Nucleic acid amplification

• US 9,487,823 B2
• Filed: 12/20/2002
• Issued: 11/08/2016
• Est. Priority Date: 12/20/2002
• Status: Active Grant

• Current Assignee: Qiagen GmbH (Qiagen NV)
• Original Assignee: Qiagen GmbH (Qiagen NV)
• Inventors: Lasken, Roger S., Egholm, Michael, Alsmadi, Osama A.
• Primary Examiner(s): Priest, Aaron
• Application Number:
  US10/327,602
• Publication Number:
  US 20040126764A1
• Time in Patent Office: 5,072 Days
• Field of Search: 435/6, 435/91.2, 536/22.1, 536/24.33
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]

Abstract

Disclosed are compositions and a method for amplification of nucleic acid sequences of interest. The disclosed method generally involves replication of a complex nucleic acid sample such as genomic samples using one, a few, or more primers such that, during replication, the replicated strands are displaced from the nucleic acid molecules in the sample by strand displacement replication of another replicated strand. It was discovered that highly complex nucleic acid samples can be efficiently amplified using only one or a few primers having specific nucleic acid sequences. The one or few primers are complementary to nucleic acid sequences distributed throughout nucleic acid in the sample.

Claims

1. A method of amplifying human genomes, the method comprising,

bringing in to contact a single DNA primer of at least 6 nucleotides in length which is non-degenerate and non-random, a non-human, strand displacement DNA polymerase, and a human genomic nucleic acid sample to form a mixture, and incubating the mixture under conditions that promote replication of nucleic acid molecules in the human genomic nucleic acid sample,

wherein the primer hybridizes to nucleic acid molecules in the genomic nucleic acid sample, and wherein the primer has a specific nucleotide sequence, wherein the genomic nucleic acid sample comprises all or a substantial portion of a human genome,

replicating the nucleic acid molecules in the human genomic nucleic acid sample under isothermal conditions, wherein replication of nucleic acid molecules in the genomic nucleic acid sample proceeds by strand displacement replication, wherein replication of the nucleic acid molecules in the genomic nucleic acid sample results in replication of all or a substantial fraction of the nucleic acid molecules in the genomic nucleic acid sample.

2. The method of claim 1 wherein the primer has a length of 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides.

3. The method of claim 1 wherein the primer has one of the sequences AGTGGG or AGAGAG.

4. The method of claim 1 wherein the primer has one of the sequences AGCCGG, AGTAGG, or AGTTGG.

5. The method of claim 1 wherein the primer has one of the sequences AGGCGG, AGTGGG, AGGGAG, or AGTGAG.

6. The method of claim 1 wherein the primer has one of the sequences AGTGGG, AGCCAG, AGTTAG, AGTCAG, or AGACAG.

7. The method of claim 1 wherein the primer has one of the sequences AGAGGG, AGGCAG, AGCCAG, AGTCAG, or AGACAG.

8. The method of claim 1 wherein the primer has one of the sequences CGGTGG, TCACGC, CGAGCG, GCGTGG, ACTCGG, AATCGC, CGGAGG, CCGAGA, GATCGC, AGAGCG, AGCGAG, or ACTCCG.

9. The method of claim 1 wherein the primer is complementary to a sequence in a repeat sequence.

10. The method of claim 9 wherein the repeat sequence is a microsatellite sequence, a minisatellite sequence, a satellite sequence, a transposon sequence, a ribosomal RNA sequence, a short interspersed nuclear element (SINE), or a long interspersed nuclear element (LINE).

11. The method of claim 1 wherein the primer is complementary to a sequence in a functional consensus sequence.

12. The method of claim 11 wherein the functional consensus sequence is a promoter sequence, an enhancer sequence, a silencer sequence, an upstream regulatory element sequence, a transcription termination site sequence, a transposon regulatory sequence, a ribosomal RNA regulatory sequence, or a polyadenylation site sequence.

13. The method of claim 12 wherein the functional consensus sequence is a microbial promoter sequence, a microbial enhancer sequence, a microbial silencer sequence, a microbial upstream regulatory element sequence, a microbial transcription termination site sequence, a microbial transposon regulatory sequence, a microbial ribosomal RNA regulatory sequence, or a microbial polyadenylation site sequence.

14. The method of claim 1 wherein the primer is a broad coverage primer.

15. The method of claim 14 wherein the primer has a G+C percentage within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, or within 1% of the G+C percentage of the genomic nucleic acid sample.

