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 difficu lt. 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 suppress or 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-singl e-strand conformation polymorphism and DNA sequence by coupling them with c rypt isolation. Results.-Mutations of p53 and APC: genes were found in 24 and 22 of 45 colo rectal carcinomas, respectively. No mutation of the NF-2 gene was observed in this cancer. Single-strand conformation polymorphism using a crypt isola tion technique showed a clear migrating band and no false-positive data. Conclusions.-The crypt isolation technique is a useful method for accuratel y analyzing genetic alterations. Furthermore, our proposed method confirmed the morphological findings obtained before the genetic analysis.