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A unique method for mutation analysis of tumor suppressor genes in colorectal carcinomas using a crypt isolation
T Sugai1, W Habano, S i Nakamura
1Division of Pathology, Central Clinical Laboratory, School of Medicine, Iwate Medical University, Morioka, Japan.
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.