Journal
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
Volume 375, Issue 5, Pages 333-343Publisher
SPRINGER HEIDELBERG
DOI: 10.1007/s00216-002-1683-2
Keywords
multiplex PCR design; Y chromosome; short tandem repeats; primer design
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The simultaneous amplification of multiple regions of a DNA template is routinely performed using the polymerase chain reaction (PCR) in a process termed multiplex PCR. A useful strategy involving the design, testing, and optimization of multiplex PCR primer mixtures will be presented. Other multiplex design protocols have focused on the testing and optimization of primers, or the use of chimeric primers. The design of primers, through the close examination of predicted DNA oligomer melting temperatures (T(m)) and primer-dimer interactions, can reduce the amount of testing and optimization required to obtain a well-balanced set of amplicons. The testing and optimization of the multiplex PCR primer mixture constructed here revolves around varying the primer concentrations rather than testing multiple primer combinations. By solely adjusting primer concentrations, a wellbalanced set of amplicons should result if the primers were designed properly. As a model system to illustrate this multiplex design protocol, a 10-loci multiplex (10-plex) Y chromosome short tandem repeat (STR) assay is used.
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