Flow cytometry-based minisequencing: A new platform for high-throughput single-nucleotide polymorphism scoring

Single-nucleotide polymorphisms (SNPs) are the most abundant type of human genetic variation. These variable sites are present at high density in the genome, making them powerful tools for mapping and diagnosing disease-relat ed alleles. We have developed a sensitive and rapid how cytometry-based ass ay for the multiplexed analysis of SNPs based on polymerase-mediated primer extension, or minisequencing, using microspheres as solid supports. The ne w method involves subnanomolar concentrations of sample in small volumes (s imilar to 10 mu l) which can be analyzed at rates of one sample per minute or faster, without a wash step. Further, genomic analysis using multiplexin g microsphere arrays (GAMMArrays), enables the simultaneous analysis of doz ens, and potentially hundreds of SNPs per sample. We have tested the new me thod by genotyping the Glu69 variant from the HLA DPB1 locus, a SNP associa ted with chronic beryllium disease, as well as HLA DPA1 alleles using the m ultiplexed method. The results demonstrate the sensitivity and accuracy of flow cytometry-based minisequencing, a powerful new tool for genome- and gl obal-scale SNP analysis.