Simplified rapid non-radioactive PCR-SSCP method applied to K-ras mutation analysis.

A newly modified non-RI, PCR-SSCP method is presented and applied to K-ras analysis of colorectal tumors. It comprises five steps: (1) sampling of tiny pieces of fresh solid tissue by scraping with disposable bamboo combs (thin bars made of bamboo, 3 x 3 x 120 mm in size); (2) one-step DNA extraction with lysis buffer containing proteinase K, Nonidet P-40 and Tween 20; (3) PCR with 108 bp, c-ki-ras 2 gene primers; (4) SSCP analysis with 10% formamide-added polyacrylamide gels; and (5) detection with silver staining. In comparison with conventional RI- or non-RI-PCR-SSCP, It can give reliable and clear results in a much shorter time (within a total of 6 h). This novel approach should allow more frequent use of molecular diagnosis in biopsies and surgical pathology.