim 1 wherein the pH of the cell lysate is reduced to the range of about pH 9.0 to about pH 7.5.

37. The method of claim 1 wherein the pH of the cell lysate is reduced to the range of about pH 9.0 to about pH 8.0.

38. The method of claim 1 wherein the pH of the cell lysate is reduced to the range of about pH 8.5 to about pH 6.0.

39. The method of claim 1 wherein the pH of the cell lysate is reduced to the range of about pH 8.5 to about pH 7.0.

40. The method of claim 1 wherein the pH of the cell lysate is reduced to the range of about pH 8.5 to about pH 7.5.

41. The method of claim 1 wherein the pH of the cell lysate is reduced to the range of about pH 8.5 to about pH 8.0.

42. The method of claim 1 wherein the pH of the cell lysate is reduced to the range of about pH 8.0 to about pH 6.0.

43. The method of claim 1 wherein the pH of the cell lysate is reduced to the range of about pH 8.0 to about pH 6.5.

44. The method of claim 1 wherein the pH of the cell lysate is reduced to the range of about pH 8.0 to about pH 7.0.

45. The method of claim 1 wherein the pH of the cell lysate is reduced to the range of about pH 8.0 to about pH 7.5.

46. The method of claim 1 wherein the pH of the cell lysate is reduced to the range of about pH 7.5 to about pH 6.0.

47. The method of claim 1 wherein the pH of the cell lysate is reduced to the range of about pH 7.5 to about pH 7.0.

48. The method of claim 1 wherein nucleic acids in the cell lysate and the stabilized cell lysate are not separated from other material in the cell lysate.

49. The method of claim 1 wherein the cell lysate and the stabilized cell lysate are not subjected to purification prior to the incubation.

50. The method of claim 49 wherein the purification comprises separation of nucleic acids in the cell lysate from other material in the cell lysate.

51. The method of claim 49 wherein the purification comprises centrifugation, extraction, chromatography, filtration, dialysis, or a combination of these.

52. The method of claim 49 wherein the purification comprises precipitation other than precipitation caused by the alkaline conditions or by the reduction of the pH.

53. The method of claim 49 wherein the purification comprises centrifugation, phenol-chloroform extraction, column chromatography, or a combination of these.

54. The method of claim 1 wherein the cell lysate, stabilized cell lysate, or both are subjected to partial purification prior to the incubation.

55. The method of claim 54 wherein the partial purification comprises centrifugation, extraction, chromatography, precipitation, filtration, dialysis, or a combination of these.

56. The method of claim 54 wherein the partial purification comprises centrifugation, phenol-chloroform extraction, column chromatography, or a combination of these.

57. The method of claim 1 wherein the cell lysate and the stabilized cell lysate are not subjected to substantial purification prior to the incubation.

58. The method of claim 57 wherein the substantial purification does not include centrifugation, extraction, chromatography, precipitation, filtration, or dialysis.

59. The method of claim 57 wherein the substantial purification does not include centrifugation, phenol-chloroform extraction, or column chromatography.

60. The method of claim 57 wherein the cell lysate, stabilized cell lysate, or both are subjected to centrifugation, extraction, chromatography, precipitation, filtration, or dialysis prior to the incubation.

61. The method of claim 57 wherein the cell lysate, stabilized cell lysate, or both are subjected to centrifugation, phenol-chloroform extraction, or column chromatography prior to the incubation.

62. The method of claim 57 wherein the substantial purification comprises centrifugation, extraction, chromatography, filtration, dialysis, or a combination of these.

63. The method of claim 57 wherein the substantial purification comprises precipitation other than precipitation caused by the alkaline conditions or by the reduction of the pH.

64. The method of claim 57 wherein the substantial purification comprises centrifugation, phenol-chloroform extraction, column chromatography, or a combination of these.

65. The method of claim 1 wherein the cell lysate and the stabilized cell lysate are not purified prior to the incubation.

66. The method of claim 1 wherein the cell lysate, stabilized cell lysate, or both are partially purified prior to the incubation.

