Fluorescent microsphere-based readout technology for multiplexed human single nucleotide polymorphism analysis and bacterial identification

Large scale human genotyping requires technologies with a minimal number of steps, high accuracy, and the ability to automate at a reasonable cost Zn this regard, we have developed a rapid, cost-effective readout method for s ingle nucleotide polymorphism (SNP) genotyping that combines an easily auto matable single tube allele specific primer extension (ASPE) with an efficie nt high throughput flow cytometric analysis performed on a Luminex 100(TM) flow cytometer, This robust technique employs an ASPE reaction using PCR-de rived target DNA containing the SNP and a Fair of synthetic complementary c apture probes that differ at their 3' end-nucleotide defining the alleles, Each capture probe has been synthesized to contain a unique 25-nucleotide i dentifying sequence (ZipCode) at its 5' end. An array of fluorescent micros pheres, covalently coupled with complementary ZipCode sequences (cZipCodes) , was hybridized to biotin-labeled ASPE reaction products, sequestering the m for flow cytometric analysis, ASPE offers both an advantage of streamlini ng the SNP analysis protocol and an ability to perform multiplex SNP analys is on any mixture of allelic variants, All steps of the assay are simple ad ditions of the solutions, incubations, and washes. This technique was used to assay 15 multiplexed SNPs on human chromosome 12 from 96 patients. Compa rison of the microsphere-based ASPE assay results to gel-based oligonucleot ide ligation assay (OLA) results showed 99.2% agreement in genotype assignm ents. In addition, the microsphere-based multiplex SNPs assay system was ad apted for the identification of bacterial samples by both ASPE and single b ase chain extension (SBCE) assays. A series of probes designed for differen t variable sites of bacterial 16S rDNA permitted multiplex analysis acid ge nerated species or genus-specific patterns, Seventeen bacterial species rep resenting a broad range of gram-negative and gram positive bacteria were an alyzed within 16 variable sites of 16S rDNA sequence, The results were cons istent with the published sequences and confirmed by direct DNA sequencing, Hum Mutat 17:305-316, 2001, (C) 2001 Wiley-Liss, Inc.