Nucleic acid amplification

• US 20030228613A1
• Filed: 05/02/2003
• Published: 12/11/2003
• Est. Priority Date: 10/15/2001
• Status: Active Grant

• Current Assignee: Qiagen GmbH (Qiagen NV)
• Original Assignee: Qiagen GmbH (Qiagen NV)
• Inventors: Lasken, Roger S., Bornarth, Carole, Hosono, Seiyu, Wisniewski, Michele, Raghunathan, Arumugham
• Granted Patent: US 7,297,485 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 2523/113 Denaturating agents
• CPC Class Codes: C12Q 2523/119 Renaturing agents
• 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 2527/137 Concentration of a component of medium
• 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 methods for amplification of nucleic acid sequences of interest. It has been discovered that amplification reactions can produce amplification products of high quality, such as low amplification bias, if performed on an amount of nucleic acid at or over a threshold amount and/or on nucleic acids at or below a threshold concentration. The threshold amount and concentration can vary depending on the nature and source of the nucleic acids to be amplified and the type of amplification reaction employed. Disclosed is a method of determining the threshold amount and/or threshold concentration of nucleic acids that can be used with nucleic acid samples of interest in amplification reactions of interest. Because amplification reactions can produce high quality amplification products, such as low bias amplification products, below the threshold amount and/or concentration of nucleic acid, such below-threshold amounts and/or concentrations can be used in amplification reactions.

Claims

1. A method of amplifying nucleic acids, the method comprising,

exposing a sample suspected of containing nucleic acids to alkaline conditions,

reducing the pH of all or a portion of the sample to form a stabilized sample, and

incubating an amplification mixture under conditions that promote replication of the nucleic acids from the sample, wherein the amplification mixture comprises all or a portion of the stabilized sample,

wherein replication of the nucleic acids results in replicated strands, wherein during replication at least one of the replicated strands is displaced from nucleic acids in the sample by strand displacement replication of another replicated strand, wherein the replicated strands have low amplification bias.

2. The method of claim 1 wherein the concentration of nucleic acids in the amplification mixture favors hybridization of primers over reassociation of the nucleic acids.

3. The method of claim 2 wherein the concentration of nucleic acids in the amplification mixture is 10 ng/μ

l or less.

4. The method of claim 2 wherein the concentration of nucleic acids in the amplification mixture is 8 ng/μ

l or less, 6 ng/μ

l or less, 5 ng/μ

l or less, 4 ng/μ

l or less, 3 ng/μ

l or less, 2 ng/μ

l or less, 1 ng/μ

l or less, or 0.5 ng/μ

l or less.

5. The method of claim 2 wherein the concentration of nucleic acids in the amplification mixture is 100 ng/μ

l or less.

6. The method of claim 1 wherein the amount of nucleic acids in the amplification mixture is at or above a threshold that can result in low amplification bias in the replicated strands.

7. The method of claim 6 wherein the amplification mixture comprises at least 100 ng of nucleic acid.

8. The method of claim 7 wherein the amplification sample comprises at least 150 ng, at least 200 ng, at least 300 ng, at least 400 ng, at least 500 ng, at least 1 mg, at least 2 mg, or at least 3 mg of nucleic acid.

9. The method of claim 6 wherein the amplification mixture comprises at least 10 ng of nucleic acid.

10. The method of claim 1 wherein the amplification bias of the replicated strands is less than 20-fold for at least ten target sequences in the sample.

12. The method of claim 1 wherein the locus representation of the replicated strands is at least 10% for at least 5 different loci.

15. The method of claim 1 wherein the amplification bias of the replicated strands is less than 50-fold.

18. The method of claim 1 wherein the sample comprises cells, wherein the alkaline conditions promote lysis of the cells, wherein the alkaline conditions result in a cell lysate.

20. The method of claim 1 wherein the sample comprises nucleic acids, wherein the nucleic acids comprise a genome, wherein replication of the nucleic acids results in replication of the genome.

