Cold single-strand conformation polymorphism analysis: Optimization for detection of APC gene mutations in patients with familial adenomatous polyposis

Over 200 adenomatous polyposis coli (APC) gene mutations have been describe d in familial adenomatous polyposis (FAP) patients. Recent single-strand co nformation polymorphism (SSCP) screening methods have introduced minigel ru ns, simple ethidium bromide staining and external temperature control witho ut any loss of sensitivity (cold-SSCP). In order to test the effectiveness in APC mutation detection, cold-SSCP was employed following polymerase chai n reaction (PCR) amplification in three patients with FAR Different running parameter combinations were compared. The three mutations already known we re all diagnosed by cold-SSCP. The gel concentration was found to be essent ial in detecting the single-base substitution in fragments of different len gths. The observation of deletions was not affected by gel concentrations a nd heteroduplex bands were always produced. The temperature or glycerol add ition did not significantly affect sensitivity. This modified cold-SSCP met hod provides a simple and effective way for detecting several known APC gen e mutations without any loss of sensitivity and could be useful for large-s cale molecular diagnosis of FAP.