Amplification and analysis of whole genome and whole transcriptome libraries generated by a DNA polymerization process

• US 20040209298A1
• Filed: 03/08/2004
• Published: 10/21/2004
• Est. Priority Date: 03/07/2003
• Status: Active Grant

• Current Assignee: Takara Bio Usa, Inc. (Ningbo Junsheng Investment Group Co., Ltd.)
• Original Assignee: Rubicon Genomics, Inc (Ningbo Junsheng Investment Group Co., Ltd.)
• Inventors: Sun, Tong, Kurihara, Takao, Makarov, Vladimir L., Kamberov, Emmanuel, Bruening, Eric, Pinter, Jonathon H., Sleptsova, Irina
• Granted Patent: US 7,718,403 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/15 incorporating a consensus or conserved sequence
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 50/06 Biochemical methods, e.g. using enzymes or whole viable microorganisms

Abstract

The present invention regards a variety of methods and compositions for whole genome amplification and whole transcriptome amplification. In a particular aspect of the present invention, there is a method of amplifying a genome comprising a library generation step followed by a library amplification step. In specific embodiments, the library generating step utilizes specific primer mixtures and a DNA polymerase, wherein the specific primer mixtures are designed to eliminate ability to self-hybridize and/or hybridize to other primers within a mixture but efficiently and frequently prime nucleic acid templates.

Claims

1. A method of preparing a nucleic acid molecule, comprising:

obtaining at least one nucleic acid molecule;

subjecting said nucleic acid molecule to a plurality of primers to form a nucleic acid molecule/ primer mixture, wherein the primers comprise nucleic acid sequence that is substantially non-self-complementary and substantially non-complementary to other primers in the plurality, wherein said sequence comprises in a 5′

to 3′

orientation a constant region and a variable region; and

subjecting said nucleic acid molecule/ primer mixture to a polymerase, under conditions wherein said subjecting steps generate a plurality of molecules including all or part of the constant region at each end.

2. The method of claim 1, wherein said nucleic acid molecule is a single stranded nucleic acid molecule.

3. The method of claim 2, wherein said single stranded nucleic acid molecule is DNA, RNA, or DNA-RNA chimera.

4. The method of claim 1, wherein said nucleic acid molecule is double stranded nucleic acid molecule.

5. The method of claim 4, wherein said double stranded nucleic acid molecule is DNA, RNA, or DNA-RNA chimera.

6. The method of claim 1, wherein said nucleic acid molecule is a mixture of single stranded and double stranded nucleic acid molecules.

7. The method of claim 6, wherein said single stranded molecule is RNA and said double stranded molecule is DNA.

8. The method of claim 1, further comprising the step of designing the primers such that they purposefully are substantially non-self-complementary and substantially noncomplementary to other primers in the plurality.

9. The method of claim 1, wherein the method further comprises the step of amplifying a plurality of the molecules comprising all or part of the constant region to produce amplified molecules.

10. The method of claim 9, wherein said amplifying step comprises polymerase chain reaction.

11. The method of claim 1, wherein the constant and variable regions are each comprised of the same two non-complementary nucleotides.

12. The method of claim 11, wherein the constant and variable regions are each comprised of guanines, adenines, or both.

13. The method of claim 11, wherein the constant and variable regions are each comprised of cytosines, thymidines, or both.

14. The method of claim 11, wherein the constant and variable regions are each comprised of adenines, cytosines, or both.

15. The method of claim 11, wherein the constant and variable regions are each comprised of guanines, thymidines, or both.

16. The method of claim 11, wherein said constant region comprises about 6 to about 100 nucleotides.

17. The method of claim 11, wherein said variable region comprises about 4 nucleotides to about 20 nucleotides.

18. The method of claim 11, wherein the primer is further comprised of 0 to about 3 random bases at its distal 3′

end.

19. The method of claim 11, wherein the constant region and the variable region are each comprised of guanines and thymidines and wherein the polynucleotide comprises 0, 1, 2, or 3 random bases at its 3′

end.

20. The method of claim 11, wherein the nucleotides are base or backbone analogs.

21. The method of claim 1, wherein the polymerase is a strand-displacing polymerase.

22. The method of claim 21, wherein the strand-displacing polymerase is φ

29 Polymerase, Bst Polymerase, Vent Polymerase, 9°

Nm Polymerase, Klenow fragment of DNA Polymerase I, MMLV Reverse Transcriptase, AMV Reverse Transcriptase, Tth DNA polymerase, human HIV Reverse transcriptase, a mutant form of T7 phage DNA polymerase that lacks 3′

-5′

exonuclease activity, or a mixture thereof.

23. The method of claim 22, wherein the strand-displacing polymerase is Klenow Exo-.

24. The method of claim 22, wherein the strand-displacing polymerase is the mutant form of T7 phage DNA polymerase that lacks 3′

→

5′

exonuclease activity.

25. The method of claim 22, wherein the strand-displacing polymerase is MMLV Reverse Transcriptase.

26. The method of claim 1, wherein the method further comprises subjecting the nucleic acid molecule/primer mixture to a polymerase processivity-enhancing compound.

27. The method of claim 26, wherein said compound is single-stranded DNA binding protein or helicase.

28. The method of claim 1, wherein subjecting the mixture to a polymerase step occurs in the presence of one or more agents that facilitate polymerizarion through GC-rich DNA and/or RNA.

