Methods and compositions for multiplex amplification of nucleic acids

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.

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)