Single Nucleotide Polymorphism Analysis Based on Minisequencing Coupled with a Fluorescence Microsphere Technology

Abstract

In this paper, we describe a new method for detection of single nucleotide polymorphisms (SNPs) by applying the minisequencing principle to a fluorescence microsphere format. The specific primer, which was designed to anneal to its target of genomic DNA fragment immediately upstream of the polymorphic site, was immobilized to carboxylated Luminex microspheres as a probe. The primer was hybridized with genomic DNA fragments containing polymorphic sites and extended one base in the presence of biotin labeled ddNTP and DNA polymerase. After the extension reaction, Streptavidin-phycoerythrin was added to a reaction mixture to combine the biotin labeled with ddNTP. The final reaction products were analyzed by a Luminex 100 instrument. The fluorescence intensity of the Streptavidin-phycoerythrin combined with the extended ddNTP-biotin was used to identify the SNPs. The results showed that this method is highly sensitive, specific, and suitable for quantitative SNP detection. There was a good linear relationship between the mutant allele frequencies and the relative fluorescence intensities produced by mutant and wild-type gene fragments. A mutant allele frequency as low as 1.0% was accurately determined.