33. The method of claim 1 wherein the sample is a eukaryotic sample, a plant sample, an animal sample, a vertebrate sample, a fish sample, a mammalian sample, a human sample, a non-human sample, a bacterial sample, a microbial sample, a viral sample, a biological sample, a serum sample, a plasma sample, 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 tissue lysate sample, a tissue culture cell sample, a buccal swab sample, a mouthwash sample, a stool sample, a mummified tissue sample, a forensic sample, an autopsy 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, an inanimate object sample, an air sample, a soil sample, a sap sample, a metal sample, a fossil sample, an excavated material sample, a terrestrial sample, an extra-terrestrial sample, or a combination thereof.

35. The method of claim 1 wherein the sample is exposed to alkaline conditions by mixing the sample with a lysis solution.

43. The method of claim 1 wherein the pH of the sample is reduced to the range of about pH 7.0 to about pH 6.8.

44. The method of claim 1 wherein the pH of the sample is reduced by mixing the sample with a stabilization solution.

45. The method of claim 44 wherein the stabilization solution comprises a buffer, an acid, or a combination.

46. The method of claim 45 wherein the stabilization solution comprises a buffer, wherein the buffer is Tris-HCl.

47. The method of claim 46 wherein the stabilization solution comprises 800 mM Tris-HCl.

48. The method of claim 46 wherein the stabilization solution comprises 200 mM Tris-HCl.

49. The method of claim 46 wherein the stabilization solution comprises 20 mM Tris-HCl.

50. The method of claim 44 wherein the sample is mixed with an equal volume of the stabilization solution.

51. The method of claim 1 wherein exposing the sample to alkaline conditions, reducing the pH of the sample, and incubating the stabilized sample are performed in the same reaction chamber.

52. The method of claim 51 wherein the reaction chamber comprises a tube, a test tube, an eppendorf tube, a vessel, a micro vessel, a plate, a well, a well of a micro well plate, a well of a microtitre plate, a chamber, a micro fluidics chamber, a micro machined chamber, a sealed chamber, a hole, a depression, a dimple, a dish, a surface, a membrane, a microarray, a fiber, a glass fiber, an optical fiber, a woven fiber, a film, a bead, a bottle, a chip, a compact disk, a shaped polymer, a particle, or a microparticle.

53. The method of claim 52 wherein the surface is sealable.

54. The method of claim 51 wherein the reaction chamber comprises acrylamide, cellulose, nitrocellulose, glass, gold, polystyrene, polyethylene vinyl acetate, polypropylene, polymethacrylate, polyethylene, polyethylene oxide, glass, polysilicates, polycarbonates, teflon, fluorocarbons, nylon, silicon rubber, polyanhydrides, polyglycolic acid, polylactic acid, polyorthoesters, functionalized silane, polypropylfumerate, collagen, glycosaminoglycans, polyamino acids, or a combination.

55. The method of claim 51 wherein the nucleic acids are not purified or extracted prior to incubation of the amplification mixture.

56. The method of claim 51 wherein nucleic acids in the sample are not separated from other material in the sample.

57. The method of claim 51 wherein nucleic acids in the stabilized sample are not separated from other material in the stabilized sample.

58. The method of claim 1 wherein the nucleic acids in the stabilized sample are less than 0.01% pure, less than 0.1% pure, less than 0.5% pure, less than 1% pure, less than 5% pure, less than 10% pure, or less than 20% pure by weight excluding water.

59. The method of claim 1 wherein the sample is not subjected to substantial purification prior to the incubation.

60. The method of claim 59 wherein the sample is subjected to centrifugation, extraction, chromatography, precipitation, filtration, or dialysis prior to the incubation.

61. The method of claim 1 further comprising detecting the replicated strands.

62. The method of claim 61 wherein detecting the replicated strands comprises detecting the presence, amount, or presence and amount of replicated strands.

63. The method of claim 62 wherein the presence, amount, or presence and amount of the replicated strands is accomplished by detecting the presence, amount, or presence and amount of one or more target sequences.

