Multiplexed single nucleotide polymorphism genotyping by oligonucleotide ligation and flow cytometry

作者：Marie A. Iannone, J. David Taylor, Jingwen Chen, May-Sung Li, Philip Rivers, Kimberly Slentz‐Kesler, Michael P. Weiner · 发表于：Cytometry · 年份：2000 · DOI：10.1002/(sici)1097-0320(20000201)39:2<131::aid-cyto6>3.0.co;2-u · 被引用次数：214 · 研究领域：Advanced Biosensing Techniques and Applications、Biosensors and Analytical Detection、Gene expression and cancer classification

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 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 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.