Nucleic acid amplification
First Claim
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.
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Accused Products
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.
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Citations
92 Claims
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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.
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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.
- 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.
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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.
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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.
- 27. The method of claim 27 wherein at least two of the genomes are genomes of different organisms.
- 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.
- 31. The method of claim 31 wherein the genomes comprise at least 90% of the nucleic acids in the amplification mixture.
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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.
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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.
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39. The method of claim 39 wherein the lysis solution comprises 400 mM KOH and 10 mM EDTA.
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41. The method of claim 41 wherein the lysis solution comprises 100 mM KOH and 2.5 mM EDTA.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
Specification