General Strategy for the Design of Multiplex PCR Assays Involving Y STR Markers
R Schoske, Peter Vallone, C M. Ruitberg, John Butler
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. It can be shown how the design of primers, through the close examination of predicted DNA oligomer melting temperatures (Tm) 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 test this multiplex design protocol, a Y chromosome short tandem repeat (STR) assay is used. The Y chromosome has been recognized as a useful tool in forensic investigations and human identification. However due to the fact that no recombination occurs along the vast majority of the chromosome, the product rule cannot be used to accurately determine the power of discrimination. Thus, the most effective way to obtain higher levels of discrimination between unrelated samples is to employ Y STR multiplex assays. As the literature contains references to Y STR multiplexes that at most only amplify five or six loci simultaneously, an effort has been made to increase the level of multiplexing for Y STRs. In this work a Y STR 10 plex is presented. The 10 plex includes the following markers, DYS19, DYS435, DYS436, DYS437, DYS438, DYS439, DYS391, DYS392, A7.1, and H4. A multiplex PCR primer mixture capable of simultaneously amplify more than 17 different Y STR markers has also been developed. With rapid multiplex PCR primer mixture design and streamlined testing and optimization techniques, Y chromosome haplotypes may be more quickly generated to study the desired loci of interest.
multiplex PCR, multiplex PCR design, STRs, Y chromosome short tandem repeats
, Vallone, P.
, Ruitberg, C.
and Butler, J.
General Strategy for the Design of Multiplex PCR Assays Involving Y STR Markers, Genome Research
(Accessed February 24, 2024)