67. The method of claim 1 wherein the incubation is substantially isothermic.

68. The method of claim 67 wherein neither the cell lysate nor the stabilized cell lysate is heated substantially above the temperature of the incubation.

69. The method of claim 67 wherein neither the cell lysate nor the stabilized cell lysate is subjected to substantial heating above the temperature of the incubation.

70. The method of claim 67 wherein the cells are not heated substantially above the temperature of the incubation.

71. The method of claim 67 wherein the cells are not subjected to substantial heating above the temperature of the incubation.

72. The method of claim 67 wherein the cells are not heated substantially above the temperature at which the cells grow.

73. The method of claim 67 wherein the cells are not subjected to substantial heating above the temperature at which the cells grow.

74. The method of claim 67 wherein the cell lysate, stabilized cell lysate, and the cells are not heated substantially above the temperature of the incubation.

75. The method of claim 67 wherein the cell lysate, stabilized cell lysate, and the cells are not subjected to substantial heating above the temperature of the incubation.

76. The method of claim 67 wherein the cell lysate, stabilized cell lysate, and the cells are not heated, prior to or during the incubation, substantially above the temperature at which the cells grow.

77. The method of claim 67 wherein the cell lysate, stabilized cell lysate, and the cells are not subjected to, prior to or during the incubation, substantial heating above the temperature at which the cells grow prior.

78. The method of claim 1 wherein neither the cell lysate nor the stabilized cell lysate is heated above a temperature and for a time that would cause notable denaturation of the genome.

79. The method of claim 1 wherein neither the cell lysate nor the stabilized cell lysate is subjected to heating above a temperature and for a time that would cause notable denaturation of the genome.

80. The method of claim 1 wherein the cells are not lysed by heat.

81. The method of claim 1 wherein the cells are not heated above a temperature and for a time that would cause substantial cell lysis in the absence of the alkaline conditions.

82. The method of claim 1 wherein the cells are not subjected to heating above a temperature and for a time that would cause substantial cell lysis in the absence of the alkaline conditions.

200. The method of claim 1 further comprising

following exposure of the cells to alkaline conditions, exposing a first portion of the cell lysate to conditions that promote substantial denaturation of damaged DNA in the first portion of the cell lysate, wherein reducing the pH of the cell lysate comprises reducing the pH of the first portion of the cell lysate to form a first stabilized cell lysate and reducing the pH of a second portion of the cell lysate to form a second stabilized cell lysate,

following reducing the pH of the cell lysate, mixing the second stabilized cell lysate with the first stabilized cell lysate under conditions that promote transient denaturation of the ends of damaged DNA in the second stabilized cell lysate and that maintain substantial denaturation of the damaged DNA in the first stabilized cell lysate, thereby forming a stabilized cell lysate mixture, and

prior to incubating the stabilized cell lysate, cooling the stabilized cell lysate mixture under conditions that promote annealing of the ends of the transiently denatured damaged DNA to the substantially denatured damaged DNA,

wherein incubating the stabilized cell lysate under conditions that promote replication of the genome also promotes replication of the damaged DNA, wherein the annealed ends of the damaged DNA prime replication, wherein replication of the damaged DNA results in repair of the replicated strands.

83. A method of amplifying a whole genome, the method comprising,

exposing cells to alkaline conditions to form a cell lysate, wherein the cell lysate comprises a whole genome, wherein the cells are exposed to alkaline conditions by mixing the cells with a lysis solution,

reducing the pH of the cell lysate to form a stabilized cell lysate, wherein the pH of the cell lysate is reduced by mixing the cell lysate with a stabilization solution, and

incubating the stabilized cell lysate under conditions that promote replication of the genome,

wherein replication of the genome results in replicated strands, wherein during replication at least one of the replicated strands is displaced from the genome by strand displacement replication of another replicated strand.

