Development of Y STR Megaplex Assays

Y chromosome short tandem repeat markers have a number of applications in human identity testing including typing the perpetrator of sexual assault cases without differential extraction and tracing paternal lineages for missing person investigations. In order for Y STR systems to become more widely accepted within the forensic DNA typing community, population studies and robust assays are required. We have focused on developing new Y STR multiplexes that can provide a high degree of discrimination between unrelated males. The Y STR markers we are working with include the hexanucleotide repeat DYS448, pentanucleotides DYS438, DYS446, DYS447, DYS450, tetranucleotide repeat DYS19, DYS385 I/II, DYS389I/II, DYS390, DYS391, DYS393, DYS437, DYS439, DYS441, DYS442, GATA A7.1, GATA H4, G09411, trinucleotides DYS388, DYS392, DYS426, and the dinucleotide repeat YCAII. Primers for the markers DYS385, DYS389, and YCAII target duplicated regions of the Y chromosome and thus can produce two polymorphic peaks with each primer set. The multiplexes we are developing are the first to include all of the European “extended haplotype” (11 PCR products) in a single reaction. We have compared our results to the commercially available Y-Plex 6 kit from Reliagene that amplifies 6 Y STRs. In order to improve the power of discrimination for Y chromosome tests, we have developed strategies for rapidly preparing multiplex PCR assays that utilize both four and five dye chemistries for detection and permit simultaneous amplification of 20 or more Y STR markers in a single reaction. Primer design issues will be reviewed, as will our efforts to avoid any homology with X chromosome sequences. Prime rs have been redesigned from previously published work with these Y STR markers in order to make them more compatible in a multiplex amplification. Results will be shown using our new Y STR multiplex from 3 different laboratories. In addition, allele ranges for each of the Y STR markers have been well characterized in a diverse set of world samples.