Multiplex PCR: Critical Parameters and Step-by-Step Protocol

Multiplex PCR: Critical Parameters and Step-by-Step Protocol
BioTechniques 23:504-511 (September 1997)

O. Henegariu, N.A. Heerema, S.R. Dlouhy, G.H. Vance and
P.H. Vogt1
Indiana University, Indianapolis, IN, USA and 1Heidelberg
University, Heidelberg, Germany

ABSTRACT
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.

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