Multiplexed single nucleotide polymorphism genotyping by oligonucleotide ligation and flow cytometry

Background: We have developed a rapid, high throughput method for single nu cleotide polymorphism (SNP) genotyping that employs an oligonucleotide liga tion 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 ge nomic "targets" amplified by polymerase chain reaction and to a separate co mplementary DNA sequence that has been coupled to a microsphere. These sequ ences 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 t ube. Flow cytometric analysis of the microspheres simultaneously identifies both the microsphere 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 wit h genotyping by sequencing in all cases. Conclusions: Multiplexed SNP genotyping by OW with flow cytometric analysis of fluorescent microspheres is an accurate and rapid method for the analys is of SNPs. Cytometry 39:131-140, 2000. (C) 2000 Wiley-Liss, Inc.