Multiplex PCR design strategy used for the simultaneous amplification of 10 Y chromosome short tandem repeat (STR) loci.
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 well-balanced 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 (10plex) Y chromosome short tandem repeat (STR) assay is used.
Schoske
Richard
R
Department of Chemistry, American University, 4400 Massachusetts Ave. NW, Washington DC 20016, USA. richard.schoske@nist.gov
Vallone
Pete M
PM
Ruitberg
Christian M
CM
Butler
John M
JM
D001483:Base Sequence; D041322:Chromosomes, Human, Y / Q000235:genetics*; D017931:DNA Primers; D030561:Databases, Nucleic Acid; D005260:Female; D005456:Fluorescent Dyes; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D016415:Sequence Alignment; D012984:Software; D020080:Tandem Repeat Sequences / Q000235:genetics*
D017931:DNA Primers; D005456:Fluorescent Dyes