Jingwen Chen¹, Fei Ye¹, Philip Rivers¹, Holly King³, Quan Nguyen², David Briley², May-Sung Li¹, J. David Taylor¹, Michael Wagner², Michael P. Weiner¹, Yiwu He³. ¹Dept. of Genomic Sciences, ²Dept. of Molecular Sciences, ³Dept. of Research Technology, Glaxo Wellcome Inc., 5 Moore Drive, Research Triangle Park, NC 27709.

A rapid, high throughput read-out technology for single nucleotide polymorphism (SNP) analysis was developed employing both flow cytometric analysis of fluorescent microspheres and enzyme-based allele detection. An array of fluorescent microspheres (each population of microspheres is identified by its unique profile of red and orange fluorescence) was coupled to unique DNA oligonucleotide sequences termed complementary ZipCodes (cZipCodes). Allele detection was based on either single-base chain extension assay (SBCE) where a synthetic ‘capture’ oligonucleotide probe was designed to contain both a ZipCode sequence at the 5’ end and an SNP-specific sequence at the 3’ end. A DNA polymerase adds a fluorescently labeled nucleotide to the capture oligonucleotide which was then hybridized to its cZipCode on the microsphere. Flow cytometric analysis of the microspheres simultaneously identified both the microsphere type and the fluorescent signal associated with the SNP genotype. The multiplexed analysis of SNPs was made possible by the fluorescent microspheres and the use of ZipCode sequence coupled to microspheres allowed multiple sets of SNPs to be analyzed by a limited set of cZipCode attached microspheres. We will describe several improvements to this semi-automated system that allows for an efficient and cost-effective high-throughput analysis