29. The method of claim 28, wherein said agents comprise dimethyl sulfoxide (DMSO), 7-Deaza-dGTP, or a mixture thereof.

30. The method of claim 9, wherein said amplifying step occurs in the presence of one or more agents that facilitate polymerization through GC-rich DNA.

31. The method of claim 30, wherein said agents comprise DMSO, 7-Deaza-dGTP, betaine, or a mixture thereof.

32. The method of claim 1, wherein the primers in the plurality each comprise the same constant region.

33. The method of claim 1, wherein two or more constant regions are represented in a mixture of the primers of the plurality.

34. The method of claim 9, wherein said method further comprises the steps of:

modifying the amplified molecules to incorporate modified nucleotide bases, thereby producing labeled molecules, said amplified molecules further defined as single stranded DNA, double stranded DNA, or a mixture thereof;

generating single stranded molecules from the labeled molecules, said single stranded molecules capable of hybridizing to complementary sequences arrayed in known locations on a substrate; and

analyzing at least one hybridization signal.

35. The method of claim 34, wherein said said modifying step comprises chemical, enzymatic, or physical incorporation of the modified nucleotide bases.

36. The method of claim 34, wherein the modified bases are radioactive or fluorescent.

37. The method of claim 34, wherein the generating step comprises denaturation of the double stranded molecules.

38. The method of claim 34, wherein the substrate comprises a microarray substrate.

39. The method of claim 34, wherein the analyzing step comprises measuring the background subtracted intensity of the at least one hybridization signal.

40. The method of claim 34, wherein the analyzing step comprises measurement of copy number, representation, or both of the amplified library.

41. The method of claim 9, wherein a tag is incorporated on the ends of the amplified molecules and wherein said constant region is penultimate to the tags on each end of the amplified molecules.

42. The method of claim 41, wherein said tag is a homopolymeric sequence.

43. The method of claim 42, wherein the homopolymeric sequence comprises poly cytosine (poly C) or poly guanine (poly G).

44. The method of claim 43, wherein the incorporation of the homopolymeric poly G comprises terminal deoxynucleotidyl transferase activity.

45. The method of claim 43, wherein the incorporation of the homopolymeric poly G or poly C comprises ligation of an adaptor comprising the homopolymeric poly G or poly C to the ends of the amplified molecules.

46. The method of claim 43, wherein the incorporation of the homopolymeric poly C comprises replicating the amplified molecules with DNA polymerase, said replicating utilizing a primer comprising in a 5′

to 3′

orientation;

the homopolymeric poly C; and

the constant region.

47. The method of claim 43, wherein the amplified molecules comprising the homopolymeric poly C are further amplified using a primer complementary to a desired sequence in the nucleic acid molecule and a poly C.

48. The method of claim 47, wherein at least some of the amplified DNA is further subjected to sequencing, hybridization, or both.

49. The method of claim 43, wherein the amplified molecules comprising the homopolymeric sequence poly C are further amplified using a mixture of primers complementary to different desired sequences in the nucleic acid molecule and a poly C primer.

50. The method of claim 49, wherein a mixture of several amplified desired DNA molecules is further subjected to sequencing, hybridization, or both.

51. A method of amplifying a DNA molecule, comprising:

obtaining at least one double stranded or single stranded DNA molecule;

subjecting said double stranded DNA molecule to heat to produce at least one single stranded DNA molecule;

subjecting said single stranded DNA molecule to a plurality of primers to form a DNA molecule/primer mixture, wherein the primers comprise nucleic acid sequence that is substantially non-self-complementary and substantially non-complementary to other primers in the plurality, wherein said sequence comprises in a 5′

to 3′

orientation a constant region and a variable region;

subjecting said DNA molecule/primer mixture to a polymerase, under conditions wherein said subjecting steps generate a plurality of DNA molecules comprising the constant region at each end; and

amplifying a plurality of the DNA molecules through polymerase chain reaction, said reaction utilizing a primer complementary to the constant region.

52. The method of claim 51, wherein the DNA molecule is further defined as comprising genomic DNA.

53. The method of claim 51, wherein the DNA molecule is obtained from a human sample.

54. The method of claim 53, wherein the sample comprises blood, serum, or plasma.

55. The method of claim 53, wherein the sample comprises a biopsy.

56. The method of claim 53, wherein the sample comprises a single cell.

57. The method of claim 53, wherein the sample comprises a hair follicle, nipple aspirate, semen, cerebrospinal fluid, urine, sputum, saliva, bronchial lavage, uterine lavage, feces, sweat, cheek scrapings, immunoprecipitated chromatin, physically isolated chromatin, or formalin-fixed tissue.

58. The method of claim 51, wherein the polymerase is a strand-displacing polymerase.

59. The method of claim 58, wherein the strand-displacing polymerase is f29 Polymerase, Bst Polymerase, Vent Polymerase, 9°

Nm Polymerase, Klenow fragment of DNA Polymerase I, MMLV Reverse Transcriptase, AMV Reverse Transcriptase, Tth DNA polymerase, human HIV Reverse transcriptase, a mutant form of T7 phage DNA polymerase that lacks 3′

-5′

exonuclease activity, or a mixture thereof.

60. The method of claim 59, wherein the strand-displacing polymerase is Klenow Exo−

.

61. The method of claim 59, wherein the strand-displacing polymerase is the mutant form of T7 phage DNA polymerase that lacks 3′

→

5′

exonuclease activity.