84. The method of claim 83 wherein the lysis solution comprises potassium hydroxide.

85. The method of claim 84 wherein the lysis solution comprises 400 mM KOH.

86. The method of claim 85 wherein the lysis solution comprises 400 mM KOH, 100 mM dithiothreitol, and 10 mM EDTA.

87. The method of claim 86 wherein the lysis solution consists of 400 mM KOH, 100 mM dithiothreitol, and 10 mM EDTA.

88. The method of claim 83 wherein the cells are mixed with an equal volume of the lysis solution.

89. The method of claim 88 wherein the lysis solution comprises 400 mM KOH, 100 mM dithiothreitol, and 10 mM EDTA.

90. The method of claim 83 wherein the stabilization solution comprises Tris-HCl at pH 4.1.

91. The method of claim 90 wherein the stabilization solution comprises 800 mM Tris-HCl, pH 4.1.

92. The method of claim 91 wherein the stabilization solution consists of 800 mM Tris-HCl, pH 4.1.

93. The method of claim 83 wherein the cell lysate is mixed with an equal volume of the stabilization solution.

94. The method claim 93 wherein the stabilization solution comprises 800 mM Tris-HCl, pH 4.1.

95. The method of claim 83 wherein the lysis solution consists of 400 mM KOH and 10 mM EDTA, wherein the stabilization solution consists of 800 mM Tris-HCl, pH 4, wherein the stabilized cell lysate is incubated in the presence of 37.5 mM Tris-HCl, 50 mM KCl, 10 mM MgCl₂, 5 mM (NH₄)₂SO₄, 1 mM deoxynucleotide triphosphates, 50 μ

M primers, and φ

29 DNA Polymerase.

96. The method of claim 95 wherein the stabilized cell lysate is incubated in the presence of 37.5 mM Tris-HCl, 50 mM KCl, 10 mM MgCl₂, 5 mM (NH₄)₂SO₄, 1 mM deoxynucleotide triphosphates, 50 μ

M primers, and φ

29 DNA Polymerase by mixing the stabilized cell lysate with one quarter volume of reaction mix, and φ

29 DNA Polymerase, wherein the reaction mix consists of 150 mM Tris-HCl, 200 mM KCl, 40 mM MgCl₂, 20 mM (NH₄)₂SO₄, 4 mM deoxynucleotide triphosphates, and 0.2 mM primers.

97. A method of amplifying a whole genome, the method comprising,

exposing cells to alkaline conditions to form a cell lysate, wherein the cell lysate comprises a whole genome, wherein the cells are exposed to alkaline conditions by mixing the cells with a lysis solution, wherein the lysis solution comprises 400 mM KOH, 100 mM dithiothreitol, and 10 mM EDTA,

reducing the pH of the cell lysate to form a stabilized cell lysate, wherein the pH of the cell lysate is reduced by mixing the cell lysate with a stabilization solution, wherein the stabilization solution comprises 800 mM Tris-HCl, pH 4.1, and

incubating the stabilized cell lysate under conditions that promote replication of the genome,

wherein replication of the genome results in replicated strands, wherein during replication at least one of the replicated strands is displaced from the genome by strand displacement replication of another replicated strand.

98. The method of claim 97 wherein the cells are mixed with an equal volume of the lysis solution.

99. The method of claim 97 wherein the cell lysate is mixed with an equal volume of the stabilization solution.

100. A kit for amplifying a whole genome, the kit comprising

a lysis solution,

a stabilization solution,

a set of primers, and

a DNA polymerase.

101. The kit of claim 100 wherein the lysis solution comprises potassium hydroxide.

102. The kit of claim 101 wherein the lysis solution comprises 400 mM KOH.

103. The kit of claim 102 wherein the lysis solution comprises 400 mM KOH, 100 mM dithiothreitol, and 10 mM EDTA.

104. The kit of claim 103 wherein the lysis solution consists of 400 mM KOH, 100 mM dithiothreitol, and 10 mM EDTA.

105. The kit of claim 100 wherein the stabilization solution comprises Tris-HCl at pH 4.1.

106. The kit of claim 105 wherein the stabilization solution comprises 800 mM Tris-HCl, pH 4.1.

107. The kit of claim 106 wherein the stabilization solution consists of 800 mM Tris-HCl, pH 4.1.

108. The kit of claim 100 further comprising deoxynucleotide triphosphates.

109. The kit of claim 100 further comprising dithiotheitol,

1×

Phosphase-Buffered Saline, pH 7.5, and control DNA template,

wherein the lysis solution comprises 400 mM KOH and 10 mM EDTA,

wherein the stabilization solution comprises 800 mM Tris-HCl, pH 4,

wherein the set of primers comprises a reaction mix, wherein the reaction mix comprises 150 mM Tris-HCl, 200 mM KCl, 40 mM MgCl₂, 20 mM (NH₄)₂SO₄, 4 mM deoxynucleotide triphosphates, and 0.2 mM random hexamer primers, wherein the DNA polymerase is φ

