A Unique Method For Mutation Analysis Of Tumor Suppressor Genes In Colorectal Carcinomas Using A Crypt Isolation Technique

Background.-Contamination of nontumor tissue makes genetic analysis difficult. For this reason, it is important to obtain pure tumor tissue to ensure accurate genetic analysis.Objective.-To accurately assess the incidence of mutation of tumor suppressor genes (p53: exon 5-8; APC: mutated cluster region; NF-2 gene: all exons) in 45 colorectal carcinomas.Methods.-We developed an application of the polymerase chain reaction-single-strand conformation polymorphism and DNA sequence by coupling them with crypt isolation.Results.-Mutations of p53 and APC: genes were found in 24 and 22 of 45 colorectal carcinomas, respectively. No mutation of the NF-2 gene was observed in this cancer. Single-strand conformation polymorphism using a crypt isolation technique showed a clear migrating band and no false-positive data.Conclusions.-The crypt isolation technique is a useful method for accurately analyzing genetic alterations. Furthermore, our proposed method confirmed the morphological findings obtained before the genetic analysis.