A simplified procedure for developing multiplex PCRs.

We have developed a simplified method for multiplex PCR based on the use 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, cycling times, and annealing temperatures have been established for any PCR primer pair comprising the chimeric motif. Under these conditions, efficient multiplex amplification is achieved easily and reproducibly by simple adjustment of the individual primer concentrations. No additional modification of either the reaction components or annealing temperatures is required. The use of tagged primers provides a method for primer design that eliminates the multiple optimization steps involved in developing multiplex PCR.