Non-radioactive single-strand conformation polymorphism (SSCP) analysis of relatively long PCR products

Abstract

The SSCP technique is based on the appearance of new “refolding” conformations during electro-phoresis due to mutation. In order to develop a simple, non-radioactive SSCP analysis method so that it can reliably detect single nucleotide changes in PCR products up to 500 bp in length, extensive optimisation trials were performed. The best separation of SSCP bands of PCR products up to 500 bp in length was obtained with 14.5 to 15.5% polyacrylamide gels that had been run in the cold room at 15 V/cm for 65 to 70 hours. After the pre-run, 5 ml of PCR product was mixed with 10 ml of denaturing-loading dye and the mixture was heated to 94 °C for 10 min. Total mixture volume of the 15 ml was loaded into the well without quenching. After electrophoresis, the gels were stained with SYBR^® Gold nucleic acid stain for 30-40 minutes and photographed under UV light using a SYBR^® gel stain photographic filter. This method alongside with confirmatory sequencing has been utilised to identify three novel mutations in the 5¢-regulatory region of the human CYP3A4 gene. The final results were very satisfactory and the optimised method was able to reliably reveal new mutations in amplified DNA from blood samples. In fact the non-radioactive SSCP method developed here seems to be robust enough to analyse PCR products up to 500 bp long.