Methods and compositions for multiplex amplification of nucleic acids
AssigneeAffymetrix, Inc.
InventorsHajime Matsuzaki, Eric A. Murphy
Life of this patent
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Abstract
A method is described for predetermining ratios of primer pairs present in a single reaction vessel so as to achieve approximately equimolar yield of products. The ratios are determined as a function of the length of the amplicon and the length of other amplicons being simultaneously tested. The primers may desirably be for p53 gene sequences.
Claims24
A method of performing multiple polymerase chain reactions in a single vessel, comprising:
- priming DNA synthesis on a template in a vessel with at least two sets of primers, wherein the primers are present in the vessel at a predetermined ratio, wherein the ratio is described by:
- CA=CL(LA÷LL)2
- wherein CA is the concentration of primers for an amplicon A;
- wherein CL is the concentration of primer for the longest amplicon;
- wherein LA is the length of the amplicon A;
- and wherein LL is the length of the longest amplicon:
A method of performing multiple polymerase chain reactions in a single vessel, comprising:
- priming DNA synthesis on a genomic p53 template in a vessel with ten sets of primers which amplify exons 2-11 of p53, wherein the primers are shown in SEQ ID NOS:
- 1-20, wherein the primers are present in the vessel at the following ratios:
- exon 2 (89.4), exon 3 (26.9), exon 4 (450), exon 5 (245.8), exon 6 (138.3), exon 7 (101.8), exon 8 (193.0), exon 9 (70.8), exon 10 (146.5), exon 11 (177.3).
A kit comprising a set of primers for performing multiple polymerase chain reactions in a single vessel, comprising:
- twenty primers having sequences as shown in SEQ ID NO:
- ID NOS:
- 1-20.
A mixture of primers for performing multiplex polymerase chain reaction, wherein the primers are present in the mixture at a predetermined ratio to each other, wherein the ratio of the concentrations of the primers is described by:
- CA=CL(LA÷LL)2
- wherein CA is the concentration of primers for an amplicon A;
- wherein CL is the concentration of primer for the longest amplicon;
- wherein LA is the length of the amplicon A;
- and wherein LL is the length of the longest amplicon.
Dependencies are inferred from the claim text.