Modified Multiple Primer Extension Method

A multiple primer extension (MPEX) was originally developed for the hybridization, extension, and amplification of a DNA template on a planar substrate by Kinoshita et al. in 2006. Herein we present a modified MPEX method refined by our group for single nucleotide polymorphism (SNP) detection. In this method, hybridization and extension reactions are performed on a plastic S-BIO� PrimeSurface� substrate, with a biocompatible polymer. Its surface chemistry offers extraordinarily stable thermal properties, as well as chemical properties advantageous for enzymatic reactions on the surface. To visualize allele-specific PCR products on the surface, biotin–dUTP was incorporated into newly synthesized complementary strands during the extension reaction. The products were ultimately detected by carrying out a colorimetric reaction with a substrate solution containing 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium. We have further successfully combined this method with multiplex PCR. We demonstrate the advantages of this combined method by analyzing representative SNPs on different linkage disequilibrium blocks of the μ opioid receptor gene (OPRM1 ), which is a marker gene for pain threshold.