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US 12,680,123 B2Granted
Polymerase chain reaction normalization through primer titration
AssigneeBecton, Dickinson and Company
InventorsKatherine Lazaruk, Eleen Shum, Christina Chang
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Abstract
Disclosed herein include systems, methods, compositions, and kits for PCR normalization. In some embodiments, after barcoding copies of a higher abundance target (e.g., a cDNA species), the barcoded copies are amplified using a pair of forward primers comprising one or more mismatches and a reverse primer. The amplified copies can be further linearly amplified using a forward primer comprising the sequence of one of the pair of forward primers, and a reverse primer.
Claims30
A method for polymerase chain reaction (PCR), comprising:
- amplifying copies of a target nucleic acid in a first amplification reaction to generate amplified nucleic acid molecules using:
- a first forward primer and a first reverse primer to generate a first plurality of amplified nucleic acid molecules, wherein the first forward primer anneals to the target nucleic acid;
- and
- a second forward primer and the first reverse primer to generate a second plurality of amplified nucleic acid molecules, wherein the second forward primer anneals to the target nucleic acid, wherein the first forward primer and the second forward primer anneal to the same sequence on the target nucleic acid,
- wherein the sequence of the first forward primer incorporated into the amplified nucleic acid molecules and the sequence of the second forward primer incorporated into the amplified nucleic acid molecules are different,
- wherein the first forward primer and the second forward primer each comprise a nucleic acid-hybridization region and an overhang region 5′ of the nucleic acid-hybridization region, and
- wherein the sequence of the nucleic acid-hybridization region of the first forward primer is identical to the sequence of the nucleic acid-hybridization region of the second forward primer, and wherein the sequence of the overhang region of the first forward primer is different from the sequence of the overhang region of the second forward primer;
- and
- subjecting the first and second pluralities of amplified nucleic acid molecules to a second amplification reaction using a third forward primer and a second reverse primer to generate a third plurality of amplified nucleic acid molecules,
- wherein amplification of the second plurality of amplified nucleic acid molecules is reduced compared to amplification of the first plurality of amplified nucleic acid molecules in the second amplification reaction due to the difference in the sequence of the first forward primer incorporated into the first plurality of amplified nucleic acid molecules and the sequence of the second forward primer incorporated into the second plurality of amplified nucleic acid molecules.
7 more elements
A method for polymerase chain reaction (PCR), comprising:
- amplifying copies of a target nucleic acid in a first amplification reaction to generate amplified nucleic acid molecules using:
- a first forward primer and a first reverse primer to generate a first plurality of amplified nucleic acid molecules, wherein the first forward primer anneals to the target nucleic acid;
- and
- a second forward primer and the first reverse primer to generate a second plurality of amplified nucleic acid molecules, wherein the second forward primer anneals to the target nucleic acid, wherein the first forward primer and the second forward primer anneal to the same sequence on the target nucleic acid,
- wherein the sequence of the first forward primer incorporated into the amplified nucleic acid molecules and the sequence of the second forward primer incorporated into the amplified nucleic acid molecules are different;
- and
- subjecting the first and second pluralities of amplified nucleic acid molecules to a second amplification reaction using a third forward primer and a second reverse primer to generate a third plurality of amplified nucleic acid molecules, wherein the third forward primer comprises a nucleic acid-hybridization region and an overhang region 5′ of the nucleic acid-hybridization region, and
- wherein amplification of the second plurality of amplified nucleic acid molecules is reduced compared to amplification of the first plurality of amplified nucleic acid molecules in the second amplification reaction due to the difference in the sequence of the first forward primer incorporated into the first plurality of amplified nucleic acid molecules and the sequence of the second forward primer incorporated into the second plurality of amplified nucleic acid molecules.
5 more elements
Dependencies are inferred from the claim text.