title: Cold single-strand conformation polymorphism analysis: optimization for detection of APC gene mutations in patients with familial adenomatous polyposis.
abstract: Over 200 adenomatous polyposis coli (APC) gene mutations have been described in familial adenomatous polyposis (FAP) patients. Recent single-strand conformation polymorphism (SSCP) screening methods have introduced minigel runs, simple ethidium bromide staining and external temperature control without any loss of sensitivity (cold-SSCP). In order to test the effectiveness in APC mutation detection, cold-SSCP was employed following polymerase chain reaction (PCR) amplification in three patients with FAP. Different running parameter combinations were compared. The three mutations already known were all diagnosed by cold-SSCP. The gel concentration was found to be essential in detecting the single-base substitution in fragments of different lengths. The observation of deletions was not affected by gel concentrations and heteroduplex bands were always produced. The temperature or glycerol addition did not significantly affect sensitivity. This modified cold-SSCP method provides a simple and effective way for detecting several known Apc gene mutations without any loss of sensitivity and could be useful for large-scale molecular diagnosis of FAP.
authors.affiliation: Institute of Histology and Embryology, University of Bologna, I-40138 Bologna, Italy.
authors.forename: P
authors.initials: P
authors.lastname: Strippoli
authors.forename: S
authors.initials: S
authors.lastname: Sarchielli
authors.forename: R
authors.initials: R
authors.lastname: Santucci
authors.forename: G P
authors.initials: GP
authors.lastname: Bagnara
authors.forename: G
authors.initials: G
authors.lastname: Brandi
authors.forename: G
authors.initials: G
authors.lastname: Biasco
mesh_terms: D011125:Adenomatous Polyposis Coli / Q000235:genetics; D025601:Adenomatous Polyposis Coli Protein / Q000235:genetics; D003080:Cold Temperature; D004247:DNA / Q000737:chemistry / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D012680:Sensitivity and Specificity; D013696:Temperature
chemical_list: D025601:Adenomatous Polyposis Coli Protein; D004247:DNA