Conditions for single-strand conformation polymorphism (SSCP) analysis of BRCA1 gene using an automated electrophoresis unit

The single-strand conformation polymorphism procedure has been applied in r outine testing for hereditary diseases and cancer. However, temperature, ru nning time, gel composition, fragment length, etc. can influence its sensit ivity. Mutation detection in the clinical setting depends on the developmen t of automated technology, especially for large genes such as the breast ca ncer gene BRCA1. We analysed DNA samples with BRCA1 mutations in an automat ed system (GenePhor System; Amersham-Pharmacia Biotech, Uppsala, Sweden). T he concentrations of DNA template and PCR primers, the effect of chilling a fter denaturation, and the temperature and time of the electrophoresis were investigated. All band-shifts were detected by electrophoresis at 5 degree sC for 2 h 15 min. Concentrations of DNA and samples used in the PCR did no t affect the SSCP pattern, but chilling the PCR product in ice after denatu ration was required. The type and position of mutation in the fragments did not influence the probability of a mobility shift, although SSCP analysis was more sensitive for fragments shorter than 350 bp. This automated SSCP m ethod meets the requirements of fast turnaround and sensitivity and can be readily adapted to the screening of large genes such as BRCA1.