Flow cytometric platform for high-throughput single nucleotide polymorphism analysis

We have developed a rapid, cost-effective, high-throughput readout for sing le nucleotide polymorphism (SNP) genotyping using flow cytometric analysis performed on a Luminex(TM) 100 flow cytometer. This robust technique employ s a PCR-derived target DNA containing the SNP, a synthetic SNP-complementar y ZipCode-bearing capture probe, a fluorescent reporter molecule, and a the rmophilic DNA polymerase. An array of fluorescent microspheres, covalently coupled with complementary ZipCode sequences (cZipCodes), was hybridized to the reaction products and sequestered them for flow cytometric analysis. T he single base chain extension (SBCE) reaction was used to assay 20 multipl exed SNPs for 633 patients in 96-well format. Comparison of the microsphere -based SBCE assay results to gel-based oligonucleotide ligation assay (OLA) results showed 99.3% agreement in genotype assignments. Substitution of di rect-labeled R6G dideoxynucleotide with indirect-labeled phycoerythrin dide oxynucleotide enhanced signal five- to tenfold while maintaining low noise levels. A new assay based on allele-specific primer extension (ASPE) was va lidated on a set of 15 multiplexed SNPs for 96 patients. ASPE offers both t he advantage of streamlining the SNP analysis protocol and the ability to p erform multiplex SNP analysis on any mixture of allelic variants.