16. The method of claim 14 wherein the primer produces a locus representation of at least 10% for at least 5 different loci for the type of genomic nucleic acid sample used.

17. The method of claim 16 wherein the primer produces a locus representation of at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100°

A for at least 5 different loci for the type of genomic nucleic acid sample used.

18. The method of claim 16 wherein the primer produces a locus representation of at least 10% for at least 6 different loci, at least 7 different loci, at least 8 different loci, at least 9 different loci, at least 10 different loci, at least 11 different loci, at least 12 different loci, at least 13 different loci, at least 14 different loci, at least 15 different loci, at least 16 different loci, at least 17 different loci, at least 18 different loci, at least 19 different loci, at least 20 different loci, at least 25 different loci, at least 30 different loci, at least 40 different loci, at least 50 different loci, at least 75 different loci, or at least 100 different loci for the type of genomic nucleic acid sample used.

19. The method of claim 1 wherein the primer does not have an inter-complementary 3′

end.

20. The method of claim 1 wherein significant replication products are not produced in the absence of a nucleic acid sample.

21. The method of claim 1 wherein the DNA polymerase is φ

29 DNA polymerase.

22. The method of claim 1 wherein the genomic nucleic acid sample is not subjected to denaturing conditions.

23. The method of claim 22 wherein the genomic nucleic acid sample is not subjected to heat denaturing conditions.

24. The method of claim 22 wherein the genomic nucleic acid sample is not subjected to alkaline denaturing conditions.

25. The method of claim 1 wherein the genomic nucleic acid sample is subjected to denaturing conditions.

26. The method of claim 25 wherein the genomic nucleic acid sample is subjected to heat denaturing conditions.

27. The method of claim 25 wherein the genomic nucleic acid sample is subjected to alkaline denaturing conditions.

28. The method of claim 1 wherein nucleic acids in the genomic nucleic acid sample are not separated from other material in the genomic nucleic acid sample.

29. The method of claim 1 wherein the genomic nucleic acid sample is a crude cell lysate.

30. The method of claim 1 wherein the genomic nucleic acid sample is a cell lysate, wherein the cell lysate is produced by exposing cells to alkaline conditions to form a cell lysate, wherein the cell lysate comprises a whole genome, and reducing the pH of the cell lysate to form a stabilized cell lysate.

31. The method of claim 30 wherein nucleic acids in the cell lysate and the stabilized cell lysate are not separated from other material in the cell lysate.

32. The method of claim 30 wherein the cell lysate and the stabilized cell lysate are not subjected to purification prior to incubating the mixture.

33. The method of claim 30 wherein the cell lysate, stabilized cell lysate, or both are subjected to partial purification prior to incubating the mixture.

34. The method of claim 30 wherein the cell lysate and the stabilized cell lysate are not subjected to substantial purification prior to incubating the mixture.

35. The method of claim 30 wherein neither the cell lysate nor the stabilized cell lysate is heated above the temperature of the incubation.

36. The method of claim 30 wherein the cells are not heated above the temperature of the incubation.

37. The method of claim 30 wherein the cells are not lysed by heat.

38. The method of claim 30 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.

39. The method of claim 1 further comprising, prior to bringing into contact the primer, the genomic nucleic acid sample and the DNA polymerase,

exposing the genomic nucleic acid sample to conditions that promote substantial denaturation of the nucleic acid molecules in the genomic nucleic acid sample, thereby forming a denatured genomic nucleic acid sample, and altering the conditions to conditions that do not promote substantial denaturation of nucleic acid molecules in the genomic nucleic acid sample to form a denatured genomic nucleic acid sample.

40. The method of claim 39 wherein replication of the nucleic acid molecules in the genomic nucleic acid sample results in a longer average fragment length for the replicated nucleic acid molecules than the average fragment length in the genomic nucleic acid sample.

41. The method of claim 39 wherein the genomic nucleic acid sample, the denatured genomic nucleic acid sample, or both are exposed to ionic conditions.

42. The method of claim 39 wherein the genomic nucleic acid sample is exposed to conditions that promote substantial denaturation by mixing the genomic nucleic acid sample with a denaturing solution and by heating the genomic nucleic acid sample to a temperature and for a length of time that substantially denatures the nucleic acid molecules in the genomic nucleic acid sample.

