Investigation of the medium composition for PCR-SSCP analysis of the HLA-DQA gene region

Abstract

An investigation was conducted to determine the most appropriate conditions for analysis of single-strand conformation polymorphism (SSCP) in the typing of HLA-DQA gene segments after PCR amplification. The original SSCP conditions (conformation of DNA fragments in 95% formamide and separation by electrophoresis on a gel containing 10% glycerol) were modified so that the same reagents as those used at the conformation and electrophoresis stages. could be applied for the SSCP analysis. Three reagents (10% glycerol, 10% formamide and 5% saccharose) were tested. It was found that all modifications gave more distinct band images and separation than the original technique. The clearest images were obtained when a 5% saccharose aliquot was used, whereas the most sensitive discrimination of SSCP bands in terms of Rf was achieved using glycerol.