Methods and compositions for multiplex amplification of nucleic acids
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.
1 . 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 each of the sets of primers in the vessel is at a predetermined ratio, wherein the ratio is described by:
C A =C L ( L A ÷L L ) 2
wherein C A is the concentration of primers for an amplicon A; wherein C L is the concentration of primer for the longest amplicon; wherein L A is the length of the amplicon A; and wherein L L is the length of the longest amplicon.
2 . The method of claim 1 wherein the template is genomic DNA encoding p53.
3 . The method of claim 1 wherein the template is a cDNA encoding p53.
4 . The method of claim 1 wherein the primers amplify at least 2 exons of p53 selected from the group consisting of exons 2-11.
5 . The method of claim 1 wherein the primers amplify at least 4 exons of p53 selected from the group consisting of exons 2-11.
6 . The method of claim 1 wherein the primers amplify exons 2-11 of p53.
7 . The method of claim 4 wherein the primers are selected from those shown in SEQ ID NO: 1-20.
8 . The method of claim 5 wherein the primers are selected from those shown in SEQ ID NO: 1-20.
9 . The method of claim 6 wherein the primers are shown in SEQ ID NOS: 1-20.
10 . The method of claim 9 wherein the primers are present in 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).
11 - 24 . (canceled)