62. The method of claim 59, wherein the strand-displacing polymerase is MMLV Reverse Transcriptase.

63. The method of claim 51, wherein the method further comprises subjecting the nucleic acid molecule/ primer mixture to a polymerase processivity-enhancing compound.

64. The method of claim 63, wherein said compound is single-stranded DNA binding protein or helicase.

65. The method of claim 51, wherein said polymerase subjecting step occurs in the presence of additieves known to facilitate polymerizarion through GC-rich DNA.

66. The method of claim 65, wherein said additives comprise DMSO, 7-Deaza-dGTP, or a mixture thereof.

67. The method of claim 51, wherein said amplifying step occurs in the presence of one or more agents that facilitate polymerization through GC-rich DNA.

68. The method of claim 67, wherein said agents comprise DMSO, 7-Deaza-dGTP, betain, or a mixture thereof.

69. The method of claim 53, wherein the prepared nucleic acid molecule from the sample provides genomic copy number information.

70. The method of claim 53, wherein the prepared nucleic acid molecule from the sample provides genomic sequence information.

71. The method of claim 53, wherein the prepared nucleic acid molecule from the sample provides allelic variation information.

72. The method of claim 53, wherein the prepared nucleic acid molecule from the sample provides information for disease detection, monitoring, and/or treatment.

73. The method of claim 53, wherein the prepared nucleic acid molecule from the sample provides information for cancer detection, monitoring, and/or treatment.

74. A method of amplifying a RNA molecule, comprising:

obtaining at least one RNA molecule;

optionally heating the molecule to produce at least one single stranded RNA molecule;

subjecting single stranded RNA molecule to a plurality of primers to form a RNA molecule/primer mixture, wherein the primers comprise nucleic acid sequence that is substantially non-self-complementary and substantially non-complementary to other primers in the plurality, wherein said sequence comprises in a 5′

to 3′

orientation a constant region and a variable region;

subjecting said RNA molecule/primer mixture to a polymerase, under conditions wherein said subjecting steps generate a plurality of DNA molecules comprising the constant region at each end; and

amplifying a plurality of the DNA molecules through polymerase chain reaction, said reaction utilizing a primer complementary to the constant region.

75. The method of claim 74, wherein the RNA molecule is obtained from a sample.

76. The method of claim 75, wherein the sample comprises total cellular RNA, a transcriptome, or both.

77. The method of claim 75, wherein the sample is obtained from one or more viruses.

78. The method of claim 75, wherein the sample is obtained from one or more bacteria.

79. The method of claim 75, wherein the sample is obtained from a mixture of animal cells, bacteria, and/or viruses.

80. The method of claim 75, wherein the sample is obtained from a human.

81. The method of claim 80, wherein the sample comprises blood, serum, or plasma.

82. The method of claim 80, wherein the sample comprises a hair follicle, nipple aspirate, semen, cerebrospinal fluid, urine, sputum, saliva, bronchial lavage, uterine lavage, feces, sweat, cheek scrapings, immunoprecipitated chromatin, physically isolated chromatin, or formalin-fixed tissue.

83. The method of claim 80, wherein the sample comprises a single cell.

84. The method of claim 80, wherein the sample comprises mRNA.

85. The method of claim 84, wherein the mRNA is obtained by affinity capture.

86. The method of claim 74, wherein the polymerase is reverse transcriptase.

87. The method of claim 86, wherein the reverse transcriptase is Tth DNA polymerase, AMV Reverse Transcriptase, MMLV Reverse Transcriptase, HIV Reverse transcriptase, or a mixture thereof.

88. The method of claim 86, wherein the reverse transcriptase is MMLV Reverse Transcriptase.

89. The method of claim 74, wherein the method further comprises subjecting the nucleic acid molecule/primer mixture to a polymerase processivity-enhancing compound.

90. The method of claim 89, wherein said compound is single-stranded DNA or RNA binding protein or helicase.

91. The method of claim 74, wherein said subjecting the mixture to a polymerase step occurs in the presence of one or more agents that facilitate polymerization through GC-rich RNA.

92. The method of claim 91, wherein said agents comprise DMSO, 7-Deaza-dGTP, or a mixture thereof.

93. The method of claim 74, wherein said amplifying step occurs in the presence of one ore more agents that facilitate polymerization through GC-rich DNA.

94. The method of claim 93, wherein said agents comprise DMSO, 7-Deaza-dGTP, betain, or a mixture thereof.

95. The method of claim 80, wherein the prepared nucleic acid molecule from the sample provides gene expression information.

96. The method of claim 80, wherein the prepared nucleic acid molecule from the sample provides gene sequence information.

97. The method of claim 80, wherein the prepared nucleic acid molecule from the sample provides exon allelic variation information.

98. The method of claim 80, wherein the prepared nucleic acid molecule from the sample provides information for disease detection, monitoring, and/or treatment.

99. The method of claim 80, wherein the prepared nucleic acid molecule from the sample provides information for cancer detection, monitoring, and/or treatment.

100. A method of amplifying a DNA molecule generated from at least one mRNA molecule, comprising:

obtaining a cDNA molecule from the mRNA molecule;

modifying the cDNA molecule to generate at least one ssDNA molecule;

subjecting said ssDNA molecule to a plurality of primers to form a ssDNA molecule/primer mixture, wherein said primers comprise nucleic acid sequence that is substantially non-self-complementary and substantially non-complementary to other primers in the plurality, wherein said sequence comprises in a 5′

to 3′

orientation a constant region and a variable region;

subjecting said ssDNA molecule/primer mixture to a polymerase, under conditions wherein said subjecting steps generate a plurality of DNA molecules comprising the constant region at each end; and

amplifying a plurality of the DNA molecules comprising the constant region at each end through polymerase chain reaction, said reaction utilizing a primer complementary to the constant region.