43. The method of claim 1 wherein the primer contains at least one modified nucleotide such that the primer is resistant to 3′

-5′

exonuclease.

44. The method of claim 1 wherein the primer is 6 nucleotides long, wherein the primer contains at least one modified nucleotide such that the primer is nuclease resistant, and where in the DNA polymerase is φ

29 DNA polymerase.

45. The method of claim 1 wherein the genomic nucleic acid sample is a blood sample, a urine sample, a semen sample, a lymphatic fluid sample, a cerebrospinal fluid sample, amniotic fluid sample, a biopsy sample, a needle aspiration biopsy sample, a cancer sample, a tumor sample, a tissue sample, a cell sample, a cell lysate sample, a crude cell lysate sample, a forensic sample, an archeological sample, an infection sample, a nosocomial infection sample, a production sample, a drug preparation sample, a biological molecule production sample, a protein preparation sample, a lipid preparation sample, a carbohydrate preparation sample, or a combination thereof.

46. The method of claim 1 wherein the replicated nucleic acid molecules are analyzed.

47. The method of claim 46 wherein the replicated nucleic acid molecules are analyzed using one or more DNA chips.

48. The method of claim 46 wherein the replicated nucleic acid molecules are analyzed by hybridization.

49. The method of claim 46 wherein the replicated nucleic acid molecules are analyzed by nucleic acid sequencing.

50. The method of claim 46 wherein the replicated nucleic acid molecules are stored prior to, following, or both prior to and following their analysis.

51. The method of claim 1 further comprising

bringing into contact the primer, DNA polymerase, and a second genomic nucleic acid sample, and incubating the second genomic nucleic acid sample under conditions that promote replication of nucleic acid molecules in the second genomic nucleic acid sample,

wherein the second genomic nucleic acid sample comprises all or a substantial portion of a genome, wherein replication of nucleic acid molecules in the second genomic nucleic acid sample proceeds by strand displacement replication, wherein replication of the nucleic acid molecules in the second genomic nucleic acid sample results in replication of all or a substantial fraction of the nucleic acid molecules in the second genomic nucleic acid sample.

52. The method of claim 51 wherein the second genomic nucleic acid sample is a sample from the same type of organism as the first genomic nucleic acid sample.

53. The method of claim 51 wherein the second genomic nucleic acid sample is a sample from the same type of tissue as the first genomic nucleic acid sample.

54. The method of claim 51 wherein the second genomic nucleic acid sample is obtained at a different time than the first genomic nucleic acid sample.

55. The method of claim 51 wherein the second genomic nucleic acid sample is a sample from a different organism than the first genomic nucleic acid sample.

56. The method of claim 51 wherein the second genomic nucleic acid sample is a sample from a different type of tissue than the first genomic nucleic acid sample.

57. The method of claim 51 wherein the second genomic nucleic acid sample is a sample from a different species of organism than the first genomic nucleic acid sample.

58. The method of claim 51 wherein the second genomic nucleic acid sample is a sample from a different strain of organism than the first genomic nucleic acid sample.

59. The method of claim 51 wherein the second genomic nucleic acid sample is a sample from a different cellular compartment than the first genomic nucleic acid sample.

80. The method of claim 1, wherein the genomic nucleic acid sample is substantially pure.

89. The method of claim 1, wherein the genomic nucleic acid sample is incubated at a temperature of 23°

C. to 40°

C.

98. The method of claim 1, wherein the DNA polymerase is bacteriophage φ

29 DNA polymerase, Bst large fragment DNA polymerase, Bca DNA polymerase, phage M2 DNA polymerase, phage φ

PRD1 DNA polymerase, exo(−

)VENT®

DNA polymerase, Klenow fragment of DNA polymerase I, T5 DNA polymerase, Sequenase, PRD1 DNA polymerase, or T4 DNA polymerase holoenzyme.

60. A method of amplifying human nucleic acid samples of notable sequence complexity, the method comprising,

bringing into contact a single, non-random, non-degenerate, DNA primer, a non-human, strand displacement DNA polymerase, and a human nucleic acid sample to form a mixture, and incubating the mixture under conditions that promote replication of nucleic acid molecules in the nucleic acid sample,

wherein the primer hybridizes to the nucleic acid sample of notable sequence complexity, and wherein the primer has a specific nucleotide sequence, wherein the nucleic acid sample has a sequence complexity of at least 1×

10⁴ nucleotides,

replicating the nucleic acid molecules in the human genomic nucleic acid sample, wherein replication of nucleic acid molecules in the nucleic acid sample proceeds by strand displacement replication, wherein replication of the nucleic acid molecules in the nucleic acid sample results in replication of all or a substantial fraction of the nucleic acid molecules in the nucleic acid sample, and wherein the primer is 6 or more nucleotides in length.