29 DNA polymerase.

110. The kit of claim 100 further comprising one or more detection probes, wherein the detection probes each comprise a complementary portion, wherein the complementary portion is complementary to a nucleic acid sequence of interest.

111. The method of claim 110 wherein the kit is designed to detect nucleic acid sequences of interest in the genome.

112. The method of claim 111 wherein the kit is designed to assess a disease, condition or predisposition of an individual based on the nucleic acid sequences of interest.

113. A method of amplifying damaged DNA, the method comprising

exposing a damaged DNA sample to conditions that promote substantial denaturation of damaged DNA in the damaged DNA sample, thereby forming a denatured damaged DNA sample,

altering the conditions to conditions that do not promote substantial denaturation of damaged DNA in the damaged DNA sample to form a stabilized damaged DNA sample,

incubating damaged DNA in the stabilized damaged DNA sample under conditions that promote replication of the damaged DNA, wherein replication of the damaged DNA results in a longer average fragment length for the replicated damaged DNA than the average fragment length in the damaged DNA sample, wherein during replication at least one of the replicated strands is displaced by strand displacement replication of another replicated strand.

114. The method of claim 113 wherein the damaged DNA sample, the denatured damaged DNA sample, or both are exposed to ionic conditions.

115. The method of claim 114 wherein the damaged DNA sample and denatured damaged DNA sample are exposed to ionic conditions by mixing an ionic solution with the damaged DNA sample.

116. The method of claim 115 wherein the ionic solution is mixed with the damaged DNA sample prior to or during exposure of the damaged DNA sample to conditions that promote substantial denaturation of the damaged DNA.

117. The method of claim 115 wherein the ionic solution is a salt solution.

118. The method of claim 117 wherein the salt solution comprises one or more salts.

119. The method of claim 118 wherein the salt is Tris-HCl, Tris-EDTA, sodium chloride, potassium chloride, magnesium chloride, sodium acetate, potassium acetate, magnesium acetate, or a combination.

120. The method of claim 119 wherein the Tris-HCl is from pH 7.0 to 8.0.

121. The method of claim 119 wherein the salt is Tris-EDTA.

122. The method of claim 121 wherein the salt solution comprises about 50 mM to about 500 mM Tris and about 1 mM to about 5 mM EDTA.

123. The method of claim 122 wherein the ionic solution is diluted 2 to 5 fold when mixed with the damaged DNA sample.

124. The method of claim 114 wherein the denatured damaged DNA sample is exposed to ionic conditions by mixing an ionic solution with the denatured damaged DNA sample.

125. The method of claim 124 wherein the ionic solution is mixed with the denatured damaged DNA sample prior to or during altering of the conditions.

126. The method of claim 113 wherein the damaged DNA sample is exposed to conditions that promote substantial denaturation by mixing the damaged DNA sample with a denaturing solution and by heating the damaged DNA sample to a temperature and for a length of time that substantially denatures the damaged DNA in the damaged DNA sample.

127. The method of claim 126 wherein the damaged DNA sample is mixed with the denaturing solution after the DNA sample is heated.

128. The method of claim 126 wherein the damaged DNA sample is mixed with the denaturing solution before the DNA sample is heated.

129. The method of claim 126 wherein the damaged DNA sample is mixed with the denaturing solution at the same time the DNA sample is heated.

130. The method of claim 126 wherein the damaged DNA sample is mixed with the denaturing solution during heating of the DNA sample.

131. The method of claim 126 wherein the damaged DNA sample is mixed with the denaturing solution when heating of the DNA sample begins.

