This report describes how to construct a large short tandem repeat (STR) multiplex so that others can understand the technology as well as how to resolve difficulties encountered throughout the process.
Researchers developed a single amplification 5-dye multiplex that contains 25 unlinked autosomal loci plus the sex-typing locus AMEL. The primary purpose of developing this multiplex is for use with reference samples in a variety of situations, such as paternity testing/kinship analysis, immigration testing, and for use in missing persons/mass disaster cases. Primers for a majority of the 25 autosomal STR loci were redesigned from the miniSTR loci in previous reports; they have already been determined to be unlinked from the 13 CODIS (Combined DNA Index System), as well as to be diverse with moderate-to-high heterozygosity values. They were comparable with the CODIS loci regarding heterozygosities with slightly lower mutation rates. This suggests that this STR multiplex will be a useful complement to the currently available commercial STR typing kits for the forensic analysis of DNA samples. Multiple strategies were used to overcome various challenges that arose while building this multiplex, such as incomplete adenylation, nonspecific products, artifacts, and simple primer incompatibility. To date, this multiplex represents the most STR loci typed together in a single PCR amplification assay. The development of the design process and optimization of the 26plex led to the identification of useful strategies for developing future large multiplexes. The descriptions of materials and methods address the multiplex design, multiplex design software, PCR amplification, analysis on the ABI 3130xl Genetic Analyzer, and sensitivity study. 2 tables, 5 figures, and 24 references