Fragment size difference between multiplex and singleplex PCR products and their practical implications

By simultaneously amplifying several loci in the same reaction, multiplex PCR has been used in gene mapping and DNA typing with polymorphic short tandem repeat loci. Previous studies have discussed in detail the various parameters and conditions that influence the quality of individual products generated by multiplex PCR. In practice, when a primer pair fails to amplify in a multiplex PCR for some individuals, singleplex PCR is often employed as a supplement to amplify the primer pair. However, the reliability of this procedure is unknown. In this study, we used six primer pairs from ABI PRISM(TM) Linkage Mapping Set version 2 to perform mutiplex and singleplex reactions. The flourescence-labeled amplification products were separated and detected on ABI PRISM 310 Genetic Analyzer. We found that for the marker D1S468, multiplex and singleplex reactions of different sizes. Therfore, the potential size difference between multiplex and singleplex reactions needs to be investigated. This investigation is essential to employ multiplex PCR supplemented with singplex PCR in gene mapping and DNA typing.