Simultaneous Analysis of Two-Site Gene Polymorphisms Using Time-Resolved Fluorescence Immunoassay

Abstract

We have developed a method for the simultaneous analysis of two-site gene polymorphisms. The principle developed by us is that two amplicons are produced by duplex PCR using two different primer sets (digoxigenin labeled primer A and biotinylated primer B for BsmI recognition site, biotinylated primer C and FITC labeled primer D for TaqI recognition site) and digested with two restriction enzymes, and the reaction products are analyzed by time-resolved fluorescence immunoassay (TR-FIA) using two different lanthanide ions (Eu and Sm) as labels. In this study, BsmI and TaqI recognition site polymorphisms in intron 8 and exon 9 of the vitamin D receptor (VDR) gene were selected as a target gene. VDR gene polymorphisms of 27 subjects were determined by the proposed method. The results agreed with that obtained by a restriction fragment-length polymorphism (RFLP) analysis using gel electrophoresis. The proposed method is effective, has excellent precision and is well suited to gene analysis for a large number samples.