101. The method of claim 100, wherein the mRNA is obtained from a human sample.

102. The method of claim 101, wherein the human sample comprises a biopsy.

103. The method of claim 101, wherein the sample comprises a single cell.

104. The method of claim 101, wherein the sample comprises a hair follicle, nipple aspirate, semen, cerebrospinal fluid, urine, sputum, saliva, bronchial lavage, uterine lavage, feces, sweat, cheek scrapings, immunoprecipitated chromatin, physically isolated chromatin, or formalin-fixed tissue.

105. The method of claim 100, wherein said obtaining step is further defined as comprising generation of the cDNA molecule by reverse transcribing the mRNA molecule with a reverse transcriptase.

106. The method of claim 105, wherein said reverse transcriptase is Tth DNA polymerase, AMV Reverse Transcriptase, MMLV Reverse Transcriptase, human HIV Reverse Transcriptase or a mixture thereof.

107. The method of claim 100, wherein the method further comprises subjecting the nucleic acid molecule/primer mixture to a polymerase processivity-enhancing compound.

108. The method of claim 107, wherein said compound is single-stranded DNA binding protein, helicase, or a mixture thereof.

109. The method of claim 100, wherein said subjecting the mixture to a polymerase step occurs in the presence of one or more agents that facilitate polymerizarion through GC-rich DNA.

110. The method of claim 109, wherein said agents comprise DMSO, 7-Deaza-dGTP, or a mixture thereof.

111. The method of claim 100, wherein said amplifying step occurs in the presence of agents that facilitate polymerization through GC-rich DNA.

112. The method of claim 111, wherein said agents comprise DMSO, 7-Deaza-dGTP, betain, or a mixture thereof.

113. The method of claim 101, wherein the prepared nucleic acid molecule from the sample provides gene expression information.

114. The method of claim 101, wherein the prepared nucleic acid molecule from the sample provides gene sequence information.

115. The method of claim 101, wherein the prepared nucleic acid molecule from the sample provides exon allelic variation information.

116. The method of claim 101, wherein the prepared nucleic acid molecule from the sample provides information for disease detection, monitoring, and/or treatment.

117. The method of claim 101, wherein the prepared nucleic acid molecule from the sample provides information for cancer detection, monitoring, and/or treatment.

118. A method of obtaining a total nucleic acid from a sample comprising a mixture of DNA and RNA, comprising:

providing the mixture of DNA and RNA;

optionally heating the mixture to a temperature that denatures double stranded nucleic acids; and

subjecting the mixture to a polymerase that replicates both single stranded DNA and RNA.

119. The method of claim 118, wherein said method consists essentially of said providing, optionally heating, and subjecting steps.

120. The method of claim 118, wherein said subjecting step is further defined as:

subjecting the mixture to a plurality of primers to form a nucleic acid/primer mixture, wherein the primers comprise nucleic acid sequence that is substantially non-self-complementary and substantially non-complementary to other primers in the plurality, wherein said sequence comprises in a 5′

to 3′

orientation a constant region and a variable region;

subjecting said nucleic acid/primer mixture to the polymerase that efficiently replicates both DNA and RNA, under conditions wherein said subjecting steps generate a plurality of DNA molecules comprising the constant nucleic acid sequence at each end; and

amplifying a plurality of the DNA molecules comprising the constant region at each end through polymerase chain reaction, said reaction utilizing a primer complementary to the constant region.

121. The method of claim 118, wherein the polymerase is reverse transcriptase.

122. The method of claim 121, wherein the reverse transcriptase is Tth DNA polymerase, AMV Reverse Transcriptase, MMLV Reverse Transcriptase, HIV Reverse transcriptase, or a mixture thereof.

123. The method of claim 122, wherein the reverse transcriptase is MMLV Reverse Transcriptase.

124. The method of claim 118, wherein said sample is from a human.

125. The method of claim 124, wherein the sample comprises blood, serum, or plasma.

126. The method of claim 124, wherein the sample comprises a hair follicle, nipple aspirate, semen, cerebrospinal fluid, urine, sputum, saliva, bronchial lavage, uterine lavage, feces, sweat, cheek scrapings, immunoprecipitated chromatin, physically isolated chromatin, or formalin-fixed tissue.

127. The method of claim 124, wherein the sample comprises a single cell.

128. The method of claim 118, wherein the method further comprises subjecting the nucleic acid molecule/primer mixture to a polymerase processivity-enhancing compound.

129. The method of claim 128, wherein said compound is single-stranded DNA or RNA binding protein or helicase.

130. The method of claim 120, wherein said subjecting the mixture to a polymerase step occurs in the presence of one or more agents that facilitate polymerizarion through GC-rich DNA and RNA.

131. The method of claim 130, wherein said agents comprise DMSO, 7-Deaza-dGTP, or a mixture thereof.

132. The method of claim 120, wherein said amplifying step occurs in the presence of one or more agents that facilitate polymerization through GC-rich DNA.