61. The method of claim 60 wherein the nucleic acid sample has a sequence complexity of at least 1×

10⁵ nucleotides, the nucleic acid sample has a sequence complexity of at least 1×

10⁶ nucleotides, the nucleic acid sample has a sequence complexity of at least 1×

10⁷ nucleotides, the nucleic acid sample has a sequence complexity of at least 1×

10⁸ nucleotides, or the nucleic acid sample has a sequence complexity of at least 1×

10⁹ nucleotides.

62. The method of claim 60 wherein the nucleic acid sample is or is derived from a genome, a chromosome, a chromosome fragment, or a combination.

63. The method of claim 60 wherein the nucleic acid sample is or is derived from a blood sample, a urine sample, a semen sample, a lymphatic fluid sample, a cerebrospinal fluid sample, amniotic fluid sample, a biopsy sample, a needle aspiration biopsy sample, a cancer sample, a tumor sample, a tissue sample, a cell sample, a cell lysate sample, a crude cell lysate sample, a forensic sample, an archeological sample, an infection sample, a nosocomial infection sample, a production sample, a drug preparation sample, a biological molecule production sample, a protein preparation sample, a lipid preparation sample, a carbohydrate preparation sample, or a combination thereof.

81. The method of claim 60, wherein the nucleic acid sample is substantially pure.

90. The method of claim 60, wherein the genomic nucleic acid sample is incubated at a temperature of 23°

C. to 40°

C.

64. A method of amplifying human genomes, the method comprising,

bringing in to contact a single, non-random, non-degenerate, DNA primer, a non-human, strand displacement DNA polymerase, and a human genomic nucleic acid sample to form a mixture, and incubating the mixture under conditions that promote replication of nucleic acid molecules in the genomic nucleic acid sample,

wherein the primer hybridizes to nucleic acid molecules in the genomic nucleic acid sample, and wherein the primer has a specific nucleotide sequence, wherein the genomic nucleic acid sample comprises all or a substantial portion of a genome,

replicating the nucleic acid molecules in the human genomic nucleic acid sample, wherein replication of nucleic acid molecules in the genomic nucleic acid sample proceeds by strand displacement replication, wherein the genomic nucleic acid sample has a sequence complexity of at least 1×

10⁹ nucleotides, wherein replication of the nucleic acid molecules in the genomic nucleic acid sample results in replication of at least 0.01% of the nucleic acid sequences in the genomic nucleic acid sample, and wherein the primer is 6 or more nucleotides in length.

65. The method of claim 64 wherein replication of the nucleic acid molecules in the genomic nucleic acid sample results in replication of at least 0.1% of the nucleic acid sequences in the genomic nucleic acid sample, at least 1% of the nucleic acid sequences in the genomic nucleic acid sample, at least 5% of the nucleic acid sequences in the genomic nucleic acid sample, at least 10% of the nucleic acid sequences in the genomic nucleic acid sample, at least 20% of the nucleic acid sequences in the genomic nucleic acid sample, at least 30% of the nucleic acid sequences in the genomic nucleic acid sample, at least 40% of the nucleic acid sequences in the genomic nucleic acid sample, at least 50% of the nucleic acid sequences in the genomic nucleic acid sample, at least 60% of the nucleic acid sequences in the genomic nucleic acid sample, at least 70% of the nucleic acid sequences in the genomic nucleic acid sample, at least 80% of the nucleic acid sequences in the genomic nucleic acid sample, at least 90% of the nucleic acid sequences in the genomic nucleic acid sample, at least 95% of the nucleic acid sequences in the genomic nucleic acid sample, at least 96% of the nucleic acid sequences in the genomic nucleic acid sample, at least 97% of the nucleic acid sequences in the genomic nucleic acid sample, at least 98% of the nucleic acid sequences in the genomic nucleic acid sample, or at least 99% of the nucleic acid sequences in the genomic nucleic acid sample.

82. The method of claim 64, wherein the genomic nucleic acid sample is substantially pure.

91. The method of claim 64, wherein the genomic nucleic acid sample is incubated at a temperature of 23°

C. to 40°

C.

