In order to increase significantly the discriminatory potential of Y-STR systems available to the forensic community, we have developed and validated a 21-locus Y-STR multiplex system. Since the system was designed specifically to augment the European Y chromosome typing community's “minimal haplotype” Y-STR set (MHL) for forensic casework, it contains a novel constellation of markers not contained therein. The system, which we refer to as Multiplex IV (MPIV), permits the co-amplification of DYS 443, DYS 444, DYS 445, DYS 447, DYS 448, DYS 449, DYS 452, DYS 453, DYS 454, DYS 455, DYS 456, DYS 458, DYS 463, DYS 464, DYS 468, DYS 484, DYS 522, DYS 527, DYS 531, DYS 557, and DYS 588. Although the multiplex contains 21 Y-STR loci, of which one is bi-local and one is tetra-local, there are actually 25 sites exhibiting allelic variation, and this has prompted us to use the descriptor “megaplex” to describe the system.
This report describes a number of performance checks that were employed to characterize the system including sensitivity, specificity, discriminatory capacity, and nonprobative casework studies. Although 1 ng of male DNA was found to be the optimal amount of input template, a complete 21-locus profile was obtained with as little as 50 pg of male DNA (i.e., ∼8 to 9 diploid cells). The specificity of the system was demonstrated by the lack of significant female DNA derived artifacts when tested using either 300 ng of female DNA alone or an admixture of male/female DNA in which the female component was present in a 100-fold excess. The ability of the system to determine the number of male donors was demonstrated by testing different admixtures of DNA at different ratios from two male donors. Cervicovaginal samples taken up to 48 h post coitus yielded a complete 21-locus Y-STR profile of the semen donor, thus confirming the potential utility of the system for forensic casework. Preliminary estimates of the gene diversity (h) of the individual loci for the Caucasian and African-American population indicated that 15 of the 21 loci possessed an h of ⩾0.5 in at least one population. Multi-locus haplotype analysis revealed that the 21-plex system could augment the use of the minimal haplotype loci and increase significantly the discriminatory capacity of Y-STR analysis.