Premium
Simplified rapid non‐radioactive PCR‐SSCP method applied to K‐ras mutation analysis
Author(s) -
Yamashita Kazuya,
Tatebayash Taeko,
Shinoda Hiroshi,
Okayasu Isao
Publication year - 1996
Publication title -
pathology international
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.73
H-Index - 74
eISSN - 1440-1827
pISSN - 1320-5463
DOI - 10.1111/j.1440-1827.1996.tb03553.x
Subject(s) - microbiology and biotechnology , silver stain , staining , single strand conformation polymorphism , proteinase k , dna , pathology , dna extraction , polymerase chain reaction , chemistry , biology , nuclear medicine , gene , medicine , genetics
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, 3×3×120 mrn In size); (2) one‐step DNA extraction with lysls buffer containing proteinase K, Nonidet P40 and Tween 20; (3) PCR with 108bp, c‐ki‐ras 2 gene primers; (4) SSCP analysis with 10% forrnamlde‐added poly‐actylamide 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 6h). This novel approach should allow more frequent use of molecular diagnosis in biopsies and surgical pathology.