133. The method of claim 132, wherein said agents comprise DMSO, 7-Deaza-dGTP, betain, or a mixture thereof.

134. The method of claim 124, wherein the prepared nucleic acid molecule from the sample provides nucleic acoid sequence information.

135. The method of claim 124, wherein the prepared nucleic acid molecule from the sample provides allelic variation information.

136. The method of claim 124, wherein the prepared nucleic acid molecule from the sample provides information for disease detection, monitoring, and/or treatment.

137. The method of claim 124, wherein the prepared nucleic acid molecule from the sample provides information for cancer detection, monitoring, and/or treatment.

138. A method of differentially obtaining DNA or RNA, respectively, from a sample comprising a mixture of DNA and RNA, comprising:

providing the mixture of DNA and RNA;

heating the mixture to a temperature that selectively affects the DNA or RNA; and

subjecting the mixture to a polymerase that selectively replicates the respective DNA or RNA.

139. The method of claim 138, wherein said subjecting step is further defined as:

subjecting the mixture to a plurality of primers to form a nucleic acid/primer mixture, wherein the primers comprise nucleic acid sequence that is substantially non-self-complementary and substantially non-complementary to other primers in the plurality, wherein said sequence comprises in a 5′

to 3′

orientation a constant region and a variable region;

subjecting said nucleic acid/primer mixture to the polymerase that selectively replicates the respective DNA or RNA, under conditions wherein said subjecting steps generate a plurality of DNA molecules comprising the known nucleic acid sequence at each end; and

amplifying a plurality of the DNA molecules comprising the constant region at each end through polymerase chain reaction, said reaction utilizing a primer complementary to the constant region.

140. The method of claim 138, wherein said sample is from a human.

141. The method of claim 138, wherein the sample comprises blood, serum, or plasma.

142. The method of claim 138, wherein the sample comprises a hair follicle, nipple aspirate, semen, cerebrospinal fluid, urine, sputum, saliva, bronchial lavage, uterine lavage, feces, sweat, cheek scrapings, immunoprecipitated chromatin, physically isolated chromatin, or formalin-fixed tissue.

143. The method of claim 138, wherein the sample comprises a single cell.

144. A method of differentially obtaining DNA from a sample comprising DNA and RNA, comprising:

providing the mixture of DNA and RNA;

heating said mixture to a temperature of at least about 94°

C. to about 100°

C. to generate single stranded nucleic acids; and

subjecting the mixture to a polymerase that replicates only DNA templates.

145. The method of claim 144, further comprising the steps of:

subjecting the mixture to a plurality of primers to form a nucleic acid/primer mixture, wherein the primers comprise nucleic acid sequence that is substantially non-self-complementary and substantially non-complementary to other primers in the plurality, wherein said sequence comprises in a 5′

to 3′

orientation a constant region and a variable region;

subjecting said nucleic acid/primer mixture to a polymerase that selectively replicates DNA, under conditions wherein said subjecting steps generate a plurality of DNA molecules comprising the constant nucleic acid sequence at each end; and

amplifying a plurality of the DNA molecules comprising the constant nucleic acid sequence at each end through polymerase chain reaction, said reaction utilizing a primer complementary to the constant nucleic acid sequence.

146. The method of claim 145, wherein the polymerase is DNA-dependent DNA polymerase.

147. The method of claim 146, wherein said DNA-dependent DNA polymerase is φ

29 Polymerase, Bst Polymerase, Vent Polymerase, 9°

Nm Polymerase, Klenow Exo⁻

fragment of DNA Polymerase I, a mutant form of T7 phage DNA polymerase that lacks 3′

-5′

exonuclease activity, or a mixture thereof.

148. The method of claim 147, wherein the polymerase is Klenow Exo⁻

fragment of DNA Polymerase I.

149. The method of claim 145, wherein the method further comprises subjecting the nucleic acid molecule/primer mixture to a polymerase processivity-enhancing compound.

150. The method of claim 149, wherein said compound is single-stranded DNA binding protein or helicase.

151. The method of claim 145, wherein said subjecting the mixture to a polymerase step occurs in the presence of one or more agents that facilitate polymerizarion through GC-rich DNA.

152. The method of claim 151, wherein said agents comprise DMSO, 7-Deaza-dGTP, or a mixture thereof.

153. The method of claim 145, wherein said amplifying step occurs in the presence of one or more agents that facilitate polymerization through GC-rich DNA.

154. The method of claim 153, wherein said agents comprise DMSO, 7-Deaza-dGTP, betain, or a mixture thereof.

155. The method of claim 144, wherein the sample is from a human.

156. The method of claim 155, wherein the sample comprises blood, serum, or plasma.

157. The method of claim 155, wherein the sample comprises a hair follicle, nipple aspirate, semen, cerebrospinal fluid, urine, sputum, saliva, bronchial lavage, uterine lavage, feces, sweat, cheek scrapings, immunoprecipitated chromatin, physically isolated chromatin, or formalin-fixed tissue.

158. The method of claim 155, wherein the sample comprises a single cell.

159. The method of claim 155, wherein the prepared nucleic acid molecule from the sample provides genomic copy number information.

160. The method of claim 155, wherein the prepared nucleic acid molecule from the sample provides genomic sequence information.

161. The method of claim 155, wherein the prepared nucleic acid molecule from the sample provides allelic variation information.

162. The method of claim 155, wherein the prepared nucleic acid molecule from the sample provides information for disease detection, monitoring, and/or treatment.

