Methods for normalized amplification of nucleic acids
AssigneeAffymetrix, Inc.
InventorsFrederick Christians, Sean Walsh, Rui Mei
Life of this patent
Estimating the term…
Abstract
Methods of preparing normalized mixtures from a plurality of nucleic acid samples are disclosed. Nucleic acids are amplified so that similar amounts of a target nucleic acid are generated in a plurality of different reactions. Separate amplification reactions are performed to amplify the same or different targets in a plurality of different reactions. The amounts of amplified product are approximately normalized during the amplification without the need to empirically measure the amount of amplified target.
Claims22
A method of amplifying a target sequence from a complex nucleic acid sample comprising:
- obtaining a first amplification product that is enriched for the target sequence by incubating the complex nucleic acid sample in a first amplification reaction, wherein said first amplification reaction comprises polymerase chain reaction with a pair of primers that are specific for said target;
- and,
- obtaining a second amplification product by amplifying an aliquot of the first amplification product with a strand displacing DNA polymerase, in the presence of dNTPs and at least one primer in a second amplification reaction.
A method for obtaining a pooled sample comprising approximately equimolar amounts of a first amplified target sequence and a second amplified target sequence comprising
- Amplifying said first target sequence in a first amplification reaction to generate a first amplification product wherein the first target is amplified by PCR with a pair of primers that are specific for said first target sequence;
- amplifying said second target sequence in a second reaction to generate a second amplification product wherein the second target sequence is amplified by PCR with a pair of primers that are specific for said second target sequence;
- amplifying an aliquot of said first amplification product in a third amplification reaction, to generate a third amplification product, wherein the third amplification reaction comprises a mixture of random primers, a strand displacing DNA polymerase, and a first amount of dNTPs;
- amplifying an aliquot of said second amplification product in a fourth amplification reaction, to generate a fourth amplification product, wherein the fourth amplification reaction comprises a mixture of random primers, a strand displacing DNA polymerase, and a second amount of dNTPs;
- and
- mixing a volume of the third amplification product with a volume of the fourth amplification product to generate a mixture of amplified first and second target sequences wherein the first and second target amplicons are present in approximately equal molar amounts in the mixture.
Dependencies are inferred from the claim text.