66. A method of amplifying human genomes, the method comprising,

bringing into contact a single, non-random, non-degenerate, DNA primer, a non-human, strand displacement DNA polymerase, and a human genomic nucleic acid sample to form a mixture, and incubating the mixture under conditions that promote replication of nucleic acid molecules in the genomic nucleic acid sample,

wherein the primer hybridizes to nucleic acid molecules in the genomic nucleic acid sample, and wherein the primer has a specific nucleotide sequence, wherein the genomic nucleic acid sample comprises all or a substantial portion of a genome,

replicating the nucleic acid molecules in the human genomic nucleic acid sample, wherein replication of nucleic acid molecules in the genomic nucleic acid sample proceeds by strand displacement replication, wherein the genomic nucleic acid sample has a sequence complexity of at least 1×

10⁸ nucleotides, wherein replication of the nucleic acid molecules in the genomic nucleic acid sample results in replication of at least 0.1% of the nucleic acid sequences in the genomic nucleic acid sample, and wherein the primer is 6 or more nucleotides in length.

67. The method of claim 66 wherein replication of the nucleic acid molecules in the genomic nucleic acid sample results in replication of at least 1% of the nucleic acid sequences in the genomic nucleic acid sample, at least 5% of the nucleic acid sequences in the genomic nucleic acid sample, at least 10% of the nucleic acid sequences in the genomic nucleic acid sample, at least 20% of the nucleic acid sequences in the genomic nucleic acid sample, at least 30% of the nucleic acid sequences in the genomic nucleic acid sample, at least 40% of the nucleic acid sequences in the genomic nucleic acid sample, at least 50% of the nucleic acid sequences in the genomic nucleic acid sample, at least 60% of the nucleic acid sequences in the genomic nucleic acid sample, at least 70% of the nucleic acid sequences in the genomic nucleic acid sample, at least 80% of the nucleic acid sequences in the genomic nucleic acid sample, at least 90% of the nucleic acid sequences in the genomic nucleic acid sample, at least 95% of the nucleic acid sequences in the genomic nucleic acid sample, at least 96% of the nucleic acid sequences in the genomic nucleic acid sample, at least 97% of the nucleic acid sequences in the genomic nucleic acid sample, at least 98% of the nucleic acid sequences in the genomic nucleic acid sample, or at least 99% of the nucleic acid sequences in the genomic nucleic acid sample.

83. The method of claim 66, wherein the genomic nucleic acid sample is substantially pure.

92. The method of claim 66, wherein the genomic nucleic acid sample is incubated at a temperature of 23°

C. to 40°

C.

68. A method of amplifying human genomes, the method comprising,

bringing into contact a single, non-random, non-degenerate, DNA primer, a non-human, strand displacement DNA polymerase, and a human genomic nucleic acid sample to form a mixture, and incubating the mixture under conditions that promote replication of nucleic acid molecules in the genomic nucleic acid sample,

wherein the primer hybridizes to nucleic acid molecules in the genomic nucleic acid sample, and wherein the primer has a specific nucleotide sequence, wherein the genomic nucleic acid sample comprises all or a substantial portion of a genome,

replicating the nucleic acid molecules in the human genomic nucleic acid sample, wherein replication of nucleic acid molecules in the genomic nucleic acid sample proceeds by strand displacement replication, wherein the genomic nucleic acid sample has a sequence complexity of at least 1×

10⁷ nucleotides, wherein replication of the nucleic acid molecules in the genomic nucleic acid sample results in replication of at least 1% of the nucleic acid sequences in the genomic nucleic acid sample, and wherein the primer is 6 or more nucleotides in length.

69. The method of claim 68 wherein replication of the nucleic acid molecules in the genomic nucleic acid sample results in replication of at least 5% of the nucleic acid sequences in the genomic nucleic acid sample, at least 10% of the nucleic acid sequences in the genomic nucleic acid sample, at least 20% of the nucleic acid sequences in the genomic nucleic acid sample, at least 30% of the nucleic acid sequences in the genomic nucleic acid sample, at least 40% of the nucleic acid sequences in the genomic nucleic acid sample, at least 50% of the nucleic acid sequences in the genomic nucleic acid sample, at least 60% of the nucleic acid sequences in the genomic nucleic acid sample, at least 70% of the nucleic acid sequences in the genomic nucleic acid sample, at least 80% of the nucleic acid sequences in the genomic nucleic acid sample, at least 90% of the nucleic acid sequences in the genomic nucleic acid sample, at least 95% of the nucleic acid sequences in the genomic nucleic acid sample, at least 96% of the nucleic acid sequences in the genomic nucleic acid sample, at least 97% of the nucleic acid sequences in the genomic nucleic acid sample, at least 98% of the nucleic acid sequences in the genomic nucleic acid sample, or at least 99% of the nucleic acid sequences in the genomic nucleic acid sample.