64. The method of claim 63 wherein the amount of a plurality of alleles, loci, or both are detected.

65. The method of claim 61 wherein detection of replicated strands indicates that the sample contains nucleic acids.

66. The method of claim 1 wherein the replicated strands are analyzed for allele dropout, wherein there is no allele dropout for 500 or more loci, 400 or more loci, 300 or more loci, 200 or more loci, 100 or more loci, 50 or more loci, 40 or more loci, 30 or more loci, 20 or more loci, 15 or more loci, 10 or more loci, 8 or more loci, 6 or more loci, 5 or more loci, 4 or more loci, 3 or more loci, 2 or more loci, or 1 or more loci.

67. The method of claim 1 wherein the replicated strands are analyzed for allele dropout, wherein allele dropout is less than 5% for 500 or more loci, 400 or more loci, 300 or more loci, 200 or more loci, 100 or more loci, 50 or more loci, 40 or more loci, 30 or more loci, 20 or more loci, 15 or more loci, 10 or more loci, 8 or more loci, 6 or more loci, 5 or more loci, 4 or more loci, 3 or more loci, 2 or more loci, or 1 or more loci.

11. The method of claim 11 wherein the amplification bias of the replicated strands is less than 10-fold for at least ten target sequences in the sample.

13. The method of claim 13 wherein the locus representation of the replicated strands is 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.

14. The method of claim 13 wherein the locus representation of the replicated strands is 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.

16. The method of claim 16 wherein the amplification bias of the replicated strands is less than 45-fold, less than 40-fold, less than 35-fold, less than 30-fold, less than 25-fold, less than 20-fold, less than 19-fold, less than 18-fold, less than 17-fold, less than 16-fold, less than 15-fold, less than 14-fold, less than 13-fold, less than 12-fold, less than 11-fold, less than 10-fold, less than 9-fold, less than 8-fold, less than 7-fold, less than 6-fold, less than 5-fold, or less than 4-fold.

17. The method of claim 16 wherein the amplification bias of the replicated strands is less than 50-fold for at least 5 different loci, 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.

19. The method of claim 19 wherein the sample is not subjected to heating above a temperature or for a time that would cause substantial cell lysis in the absence of the alkaline conditions.

21. The method of claim 21 wherein replication of the nucleic acids in the sample results in replication of all or a substantial fraction of the genome.

23. The method of claim 21 wherein the genome comprises at least 50% of the nucleic acids in the amplification mixture.

26. The method of claim 21 wherein the nucleic acids comprise a plurality of genomes, wherein replication of the nucleic acids results in replication of a plurality of the genomes.

22. The method of claim 22 wherein the genome is a eukaryotic genome, a plant genome, an animal genome, a vertebrate genome, a fish genome, a mammalian genome, a human genome, a bacterial genome, a microbial genome, or a viral genome.

24. The method of claim 24 wherein the genome comprises at least 90% of the nucleic acids in the amplification mixture.

25. The method of claim 24 wherein the genome comprises at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the nucleic acids in the amplification mixture.

27. The method of claim 27 wherein at least two of the genomes are genomes of different organisms.

30. The method of claim 27 wherein the genomes comprise at least 50% of the nucleic acids in the amplification mixture.

28. The method of claim 28 wherein at least one genome is a human genome and at least one genome is a bacterial genome, viral genome, microbial genome, or pathogen genome.

29. The method of claim 28 wherein at least one genome is a eukaryotic genome, a plant genome, an animal genome, a vertebrate genome, a fish genome, a mammalian genome, a human genome, a bacterial genome, a microbial genome, or a viral genome, and at least one genome is a eukaryotic genome, a plant genome, an animal genome, a vertebrate genome, a fish genome, a mammalian genome, a human genome, a bacterial genome, a microbial genome, or a viral genome.

31. The method of claim 31 wherein the genomes comprise at least 90% of the nucleic acids in the amplification mixture.

32. The method of claim 31 wherein the genomes comprise at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the nucleic acids in the amplification mixture.