132. The method of claim 126 wherein mixing the damaged DNA sample with a denaturing solution produces alkaline conditions in the damaged DNA sample.

133. The method of claim 132 wherein the denaturing solution comprises a base.

134. The method of claim 133 wherein the base is an aqueous base.

135. The method of claim 133 wherein the base is sodium hydroxide, potassium hydroxide, potassium acetate, sodium acetate, ammonium hydroxide, lithium hydroxide, calcium hydroxide, magnesium hydroxide, sodium carbonate, sodium bicarbonate, calcium carbonate, ammonia, aniline, benzylamine, n-butylamine, diethylamine, dimethylamine, diphenylamine, ethylamine, ethylenediamine, methylamine, N-methylaniline, morpholine, pyridine, triethylamine, trimethylamine, aluminum hydroxide, rubidium hydroxide, cesium hydroxide, strontium hydroxide, barium hydroxide, or DBU (1,8-diazobicyclo[5,4,0]undec-7-ene).

136. The method of claim 135 wherein the base is sodium hydroxide.

137. The method of claim 136 wherein the denaturing solution comprises about 150 mM to about 1 M NaOH.

138. The method of claim 132 wherein the denaturing solution is 10×

concentration, wherein the damaged DNA sample is mixed with the denaturing solution to create a 1×

concentration.

139. The method of claim 132 wherein the alkaline conditions comprise 15 to 50 mM NaOH.

140. The method of claim 126 wherein the damaged DNA in the damaged DNA sample is substantially denatured without further damaging the DNA.

141. The method of claim 126 wherein the damaged DNA sample is heated to a temperature of about 70°

C. or less and for a length of time of about 5 minutes or less.

142. The method of claim 126 wherein the temperature is about 60°

C. to about 70°

C.

143. The method of claim 126 wherein the temperature is about 50°

C. to about 60°

C.

144. The method of claim 126 wherein the temperature is about 40°

C. to about 50°

C.

145. The method of claim 126 wherein the temperature is about 25°

C. to about 40°

C.

146. The method of claim 126 wherein the temperature is about 60°

C. to about 70°

C. and the length of time is about 3 minutes.

147. The method of claim 126 wherein the temperature is about 25°

C. to about 50°

C. and the length of time is about 5 minutes or more.

188. The method of claim 126 wherein the denaturing solution comprises one or more salts.

189. The method of claim 188 wherein the salt is Tris-HCl, Tris-EDTA, sodium chloride, potassium chloride, magnesium chloride, sodium acetate, potassium acetate, magnesium acetate, or a combination.

190. The method of claim 189 wherein the Tris-HCl is from pH 7.0 to 8.0.

191. The method of claim 189 wherein the salt is Tris-EDTA.

192. The method of claim 191 wherein the denaturing solution comprises about 50 mM to about 500 mM Tris and about 1 mM to about 5 mM EDTA.

193. The method of claim 113 wherein the damaged DNA sample is comprised of degraded DNA fragments of genomic DNA.

194. The method of claim 113 wherein replication and repair of the damaged DNA is accomplished by incubating the damaged DNA in the presence of a DNA polymerase.

195. The method of claim 194 wherein the polymerase is a DNA polymerase that can extend the 3′

-ends of the damaged DNA.

196. The method of claim 195 wherein the DNA polymerase is φ

29 DNA polymerase, BST DNA polymerase, Taq DNA polymerase, a modified form of Taq DNA polymerase, a Reverse Transcriptase, T4 DNA polymerase, T7 DNA polymerase, Pol I DNA polymerase, or a modified form of DNA Polymerase I.

197. The method of claim 194 wherein the DNA polymerase is φ

29 DNA Polymerase.

198. The method of claim 194 wherein the damaged DNA is amplified using a kit, wherein the kit comprises

a denaturing solution,

a stabilization solution,

a set of primers, and

a DNA polymerase.

199. The method of claim 113 wherein the damaged DNA sample is a cell lysate, wherein the cell lysate is produced by exposing cells to alkaline condition, wherein the cell lysate comprises a whole genome.

148. The method of 126 wherein altering the conditions comprises reducing the pH of and cooling the denatured damaged DNA sample.