84. The method of claim 68, wherein the genomic nucleic acid sample is substantially pure.

93. The method of claim 68, wherein the genomic nucleic acid sample is incubated at a temperature of 23°

C. to 40°

C.

70. A method of amplifying human genomes, the method comprising,

bringing into contact a single, non-random, non-degenerate, DNA primer, a non-human, strand displacement DNA polymerase, and a human genomic nucleic acid sample to form a mixture, and incubating the mixture under conditions that promote replication of nucleic acid molecules in the genomic nucleic acid sample, wherein the primer hybridizes to nucleic acid molecules in the genomic nucleic acid sample, and wherein the primer has a specific nucleotide sequence, wherein the genomic nucleic acid sample comprises all or a substantial portion of a genome,

replicating the nucleic acid molecules in the human genomic nucleic acid sample, wherein replication of nucleic acid molecules in the genomic nucleic acid sample proceeds by strand displacement replication, wherein the genomic nucleic acid sample has a sequence complexity of at least 1×

10⁶ nucleotides, wherein replication of the nucleic acid molecules in the genomic nucleic acid sample results in replication of at least 10% of the nucleic acid sequences in the genomic nucleic acid sample, and wherein the primer is 6 or more nucleotides in length.

71. The method of claim 70 wherein replication of the nucleic acid molecules in the genomic nucleic acid sample results in replication of at least 20% of the nucleic acid sequences in the genomic nucleic acid sample, at least 30% of the nucleic acid sequences in the genomic nucleic acid sample, at least 40% of the nucleic acid sequences in the genomic nucleic acid sample, at least 50% of the nucleic acid sequences in the genomic nucleic acid sample, at least 60% of the nucleic acid sequences in the genomic nucleic acid sample, at least 70% of the nucleic acid sequences in the genomic nucleic acid sample, at least 80% of the nucleic acid sequences in the genomic nucleic acid sample, at least 90% of the nucleic acid sequences in the genomic nucleic acid sample, at least 95% of the nucleic acid sequences in the genomic nucleic acid sample, at least 96% of the nucleic acid sequences in the genomic nucleic acid sample, at least 97% of the nucleic acid sequences in the genomic nucleic acid sample, at least 98% of the nucleic acid sequences in the genomic nucleic acid sample, or at least 99% of the nucleic acid sequences in the genomic nucleic acid sample.

85. The method of claim 70, wherein the genomic nucleic acid sample is substantially pure.

94. The method of claim 70, wherein the genomic nucleic acid sample is incubated at a temperature of 23°

C. to 40°

C.

72. A method of amplifying human genomes, the method comprising,

bringing into contact a single, non-random, non-degenerate, DNA primer, a non-human, strand displacement DNA polymerase, and a human genomic nucleic acid sample to form a mixture, and incubating the mixture under conditions that promote replication of nucleic acid molecules in the genomic nucleic acid sample,

wherein the primer hybridizes to nucleic acid molecules in the genomic nucleic acid sample, and wherein the primer has a specific nucleotide sequence, wherein the genomic nucleic acid sample comprises all or a substantial portion of a genome,

replicating the nucleic acid molecules in the human genomic nucleic acid sample, wherein replication of nucleic acid molecules in the genomic nucleic acid sample proceeds by strand displacement replication, wherein the genomic nucleic acid sample has a sequence complexity of at least 1×

10⁵ nucleotides, wherein replication of the nucleic acid molecules in the genomic nucleic acid sample results in replication of at least 80% of the nucleic acid sequences in the genomic nucleic acid sample, and wherein the primer is 6 or more nucleotides in length.

