DOE Human Genome Program Contractor-Grantee Workshop VIII
February 27-March 2, 2000  Santa Fe, NM

32. High Throughput Multiplexed mtDNA SNP Scoring Using Microsphere-Based Flow Cytometry

P. Scott White, Alina Deshpande, Lance Green, Yolanda Valdez, David C. Torney, and John P. Nolan

Bioscience Division and DOE Joint Genome Institute, Los Alamos National Laboratory, Los Alamos, NM 87545

white_paul_scott@lanl.gov

We have developed a flow cytometry-based minisequencing platform capable of extremely high-throughput, low cost assays. These are no-wash assays analyzed at less than 1 minute/sample, with superior sensitivities. Furthermore, with commercially available multiplexed microspheres we can score dozens to hundreds of SNPs simultaneously. Multiplexing, coupled with high throughput rates, makes it possible to score several million SNPs/day at costs that are a fraction of competing technologies. In addition, these assays can easily be integrated into conventional liquid handling automation systems, and require no unique instrumentation for setup and analysis.

Multiplexing is enhanced by universal capture tags consisting of carefully designed, unique DNA tails incorporated into each minisequencing primer. These are complementary to address tags attached to discrete populations of microspheres in multiplexed sets. This enables simultaneous minisequencing of many SNPs in solution, followed by capture onto the appropriate microspheres for multiplexed analysis by flow cytometry. High signal-to-noise ratios, ease of setup, flexibility in format and scale, and low cost of these assays make them versatile and valuable tools for studies at a wide range of scales where SNP scoring is needed.

A typing method that could rapidly and inexpensively score 100 or more SNPs located in the mitochondrial genome would be extremely valuable in population and evolutionary biological studies, as well as a powerful forensics tool. We present results from multiplexed analyses of mtDNA and HLA SNPs, performed on a few large PCR amplicons, each containing numerous SNPs that have been scored simultaneously.