Multiplex PCR: critical parameters and step-by-step protocol.

By simultaneously amplifying more than one locus in the same reaction, multiplex PCR is becoming a rapid and convenient screening assay in both the clinical and the research laboratory. While numerous papers and manuals discuss in detail conditions influencing the quality of PCR in general, relatively little has been published about the important experimental factors and the common difficulties frequently encountered with multiplex PCR. We have examined various conditions of the multiplex PCR, using a large number of primer pairs. Especially important for a successful multiplex PCR assay are the relative concentrations of the primers at the various loci, the concentration of the PCR buffer, the cycling temperatures and the balance between the magnesium chloride and deoxynucleotide concentrations. Based on our experience, we propose a protocol for developing a multiplex PCR assay and suggest ways to overcome commonly encountered problems.