163. The method of claim 155, wherein the prepared nucleic acid molecule from the sample provides information for cancer detection, monitoring, and/or treatment.

164. A method of differentially obtaining RNA from a sample comprising dsDNA and RNA, comprising:

providing the mixture of dsDNA and RNA;

optionally heating said mixture to a temperature not exceeding about 75°

C. to prevent denaturation of dsDNA; and

subjecting the mixture to a polymerase that replicates only single stranded RNA templates.

165. The method of claim 164, wherein said subjecting step is further defined as:

subjecting the mixture to a plurality of primers to form a nucleic acid/primer mixture, wherein the primers comprise nucleic acid sequence that is substantially non-self-complementary and substantially non-complementary to other primers in the plurality, wherein said sequence comprises in a 5′

to 3′

orientation a constant region and a variable region;

subjecting said nucleic acid/primer mixture to a polymerase that prime and replicate only single-stranded RNA, under conditions wherein said subjecting steps generate a plurality of DNA molecules comprising the constant nucleic acid sequence at each end; and

amplifying a plurality of the DNA molecules comprising the constant region at each end through polymerase chain reaction, said reaction utilizing a primer complementary to the constant region.

166. The method of claim 165, wherein the polymerase is reverse transcriptase.

167. The method of claim 166, wherein the reverse transcriptase is Tth DNA polymerase, AMV Reverse Transcriptase, MMLV Reverse Transcriptase, HIV Reverse transcriptase, or a mixture thereof.

168. The method of claim 166, wherein the reverse transcriptase is MMLV Reverse Transcriptase.

169. The method of claim 165, wherein the method further comprises subjecting the nucleic acid molecule/primer mixture to a polymerase processivity-enhancing compound.

170. The method of claim 169, wherein said compound is single-stranded DNA or RNA binding protein or helicase.

171. The method of claim 165, wherein said polymerase subjecting step occurs in the presence of one or more agents that facilitate polymerizarion through GC-rich RNA.

172. The method of claim 171, wherein said agents comprise DMSO, 7-Deaza-dGTP, or a mixture thereof.

173. The method of claim 165, wherein said amplifying step occurs in the presence of one or more agents that facilitate polymerization through GC-rich DNA.

174. The method of claim 173, wherein said agents comprise DMSO, 7-Deaza-dGTP, betain, or a mixture thereof.

175. The method of claim 164, wherein the sample is from a human.

176. The method of claim 175, wherein the sample comprises blood, serum, or plasma.

177. The method of claim 175, wherein the sample comprises a hair follicle, nipple aspirate, semen, cerebrospinal fluid, urine, sputum, saliva, bronchial lavage, uterine lavage, feces, sweat, cheek scrapings, immunoprecipitated chromatin, or physically isolated chromatin, or formalin-fixed tissue.

178. The method of claim 175, wherein the sample comprises a single cell.

179. The method of claim 175, wherein the prepared nucleic acid molecule from the sample provides gene expression information.

180. The method of claim 175, wherein the prepared nucleic acid molecule from the sample provides gene sequence information.

181. The method of claim 175, wherein the prepared nucleic acid molecule from the sample provides exon allelic variation information.

182. The method of claim 175, wherein the prepared nucleic acid molecule from the sample provides information for disease detection, monitoring, and/or treatment.

183. The method of claim 175, wherein the prepared nucleic acid molecule from the sample provides information for cancer detection, monitoring, and/or treatment.

184. A plurality of polynucleotides, wherein the polynucleotides in the plurality comprise nucleic acid sequence that is substantially non-self-complementary and substantially non-complementary to other polynucleotides in the plurality.

185. The plurality of claim 184, wherein said nucleic acid sequence is further defined as rendering the polynucleotides substantially incapable of at least one of the following:

self-hybridization;

self-priming;

hybridization to another polynucleotide in the plurality;

or

initiation of a polymerization reaction in the plurality.

186. The plurality of claim 184, wherein the polynucleotides are further defined as having a 5′

to 3′

orientation and comprising a constant region that is 5′

to a variable region.

187. The plurality of claim 186, wherein the constant region is for subsequent amplification.

188. The plurality of claim 186, wherein the variable region is for random annealing, random priming, or both.

189. The plurality of claim 186, wherein the constant and variable regions are each comprised of two non-complementary nucleotides.

190. The plurality of claim 186, wherein the constant and variable regions are each comprised of guanines, adenines, or both.

191. The plurality of claim 186, wherein the constant and variable regions are each comprised of cytosines, thymidines, or both.

192. The plurality of claim 186, wherein the constant and variable regions are each comprised of adenines, cytosines, or both.

193. The plurality of claim 186, wherein the constant and variable regions are each comprised of guanines, thymidines, or both.

194. The plurality of claim 186, wherein said constant region comprises about 6 to about 100 nucleotides.

195. The plurality of claim 186, wherein said second region comprises about 4 nucleotides to about 20 nucleotides.

196. The plurality of claim 186, wherein the polynucleotide is further comprised of 0 to about 3 random bases at its distal 3′

end.

197. The plurality of claim 186, wherein the first region and the second region are each comprised of guanines and thymidines and wherein the polynucleotide comprises 0, 1, 2, or 3 random bases at its 3′

end.

198. The plurality of claim 184, wherein the nucleic acid sequence is comprised of base or backbone analogs.

199. The plurality of claim 184, wherein the concentration of each polynucleotide in the plurality of primers is adjusted for optimal priming of a given nucleic acid.

