Development of Novel Y-CSTR and Y-SNP Markers for Operational Use

Presently few public laboratories in the US perform Y-STR analysis in casework and those that do so employ multiplex systems/kits with somewhat limited discrimination potential. Although other Y-STR multiplex systems have been described, there is still a need to develop novel Y-STR markers to produce better discrimination ability. We have completed construction of a third multiplex system, MPIII, which comprises nine additional Y chromosome markers and the indel marker DYS287 (YAP) consisting of the presence or absence of an Alu element insertion. Construction and validation of a fourth multiplex, MPIV, is completed and comprises 21 additional Y-STR loci. These 'third generation' Y-STR systems are promising in that they demonstrate no female DNA cross reactivity under stringent conditions. Performance checks with MPIII and MPIV confirm their usefulness in that they can distinguish between several unrelated individuals who otherwise match with the nineteen MPI/MPII loci.

In a separate but related approach, we are in the process of evaluating Y chromosome single nucleotide polymorphisms (SNPs) as a forensic tool. Y-SNPs may provide information such as ethnic and geographical points of origin to investigators. The advantages of Y-SNP analysis include the ability to perform automated massively parallel analysis and the development of point-of-use instrumentation. A novel semi-automated rapid sequencing methodology, pyrosequencing, is used to analyze a number of Y-SNP loci that we have identified as suitable candidates for forensic use. We have identified, characterized and typed a panel of 16 Y-SNP markers (obtained from the published literature and from the dbSNP database) and are in the process of developing assays for an additional 10 loci. Significantly, we have confirmed the potential utility of pyrosequencing technology to quickly and efficiently type Y-SNP markers using monoplex reactions. After initial set up, pyrosequencing permits the reliable typing of 96 Y-SNPs in ten minutes. Initial studies on the haplotype diversity of 11 of our bi-allelic Y-SNP loci revealed the presence of six haplotypes in 32 individuals (14 Caucasians, 18 African Americans). Interestingly, significant ethnic stratification was observed at each of the 11 sites with the most common allele in one population invariably being the least common in the other population. The biggest challenge with pyrosequencing is to develop multiplex capabilities, which is not a requirement by other more traditional end-users of this technology. To this end, we have designed a number of duplex systems and have had success with a number of these.

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Jack Ballantyne
Department of Chemistry, CH 117
University of Central Florida
Orlando, FL 32816
Telephone: 407-823-0163
FAX: 407-823-2252
E-Mail: jballant@mail.ucf.edu