34. The method of claim 34 wherein the sample is a serum sample or a plasma sample.

36. The method of claim 36 wherein the lysis solution comprises a base, a buffer, or a combination.

42. The method of claim 36 wherein the sample is mixed with an equal volume of the lysis solution.

37. The method of claim 37 wherein the lysis solution comprises a base, wherein the base is potassium hydroxide.

38. The method of claim 38 wherein the lysis solution comprises 400 mM KOH.

40. The method of claim 38 wherein the lysis solution comprises 100 mM KOH.

39. The method of claim 39 wherein the lysis solution comprises 400 mM KOH and 10 mM EDTA.

41. The method of claim 41 wherein the lysis solution comprises 100 mM KOH and 2.5 mM EDTA.

68. A method of detecting the presence of nucleic acids in a sample, the method comprising,

exposing the sample to alkaline conditions,

reducing the pH of the sample to form a stabilized sample, and

incubating an amplification mixture under conditions that promote replication of the nucleic acids from the sample, wherein the amplification mixture comprises all or a portion of the stabilized sample,

wherein replication of the nucleic acids results in replicated strands,

detecting the replicated strands, wherein detection of replicated strands indicates that the sample comprises nucleic acids.

69. The method of claim 68 further comprising quantitating the replicated strands.

70. The method of claim 69 wherein the quantity of replicated strands is a measure of the amount of nucleic acids present in the sample.

71. The method of claim 68 wherein the sample comprises nucleic acids from more than two organisms, wherein detecting the replicated strands detects at least one organism.

72. The method of claim 68 wherein the sample comprises nucleic acids from a whole ecosystem, wherein detecting the replicated strands detects at least one organism.

73. The method of claim 68 wherein the sample is a substantially cell-free sample.

74. The method of claim 68 wherein the sample is a serum sample or a plasma sample.

75. The method of claim 68 wherein the sample is a water sample.

76. The method of claim 68 wherein during replication at least one of the replicated strands is displaced from nucleic acids in the sample by strand displacement replication of another replicated strand.

77. The method of claim 68 wherein the nucleic acids are replication using exponential rolling circle amplification (ERCA), and rolling circle amplification (RCA), multiple displacement amplification (MDA), strand displacement amplification (SDA), nucleic acid sequence based amplification (NASBA), transcription-mediated amplification (TMA), polymerase chain reaction (PCR), self-sustained sequence replication (3SR), amplification with Qβ

replicase, and cycle sequencing.

78. A method of detecting the presence of an organism in a sample, the method comprising,

exposing the sample to alkaline conditions,

reducing the pH of the sample to form a stabilized sample,

incubating an amplification mixture under conditions that promote replication of the nucleic acids from the sample, wherein the amplification mixture comprises all or a portion of the stabilized sample, wherein replication of the nucleic acids results in replicated strands, and

identifying one or more types of organism that contain one or more of the sequences of the replicated strands, thereby detecting the presence of the organism in the sample.

79. The method of claim 78 wherein the one or more types of organism that contain one or more of the sequences of the replicated strands are identified by sequencing at least a portion of the replicated strands to obtain one or more sequences of the replicated strands, searching a database of nucleic acid sequences sequentially using one or more of the sequences of the replicated strands as strings, and identifying the results of the search as sequences of organisms likely to be present in the sample and not likely to be present in the sample, thereby detecting the presence of the organism in the sample.

80. The method of claim 79 wherein the sample comprises nucleic acids from a whole ecosystem, wherein at least one organism in the sample is a variant organism, wherein the variant organism comprises a variant sequence from sequence of the same type of organism present in the database, wherein identifying a type of organism that contains one or more of the sequences of the replicated strands identifies at least one organism.

81. The method of claim 79 wherein sequencing at least a portion of the replicated strands is accomplished by hybridization to at least one of a plurality of nucleic acid probes.

82. The method of claim 81 wherein the nucleic acid probes are immobilized on a microarray.

83. The method of claim 78 wherein the sample comprises nucleic acids from two organisms, wherein identifying a type of organism that contains one or more of the sequences of the replicated strands identifies both organisms.

84. The method of claim 78 wherein the sample comprises nucleic acids from two organisms, wherein identifying a type of organism that contains one or more of the sequences of the replicated strands identifies at least one organism.