200. The plurality of claim 199, wherein the concentration of all polynucleotides in the plurality is equimolar.

201. The plurality of claim 199, wherein the given nucleic acid comprises part or all of a genome.

202. The plurality of claim 199, wherein the given nucleic acid comprises part or all of a transciptome.

203. A method of amplifying a genome, a transcriptome, or both comprising:

obtaining genomic DNA, RNA, or both;

modifying the genomic DNA, RNA, or both to generate at least one single stranded nucleic acid molecule;

subjecting said single stranded nucleic acid molecule to a plurality of primers to form a nucleic acid/primer mixture, wherein the primers comprise nucleic acid sequence that is substantially non-self-complementary and substantially non-complementary to other primers in the plurality, wherein said sequence comprises in a 5′

to 3′

orientation a constant region and a variable region;

subjecting said nucleic acid/primer mixture to a polymerase, under conditions wherein said subjecting steps generate a plurality of DNA molecules comprising the constant region at each end; and

amplifying a plurality of the DNA molecules through polymerase chain reaction, said reaction utilizing a primer complementary to the constant region.

204. The method of claim 203, wherein said method further comprises the steps of:

modifying amplified DNA molecules to produce single stranded molecules, said single stranded molecules comprising the constant region at both the 5′ and

3′

ends, wherein said DNA molecules are further defined as single stranded DNA, double stranded DNA, or a mixture thereof;

hybridizing a region of at least one of the single stranded DNA molecules to a complementary region in the 3′

end of an oligonucleotide immobilized to a support to produce a single stranded DNA/oligonucleotide hybrid; and

extending the 3′

end of the oligonucleotide to produce an extended polynucleotide.

205. The method of claim 203, wherein said method further comprises the step of removing the single stranded DNA molecule from the single stranded DNA/oligonucleotide hybrid.

206. A kit comprising a plurality of polynucleotides, wherein the polynucleotides comprise nucleic acid sequence that is substantially non-self-complementary and substantially non-complementary to other polynucleotides in the plurality, said plurality dispersed in a suitable container.

207. The kit of claim 206, further comprising a polymerase.

208. The kit of claim 207, wherein said polymerase is a strand-displacing polymerase.

209. The kit of claim 208, wherein said strand-displacing polymerase is φ

29 Polymerase, Bst Polymerase, Vent Polymerase, 9°

Nm Polymerase, Klenow fragment of DNA Polymerase I, MMLV Reverse Transcriptase, Tth DNA polymerase, AMV Reverse Transcriptase, human HIV Reverse Transcriptase, a mutant form of T7 phage DNA polymerase that lacks 3′

-5′

exonuclease activity, or a mixture thereof.

210. A method of amplifying a population of DNA molecules comprised in a plurality of populations of DNA molecules, said method comprising the steps of:

obtaining a plurality of populations of DNA molecules, wherein at least one population in said plurality comprises DNA molecules having in a 5′

to 3′

orientation the following;

a known identification sequence specific for said population; and

a known primer amplification sequence; and

amplifying said population of DNA molecules by polymerase chain reaction, said reaction utilizing a primer for said identification sequence.

211. The method of claim 210, wherein said obtaining step is further defined as:

obtaining a population of DNA molecules, said molecules comprising a known primer amplification sequence;

amplifying said DNA molecules with a primer having in a 5′

to 3′

orientation the following;

the known identification sequence; and

the known primer amplification sequence; and

mixing said population with at least one other population of DNA molecules.

212. The method of claim 210, wherein said population of DNA molecules comprises genomic DNA.

213. The method of claim 210, wherein said population of DNA molecules comprises a genome.

214. The method of claim 210, wherein said population of DNA molecules comprises a transcriptome.

215. A method of amplifying a population of DNA molecules comprised in a plurality of populations of DNA molecules, said method comprising the steps of:

obtaining a plurality of populations of DNA molecules, wherein at least one population in said plurality comprises DNA molecules, wherein the 5′

ends of said DNA molecules comprise in a 5′

to 3′

orientation the following;

a single-stranded region comprising a known identification sequence specific for said population; and

a known primer amplification sequence;

isolating said population through binding of at least part of the single stranded known identification sequence of a plurality of said DNA molecules to a surface; and

amplifying the isolated DNA molecules by polymerase chain reaction, said reaction utilizing a primer for said primer amplification sequence.

216. The method of claim 215, wherein said obtaining step is further defined as:

obtaining a population of DNA molecules, said molecules comprising a known primer amplification sequence;

amplifying said DNA molecules with a primer comprising in a 5′

to 3′

orientation the following;

the known identification sequence;

a non-replicable linker; and

the known primer amplification sequence; and

mixing said population with at least one other population of DNA molecules.

217. The method of claim 216, wherein said isolating step is further defined as binding at least part of the single stranded known identification sequence to an immobilized oligonucleotide comprising a region complementary to the known identification sequence.

218. A method of immobilizing an amplified genome, transcriptome, or both, comprising the steps of:

obtaining an amplified genome, transcriptome, or both, wherein a plurality of molecules from the genome, transcriptome, or both comprise a known primer amplification sequence at both the 5′ and

3′

ends of the molecules; and

attaching a plurality of the molecules to a support.

219. The method of claim 218, wherein said attaching step is further defined as comprising covalently attaching the plurality of molecules to the support through said known primer amplification sequence.