149. The method of claim 148 wherein the temperature to which the damaged DNA sample is heated is maintained during reduction of the pH of the denatured damaged DNA sample.

150. The method of claim 148 wherein the temperature to which the damaged DNA sample is heated is reduced before reduction of the pH of the denatured damaged DNA sample.

151. The method of claim 148 wherein the temperature to which the damaged DNA sample is heated is reduced during reduction of the pH of the denatured damaged DNA sample.

152. The method of claim 148 wherein cooling the denatured damaged DNA sample is commenced during reduction of the pH of the denatured damaged DNA sample.

153. The method of claim, 148 wherein cooling the denatured damaged DNA sample is commenced when the pH of the denatured damaged DNA sample is reduced.

154. The method of claim 148 wherein the pH of the denatured damaged DNA sample is reduced by mixing the denatured damaged DNA sample with a stabilization solution.

155. The method of claim 154 wherein the pH of the denatured damaged DNA sample is reduced to the range of about pH 7.5 to about pH 8.0.

156. The method of claim 154 wherein the pH of the denatured damaged DNA sample is reduced by mixing the denatured damaged DNA sample with a stabilization solution.

157. The method of claim 156 wherein the stabilization solution comprises a buffer.

158. The method of claim 154 wherein the buffer is phosphate buffer, Good buffer, BES, BICINE, CAPS, EPPS, HEPES, MES, MOPS, PIPES, TAPS, TES, TRICINE, sodium cacodylate, sodium citrate, triethylammonium acetate, triethylammonium bicarbonate, Tris, Bis-tris, or Bis-tris propane.

159. The method of claim 154 wherein the stabilization solution comprises 800 mM Tris-HCl, pH 4.1.

160. The method of claim 154 wherein the stabilization solution comprises an acid.

161. The method of claim 160 wherein the acid is hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, acetylsalicylic acid, ascorbic acid, carbonic acid, citric acid, formic acid, nitric acid, perchloric acid, HF, HBr, HI, H₂S, HCN, HSCN, HClO, monochloroacetic acid, dichloroacetic acid, trichloroacetic acid, or a carboxylic acid.

162. The method of claim 160 wherein the carboxylic acid is ethanoic, propanoic, or butanoic.

163. The method of claim 160 wherein the stabilization solution comprises a plurality of acidic agents.

164. The method of claim 154 wherein the pH of the denatured damaged DNA sample is reduced to the range of about pH 9.0 to about pH 6.8.

165. The method of claim 154 wherein the pH of the denatured damaged DNA sample is reduced to the range of about pH 9.0 to about pH 7.5.

166. The method of claim 154 wherein the pH of the denatured damaged DNA sample is reduced to the range of about pH 9.0 to about pH 8.0.

167. The method of claim 154 wherein the pH of the denatured damaged DNA sample is reduced to the range of about pH 8.5 to about pH 6.8.

168. The method of claim 154 wherein the pH of the denatured damaged DNA sample is reduced to the range of about pH 8.5 to about pH 7.5.

169. The method of claim 154 wherein the pH of the denatured damaged DNA sample is reduced to the range of about pH 8.5 to about pH 8.0.

170. The method of claim 154 wherein the pH of the denatured damaged DNA sample is reduced to the range of about pH 8.0 to about pH 6.8.

171. The method of claim 154 wherein the pH of the denatured damaged DNA sample is reduced to the range of about pH 8.0 to about pH 7.5.

172. The method of claim 154 wherein the pH of the denatured damaged DNA sample is reduced to about pH 9.0 or less.

173. The method of claim 154 wherein the pH of the denatured damaged DNA sample is reduced to about pH 8.5 or less.

174. The method of claim 154 wherein the pH of the denatured damaged DNA sample is reduced to about pH 8.0 or less.

175. The method of claim 154 wherein the pH of the denatured damaged DNA sample is reduced to about pH 7.5 or less.

176. The method of claim 154 wherein the stabilization solution comprises one or more salts.

177. The method of claim 176 wherein the salt is Tris-HCl, Tris-EDTA, sodium chloride, potassium chloride, magnesium chloride, sodium acetate, potassium acetate, magnesium acetate, or a combination.

