Method for nucleic acid amplification that results in low amplification bias
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, andincubating 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 the conditions that promote replication of the nucleic acids are carried out in the absence of thermal cycling,wherein the concentration of the nucleic acids in the amplification mixture favors hybridization of primers over reassociation of the nucleic acids,wherein target nucleic acids in the sample are preferentially replicated over non-target nucleic acids in the 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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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
70 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 the conditions that promote replication of the nucleic acids are carried out in the absence of thermal cycling, wherein the concentration of the nucleic acids in the amplification mixture favors hybridization of primers over reassociation of the nucleic acids, wherein target nucleic acids in the sample are preferentially replicated over non-target nucleic acids in the 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.
Specification