High-Throughput Single Nucleotide Polymorphism Genotyping by Fluorescent 5′ Exonuclease Assay

Abstract

Single nucleotide polymorphisms (SNPs) are the class of DNA variants that are most common in the genome. Technologies are needed that allow relatively highthroughput, high-quality genotyping of SNPs, with rapid assay development for individual SNPs of interest. We have implemented an accurate and high-throughput SNP genotyping system using a commercially available fluorescent 5′ exonuclease assay. Optimization of the assay system for low-volume reactions and low-probe concentrations has reduced assay costs by 75%. Using a simple assay optimization process, we successfully developed genotyping assays for 92% of assays attempted (309 out of 335) and have generated over 200 000 genotypes.