85. A method of amplifying nucleic acids, the method comprising,

exposing a sample to alkaline conditions,

reducing the pH of all or a portion of the sample to form a stabilized sample, and

incubating an amplification mixture under conditions that promote replication of the nucleic acids from the sample, wherein the amplification mixture comprises all or a portion of the stabilized sample,

wherein replication of the nucleic acids results in replicated strands, wherein the replicated strands have low amplification bias, wherein the concentration of nucleic acids in the amplification mixture favors hybridization of primers over reassociation of the nucleic acids, wherein amount of nucleic acids in the amplification mixture is at or above a threshold that can result in low amplification bias in the replicated strands.

86. The method of claim 85 wherein the nucleic acids are replicated using exponential rolling circle amplification (ERCA), and rolling circle amplification (RCA), multiple displacement amplification (MDA), strand displacement amplification (SDA), nucleic acid sequence based amplification (NASBA), transcription-mediated amplification (TMA), polymerase chain reaction (PCR), self-sustained sequence replication (3SR), amplification with Qβ

replicase, and cycle sequencing.

87. A method of identifying reaction conditions for nucleic acid amplification, the method comprising

amplifying a test sample under test conditions to produce amplified nucleic acids,

measuring amplification bias in the amplified nucleic acids,

wherein if the amplification bias is less than a threshold of interest, then the test conditions are identified as conditions for a nucleic acid amplification.

88. The method of claim 87 wherein the nucleic acids are replicated using exponential rolling circle amplification (ERCA), and rolling circle amplification (RCA), multiple displacement amplification (MDA), strand displacement amplification (SDA), nucleic acid sequence based amplification (NASBA), transcription-mediated amplification (TMA), polymerase chain reaction (PCR), self-sustained sequence replication (3SR), amplification with Qβ

replicase, and cycle sequencing.

89. A method of identifying reaction conditions for nucleic acid amplification, the method comprising

exposing a test sample to alkaline conditions,

reducing the pH of all or a portion of the test sample to form a stabilized test sample, and

incubating a test amplification mixture under conditions to produce amplified nucleic acids, wherein the test amplification mixture comprises all or a portion of the test stabilized sample,

wherein the test conditions promote replication of nucleic acids, wherein the concentration of nucleic acids in the test amplification mixture is a test nucleic acid concentration, wherein the amount of nucleic acids in the test amplification mixture is a test amount of nucleic acids,

measuring amplification bias in the amplified nucleic acids,

wherein if the amplification bias is less than a threshold of interest, then the test concentration of nucleic acids is a concentration to be used for nucleic acid amplification and the test amount of nucleic acids is threshold amount to be used for nucleic acid amplification.

90. A method of amplifying nucleic acids, the method comprising,

exposing a sample that may comprise nucleic acids to alkaline conditions,

reducing the pH of all or a portion of the sample to form a stabilized sample, and

incubating an amplification mixture under conditions that promote replication of the nucleic acids from the sample, wherein the amplification mixture comprises all or a portion of the stabilized sample,

wherein replication of the nucleic acids results in replicated strands, wherein during replication at least one of the replicated strands is displaced from nucleic acids in the sample by strand displacement replication of another replicated strand, wherein the replicated strands have an amplification bias less than a threshold of interest,

wherein the concentration of nucleic acids in the amplification mixture is at or above a predetermined concentration, wherein amount of nucleic acids in the amplification mixture is at or above a threshold amount, wherein the predetermined concentration and the threshold amount are determined by

exposing a test sample to the alkaline conditions,

reducing the pH of all or a portion of the test sample to form a stabilized test sample, and

incubating a test amplification mixture under conditions that promote replication of nucleic acids to produce amplified nucleic acids, wherein the amplification mixture comprises all or a portion of the stabilized sample,

wherein the concentration of nucleic acids in the test amplification mixture is a test nucleic acid concentration, wherein the amount of nucleic acids in the test amplification mixture is a test amount of nucleic acids,

measuring amplification bias in the amplified nucleic acids,

wherein if the amplification bias is less than the threshold of interest, then the test concentration of nucleic acids is the predetermined concentration and the test amount of nucleic acids is the threshold amount.

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

a stabilization solution,

a reaction mix comprising a set of primers, and

a DNA polymerase mix comprising a DNA polymerase.

92. The kit of claim 91 further comprising dithiotheitol,

1×

Phosphase-Buffered Saline, pH 7.5, and

control DNA template,

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

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.