73. The method of claim 72 wherein replication of the nucleic acid molecules in the genomic nucleic acid sample results in replication of at least 90% of the nucleic acid sequences in the genomic nucleic acid sample, at least 95% of the nucleic acid sequences in the genomic nucleic acid sample, at least 96% of the nucleic acid sequences in the genomic nucleic acid sample, at least 97% of the nucleic acid sequences in the genomic nucleic acid sample, at least 98% of the nucleic acid sequences in the genomic nucleic acid sample, or at least 99% of the nucleic acid sequences in the genomic nucleic acid sample.

86. The method of claim 72, wherein the genomic nucleic acid sample is substantially pure.

95. The method of claim 72, wherein the genomic nucleic acid sample is incubated at a temperature of 23°

C. to 40°

C.

74. A method of amplifying human genomes, the method comprising,

bringing into contact a single, non-random, non-degenerate, DNA primer, a non-human, strand displacement DNA polymerase, and a human genomic nucleic acid sample to form a mixture, and incubating the mixture under conditions that promote replication of nucleic acid molecules in the genomic nucleic acid sample,

wherein the primer hybridizes to nucleic acid molecules in the genomic nucleic acid sample, and wherein the primer has a specific nucleotide sequence, wherein the genomic nucleic acid sample comprises all or a substantial portion of a genome,

replicating the nucleic acid molecules in the human genomic nucleic acid sample, wherein replication of nucleic acid molecules in the genomic nucleic acid sample proceeds by strand displacement replication, wherein replication of the nucleic acid molecules in the genomic nucleic acid sample results in a locus representation of at least 10% for at least 5 different loci, and wherein the primer is 6 or more nucleotides in length.

75. The method of claim 74 wherein replication of the nucleic acid molecules in the genomic nucleic acid sample results in a locus representation of at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% for at least 5 different loci.

76. The method of claim 74 wherein replication of the nucleic acid molecules in the genomic nucleic acid sample results in a locus representation of at least 10% for at least 6 different loci, at least 7 different loci, at least 8 different loci, at least 9 different loci, at least 10 different loci, at least 11 different loci, at least 12 different loci, at least 13 different loci, at least 14 different loci, at least 15 different loci, at least 16 different loci, at least 17 different loci, at least 18 different loci, at least 19 different loci, at least 20 different loci, at least 25 different loci, at least 30 different loci, at least 40 different loci, at least 50 different loci, at least 75 different loci, or at least 100 different loci.

87. The method of claim 74, wherein the genomic nucleic acid sample is substantially pure.

96. The method of claim 74, wherein the genomic nucleic acid sample is incubated at a temperature of 23°

C. to 40°

C.

77. A method of amplifying human nucleic acid samples of high sequence complexity, the method comprising,

bringing into contact a single, non-random, non-degenerate, DNA primer, a non-human, strand displacement DNA polymerase, and a human nucleic acid sample to form a mixture, and incubating the mixture under conditions that promote replication of nucleic acid molecules in the nucleic acid sample,

wherein the primer hybridizes to the nucleic acid sample of high sequence complexity, and wherein the primer has a specific nucleotide sequence, wherein the nucleic acid sample has a sequence complexity of at least 1×

10³ nucleotides,

replicating the nucleic acid molecules in the human genomic nucleic acid sample, wherein replication of nucleic acid molecules in the nucleic acid sample proceeds by strand displacement replication, wherein replication of the nucleic acid molecules in the nucleic acid sample results in a sequence representation of at least 10% for at least 5 different target sequences, and wherein the primer is 6 or more nucleotides in length.

78. The method of claim 77 wherein replication of the nucleic acid molecules in the nucleic acid sample results in a sequence representation of at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% for at least 5 different target sequences.

79. The method of claim 77 wherein replication of the nucleic acid molecules in the nucleic acid sample results in a sequence representation of at least 10% for at least 6 different target sequences, at least 7 different target sequences, at least 8 different target sequences, at least 9 different target sequences, at least 10 different target sequences, at least 11 different target sequences, at least 12 different target sequences, at least 13 different target sequences, at least 14 different target sequences, at least 15 different target sequences, at least 16 different target sequences, at least 17 different target sequences, at least 18 different target sequences, at least 19 different target sequences, at least 20 different target sequences, at least 25 different target sequences, at least 30 different target sequences, at least 40 different target sequences, at least 50 different target sequences, at least 75 different target sequences, or at least 100 different target sequences.

88. The method of claim 77, wherein the nucleic acid sample is substantially pure.

97. The method of claim 77, wherein the genomic nucleic acid sample is incubated at a temperature of 23°

C. to 40°

C.