A Unique Method for Mutation Analysis of Tumor Suppressor Genes in Colorectal Carcinomas Using a Crypt Isolation Technique

Abstract

Abstract 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.