A SIMPLIFIED PROCEDURE FOR DEVELOPING MULTIPLEX PCRS

We have developed a simplified method for multiplex PCR based on the u se of chimeric primers. Each primer contains a 3' region complementary to sequence-specific recognition sites and a 5' region made up of an unrelated 20-nucleotide sequence. Identical reaction conditions, cycli ng times, and annealing temperatures have been established for any PCR primer pair comprising the chimeric motif. Under these conditions, ef ficient multiplex amplification is achieved easily and reproducibly by simple adjustment of the individual primer concentrations. No additio nal modification of either the reaction components or annealing temper atures Is required. The use of tagged primers provides a method for pr imer design that eliminates the multiple optimization steps involved i n developing multiplex PCR.