This study describes an advantageous, effective protocol for deflecting K-Ras mutations in human stool as a prototype screen for colorectal carcinoma (CRC), the third most common malignancy in the United States. A reliable screening test that detects early lesions would contribute to a decrease in mortality. Currently, the only noninvasive screen for CRC is the hemeoccult, test which has a high false-positive rate. Previously, several investigators have identified genetic biomarkers for CRC in stool DNA. The K-Ras oncogene, mutated in 46-50% of CRC tumors, serves as one molecular marker by which stool samples may be evaluated for early detection of adenocarcinomas. DNA was isolated from stool samples by a new method we specifically designed for extracting high-quality DNA using tetradecyltrimethylammonium oxalate [Catrimox-14, Iowa Biotechnology Corp., (currently Qiagen)]. This protocol produces an optimal yield of high-purity DNA: suitable for genotyping. Detection of the human gene in stool samples was enhanced by hybrid selection of the K-Ras sequences, polymerase chain reaction, and single-strand conformation polymorphism. Tumor tissue and preoperative stool samples for eight patients were K-Ras genotyped and compared; stool samples from two asymptomatic, healthy patients were also evaluated in a double-blind format. In seven of eight samples (87%), the genotypes of the stool and colon tissue DNA were the same. Both healthy patients showed wild-type K-Ras. This protocol shows promise for the development of an efficient and accurate screen for CRC. (C) 2001 Academic Press.
