Detection of colorectal cancer K-ras mutations using a simplified oligonucleotide ligation assay.
It has been suggested that some mutations in codons 12 and 13 of the K-ras gene are associated with the progression of colorectal adenomas to carcinomas. The aim of this study was to develop a rapid, colorimetric assay for K-ras point mutations commonly associated with colorectal cancer. K-ras exon 1 was amplified from colorectal tumor DNA and K-ras activating mutations detected using an oligonucleotide ligation assay (OLA) in combination with immunological and colorimetric detection. Using the OLA with oligonucleotides specific to individual K-ras mutations, 6 (of 17 total colorectal adenomas/carcinomas) were found to have K-ras mutations. The assay could detect as little as 10% mutant allele. A simplified OLA designed to test for either the presence (+) or absence (-) of any of the K-ras activating mutations was developed. The assay was further streamlined by use of a dipstick methodology for colorimetric development. If required, assay sensitivity can be increased by the use of the recently described EDNA-ELCA detection system. The simplified (+/-) mutation OLA in combination with a dipstick or EDNA-ELCA detection system provides a rapid, sensitive assay for K-ras point mutations suitable for use as part of the clinical assessment of colorectal cancer.
Rothschild
C B
CB
Department of Biochemistry, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC, USA.
Brewer
C S
CS
Loggie
B
B
Beard
G A
GA
Triscott
M X
MX
D000230:Adenocarcinoma / Q000235:genetics*; D000236:Adenoma / Q000235:genetics*; D000483:Alleles; D015179:Colorectal Neoplasms / Q000235:genetics*; D003124:Colorimetry; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005784:Gene Amplification; D011905:Genes, ras; D005820:Genetic Testing; D006801:Humans; D008297:Male; D015345:Oligonucleotide Probes / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D012680:Sensitivity and Specificity; D018631:ras Proteins / Q000032:analysis
D015345:Oligonucleotide Probes; D018631:ras Proteins