Single-stranded conformation polymorphism analysis of Vitamin D receptor gene by capillary electrophoresis with laser-induced fluorescence detection

Abstract

A highly sensitive single-stranded conformation polymorphism method for analysis of Vitamin D receptor ( VDR) gene polymorphism was developed employing laser-induced fluorescence capillary electrophoresis (LIF-CE). LIF-CE was conducted utilizing a linear polyacrylamide solution as entangled polymer and acridine orange as a fluorescent dye of single-stranded DNA. Effect of acridine orange, size of PCR product and running temperature were investigated by LIF-CE in order to analyze the two polymorphisms of the allelic variation of the Bsm I and Taq I sites in intron 8 and exon 9, respectively, of the VDR gene. The developed method was simple, rapid (<16 min for Taq I type, <20 min for Bsm I type) and highly sensitive for VDR gene polymorphism. VDR gene polymorphism in 32 subjects was determined by the proposed method. The results were consistent with those obtained by restriction fragment-length polymorphism (RFLP) analysis using gel electrophoresis. The proposed method can be employed among the various VDR gene polymorphism analyses related to osteoporosis.