Multiplexed single nucleotide polymorphism genotyping by oligonucleotide ligation and flow cytometry

Abstract

Background: We have developed a rapid, high throughput method for single nucleotide polymorphism (SNP) genotyping that employs an oligonucleotide ligation assay (OLA) and flow cytometric analysis of fluorescent microspheres. Methods: A fluoresceinated oligonucleotide reporter sequence is added to a 'capture' probe by OLA. Capture probes are designed to hybridize both to genomic 'targets' amplified by polymerase chain reaction and to a separate complementary DNA sequence that has been coupled to a microsphere. These sequences on the capture probes are called 'ZipCodes'. The OLA-modified capture probes are hybridized to ZipCode complement-coupled microspheres. The use of microspheres with different ratios of red and orange fluorescence makes a multiplexed format possible where many SNPs may be analyzed in a single tube. Flow cytometric analysis of the microspheres simultaneously identifies both the micro- sphere type and the fluorescent green signal associated with the SNP genotype. Results: Application of this methodology is demonstrated by the multiplexed genotyping of seven CEPH DNA samples for nine SNP markers located near the ApoE locus on chromosome 19. The microsphere- based SNP analysis agreed with genotyping by sequencing in all cases. Conclusions: Multiplexed SNP genotyping by OLA with flow cytometric analysis of fluorescent microspheres is an accurate and rapid method for the analysis of SNPs. (C) 2000 Wiley-Liss, Inc.