43. High Throughput SNP Discovery and Scoring Using Bead-Based Flow Cytometry 

P. Scott White, Hong Cai, and John P. Nolan 
Los Alamos National Laboratory, Los Alamos, New Mexico 
swhite@telomere.lanl.gov 

There is a pressing need for SNP discovery and analysis capabilities that are rapid and robust. We are developing approaches using microsphere-based flow cytometry to address these needs. 

For SNP discovery, we have developed a system that uses immobilized mismatch-binding proteins (IMBP) to detect SNPs in heteroduplexes. IMBP-coated microspheres are added to fluorescently labeled PCR amplicons, and analyzed by flow cytometry. The detection of fluorescence associated with the beads indicates the amplified region contains one or more SNPs, which are then sequenced. 

Bead-based minisequencing or oligo ligation using flow cytometry is used to score SNPs. A novel system for multiplexed analysis enables simultaneous scoring of 64 or many more different SNPs/sample. Furthermore, because of the quantitative nature of flow cytometry, pooling amplicons from large numbers of individuals will allow for the determination of the frequencies of each SNP in populations. 

These microsphere-based flow cytometric analyses have the following general advantages: 1) Intrinsic resolution between free and microsphere-bound probe, allowing homogeneous assays with no wash steps; 2) Quantitative, multicolor fluorescence detection with sensitivity surpassing microplate or microscope-based detection; 3) Soluble solid phase that can be prepared, pipetted, and handled by conventional fluidics systems; and 4) An instrument that is already available in core facilities at the vast majority of research universities, medical schools, pharmaceutical companies, and clinical diagnostic laboratories. Furthermore, the potential for multiplexing these assays will greatly enhance throughput and allow for the scanning of over one megabase/day for new SNPs, or for scoring thousands of individuals for hundreds to thousands of known SNPs/day.