178. The method of claim 177 wherein the Tris-HCl is from pH 7.0 to 8.0.

179. The method of claim 177 wherein the salt is Tris-EDTA.

180. The method of claim 179 wherein the stabilization solution comprises about 50 mM to about 500 mM Tris and about 1 mM to about 5 mM EDTA.

181. The method of claim 148 wherein the damaged DNA mixture is cooled at a rate of about 1°

C. per minute or less.

182. The method of claim 148 wherein the damaged DNA mixture is cooled at a rate of about 1% per minute or less.

183. The method of claim 182 wherein the damaged DNA mixture is cooled to room temperature or lower from 60°

C. to 70°

C.

184. The method of claim 182 wherein the damaged DNA mixture is cooled to room temperature or lower from 50 to 60°

C.

185. The method of claim 182 wherein the damaged DNA mixture is cooled to room temperature or lower from 40°

C. to 50°

C.

186. The method of claim 182 wherein the damaged DNA mixture is cooled to room temperature or lower from 30°

C. to 40°

C.

187. The method of claim 182 wherein the damaged DNA mixture is cooled to room temperature from 50°

C. to 70°

C.

201. A method of amplifying damaged DNA, the method comprising

exposing a first damaged DNA sample to conditions that promote substantial denaturation of damaged DNA in the first damaged DNA sample, thereby forming a denatured damaged DNA sample,

reducing the pH of the denatured damaged DNA sample to form a stabilized denatured damaged DNA sample,

mixing a second damaged DNA sample with the stabilized denatured damaged DNA sample under conditions that promote transient denaturation of the ends of damaged DNA in the second sample and that maintain substantial denaturation of the damaged DNA in the stabilized denatured damaged DNA sample, thereby forming a damaged DNA mixture,

cooling the damaged DNA mixture under conditions that promote annealing of the ends of the transiently denatured damaged DNA to the substantially denatured damaged DNA,

incubating the annealed damaged DNA under conditions that promote replication of the damaged DNA, wherein the annealed ends of the damaged DNA prime replication, wherein replication of the damaged DNA results in repair of the replicated strands, wherein during replication at least one of the replicated strands is displaced by strand displacement replication of another replicated strand.

202. The method of claim 201 wherein the temperature to which the first damaged DNA sample is heated is maintained during mixing of the second damaged DNA sample with the stabilized denatured damaged DNA sample.

203. The method of claim 201 wherein the pH of the stabilized denatured damaged DNA sample is not high enough nor low enough to cause further substantial denaturation upon mixing second damaged DNA sample with the stabilized denatured damaged DNA sample.

204. The method of claim 201 wherein the first damaged DNA sample is a portion of a damaged DNA sample, wherein the second damaged DNA sample is a portion of the same damaged DNA sample.

205. The method of claim 201 wherein the first damaged DNA sample is from the same source as the second damaged DNA sample.

206. The method of claim 201 wherein the first damaged DNA sample is from the same organism as the second damaged DNA sample.

207. The method of claim 201 wherein the first damaged DNA sample is from the same tissue as the second damaged DNA sample.

208. The method of claim 201 wherein the second damaged DNA sample is mixed with the stabilized denatured damaged DNA sample at a temperature and for a length of time that transiently denatures the damaged DNA in the second damaged DNA sample.

209. The method of claim 208 wherein the temperature is about 70°

C. or less and the length of time is about 30 seconds or less.

210. The method of claim 208 wherein the second damaged DNA sample is mixed with the stabilized denatured damaged DNA sample at a temperature that does not further damaging the DNA.

211. The method of claim 210 wherein the temperature is about 60°

C. to about 70°

C.

212. The method of claim 211 wherein the temperature is about 50°

C. to about 60°

C.

213. The method of claim 212 wherein the temperature is about 40°

C. to about 50°

C.

214. The method of claim 213 wherein the temperature is about 25°

C. to about 40°

C.

215. The method of claim 201 wherein the temperature is about 25°

C. to about 70°

C. and the length of time is about 30 seconds.