Detection of the mutated K-Ras biomarker in colorectal carcinoma.
This study describes an advantageous, effective protocol for detecting 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.
Doolittle
B R
BR
Department of Pathology, Yale University, School of Medicine, New Haven, Connecticut, USA.
Emanuel
J
J
Tuttle
C
C
Costa
J
J
D014408:Biomarkers, Tumor / Q000032:analysis*; D015179:Colorectal Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D004247:DNA / Q000302:isolation & purification; D004273:DNA, Neoplasm / Q000302:isolation & purification; D015246:Deoxyribonuclease EcoRI; D005243:Feces / Q000737:chemistry; D011905:Genes, ras; D006801:Humans; D007202:Indicators and Reagents; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D018807:Polymorphism, Single-Stranded Conformational; D000644:Quaternary Ammonium Compounds; D012016:Reference Values; D015203:Reproducibility of Results; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D050337:Trimethyl Ammonium Compounds
D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm; D007202:Indicators and Reagents; D000644:Quaternary Ammonium Compounds; D050337:Trimethyl Ammonium Compounds; D004247:DNA; C090528:DNA modification methylase HindIII; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D015246:Deoxyribonuclease EcoRI; C009410:tetradecyltrimethylammonium