Oligonucleotide microarray-based mutation detection of the K-ras gene in colorectal cancers with use of competitive DNA hybridization.

Abstract

In cancer research, gene expression and mutations are increasingly investigated by use of oligonucleotide microarrays, which use immobilized oligonucleotides and sequence-specific DNA probe hybridization to investigate differences between nondiseased and cancer tissues (1). In our previous works, we used oligonucleotide microarray-based mutation analysis to detect germline or somatic mutations (2)(3). Activating mutations of the K- ras gene occur in ∼20–50% of colorectal cancers, with ∼85% of the mutations restricted to codons 12 and 13 (4). K- ras gene mutations have been widely studied as markers for cancer prognosis, and population-based studies have suggested that mutated K- ras might be associated with some tumor phenotypes (4)(5)(6). Studies of associations between K- ras mutations and specific clinical features generally require the analysis of large numbers of samples (5). Thus, researchers need a high-throughput technique for assessing K- ras mutations. Oligonucleotide microarrays may provide a valid option because they allow scientists to accurately and rapidly process large numbers of samples. Because the K- ras gene is known to have two mutational hot spots (codons 12 and 13), it has been used as a target gene for testing newly developed techniques for mutation detection, including various applications of the DNA chip (7)(8). Here we describe a new method for K- ras oligonucleotide microarray analysis called competitive DNA hybridization (CDH). CDH is a novel, efficient, high-capacity hybridization technique in which various fluorescently labeled samples are mixed to compete with each other in a hybridization reaction. A total of 204 Korean patients with colorectal cancer from Seoul National University Hospital and the National Cancer Center of Korea were screened for somatic K- ras mutations in this study. Written informed consent was obtained from all patients; the cancers included 103 cancers originating from the proximal colon and 101 …