220. The method of claim 219, wherein said covalently attaching step is further defined as:

hybridizing a region of at least one single stranded molecule to a complementary region in the 3′

end of a oligonucleotide immobilized to said support; and

extending the 3′

end of the oligonucleotide to produce a single stranded molecule/extended polynucleotide hybrid.

221. The method of claim 220, wherein said method further comprises the step of removing the single stranded DNA molecule from the single stranded molecule/extended polynucleotide hybrid to produce an extended polynucleotide.

222. The method of claim 220, wherein said method further comprises the step of replicating the extended polynucleotide.

223. The method of claim 222, wherein said replicating step is further defined as:

providing to said extended polynucleotide a polymerase and a primer complementary to the known primer amplification sequence;

extending the 3′

end of said primer to form an extended primer molecule; and

releasing said extended primer molecule.

224. A method of immobilizing an amplified genome, comprising the steps of:

obtaining an amplified genome, wherein a plurality of DNA molecules from the genome and comprise;

a tag; and

a known primer amplification sequence at both the 5′ and

3′

ends of the molecules; and

attaching a plurality of the DNA molecules to a support.

225. The method of claim 224, wherein said attaching step is further defined as comprising attaching the plurality of DNA molecules to the support through said tag.

226. The method of claim 224, wherein said tag comprises biotin and said support comprises streptavidin.

227. The method of claim 224, wherein said tag comprises an amino group or a carboxy group.

228. The method of claim 224, wherein said tag comprises a single stranded region and said support comprises an oligonucleotide comprising a sequence complementary to a region of said tag.

229. The method of claim 228, wherein said single stranded region is further defined as comprising an identification sequence.

230. The method of claim 229, wherein said DNA molecules are further defined as comprising a non-replicable linker that is 3′

to said identification sequence and that is 5′

to said known primer amplification sequence.

231. The method of claim 224, wherein said method further comprises the steps of removing contaminants from the immobilized genome.

232. A plurality of ds DNA molecules comprising genomic DNA, wherein when the molecules are denatured to produce first and second strand molecules, each of which comprises a first and second end region at the respective ends of the first and second strand molecules, each of the first and second end regions of the first molecule comprise nucleic acid sequence that is substantially non-self-complementary to sequence in the first and second end regions in said first molecule, and each of the first and second end regions of the second molecule comprise nucleic acid sequence that is substantially non-self-complementary to sequence in the first and second end regions in said second molecule.

233. The plurality of claim 232, wherein each of the first and second end regions of the first strand molecule are substantially non-complementary to the first and second end regions of the first strand of other molecules in the plurality, and wherein each of the first and second end regions of the second strand molecule are substantially non-complementary to the first and second end regions of the second strand of other molecules in the plurality.

234. The plurality of claim 232, wherein the DNA molecules further comprise a homopolymeric tag at the first and second end regions, wherein said end regions are penultimate on the molecules to the homopolymeric tag.

235. The plurality of claim 232, further defined as a genomic library.

236. A method of sequencing a genome from a limited source of material, comprising the steps of:

obtaining at least one double stranded or single stranded DNA molecule from a limited source of material;

subjecting said double stranded DNA molecule to heat to produce at least one single stranded DNA molecule;

subjecting said single stranded DNA molecule to a plurality of primers to form a DNA molecule/primer mixture, wherein the primers comprise nucleic acid sequence that is substantially non-self-complementary and substantially non-complementary to other primers in the plurality, wherein said sequence comprises in a 5′

to 3′

orientation a constant region and a variable region;

subjecting said DNA molecule/primer mixture to a polymerase, under conditions wherein said subjecting steps generate a plurality of DNA molecules comprising the constant region at each end; and

amplifying a plurality of the DNA molecules through polymerase chain reaction, said reaction utilizing a primer complementary to the constant region;

providing from the plurality of the amplified molecules a first and second sample of amplified DNA molecules;

sequencing at least some of the amplified DNA molecules from the first sample to obtain at least one specific DNA sequence;

incorporating homopolymeric poly C/poly G sequence to the ends of the amplified DNA molecules from the second sample to produce homopolymeric amplified molecules;

amplifying at least some of the homopolymeric amplified molecules from the second sample with a poly C primer and a primer complementary to the specific DNA sequence; and

repeating the sequencing and amplifying steps related to additional specific sequences, thereby producing a substantially complete contig of the genome.

237. The method of claim 236, wherein said incorporating of the homopolymeric sequence comprises one of the following steps:

extending the 3′

end of the amplified DNA fragments by terminal deoxynucleotidyl transferase in the presence of dGTP;

ligating an adaptor comprising the homopolymeric poly C/poly G sequence to the ends of the amplified DNA fragments;

or

replicating the amplified DNA fragments with a primer comprising the homopolymeric poly C sequence at its 5′

end and constant region at the 3′

end.

238. The method of claim 236, wherein said sequencing step is further defined as:

cloning the amplified DNA fragments from the first sample into a vector; and

sequencing at least some of the cloned fragments.

239. The method of claim 236, wherein the specific sequence of the amplified molecule is obtained by the sequencing step of the first sample and wherein one or more of the additional specific sequences is obtained by the sequencing step of amplified molecules from the second sample.

240. The method of claim 236, wherein said limited source of material is a microorganism substantially resistant to culturing.

241. The method of claim 236, wherein said limited source of material is an extinct species.

242. The method of claim 236, wherein said sequencing a genome is achieved with minimal redundancy.