A microsphere-based assay for multiplexed single nucleotide polymorphism analysis using single base chain extension

A rapid, high throughput readout for single-nucleotide polymorphism (SNP) a nalysis was developed employing single base chain extension and cytometric analysis of an array of fluorescent microspheres. An array of fluorescent m icrospheres was coupled with uniquely identifying sequences, termed complem entary ZipCodes (cZipCodes), which allowed for multiplexing possibilities. For a given assay, querying a polymorphic base involved extending an oligon ucleotide containing both a ZipCode and a SNP-specific sequence with a DNA polymerase and a pair of fluoresceinated dideoxynucleotides. To capture the reaction products For analysis, the ZipCode portion of the oligonucleotide was hybridized with its cZipCodes on the microsphere. Flow cytometry was u sed for microsphere decoding and SNP typing by detecting the fluorescein la bel captured on the microspheres. In addition to multiplexing capability, t he ZipCode system allows multiple sets of SNPs to be analyzed by a limited set of cZipCode-attached microspheres. A standard set of non-cross reactive ZipCodes was established experimentally and the accuracy of the system was validated by comparison with genotypes determined by other technologies. F rom a total of 58 SNPs, 55 SNPs were successfully analyzed in the first pas s using this assay format and all 181 genotypes across the 55 SNPs were cor rect. These data demonstrate that the microsphere-based single base chain e xtension (SBCE) method is a sensitive and reliable assay. It can be readily adapted to an automated, high-throughput genotyping system.