BACKGROUND: We developed a rapid, precise, and accurate microarray-based method that uses a three-dimensional platform for detection of mutations. METHODS: We used the PamChip microarray to detect mutations in codons 12 and 13 of K-ras in 15 cell lines and 81 gastric or colorectal cancer tissues. Fluorescein isothiocyanate-labeled PCR products were analyzed with the microarray. We confirmed the microarray results with PCR-single-strand conformation polymorphism (SSCP) analysis and DNA sequencing. RESULTS: We could correctly identify wild-type, heterozygous, and homozygous mutant genotypes with the PamChip microarray in <3.5 h. The array data were consistent with those of PCR-SSCP analysis and DNA sequencing. All 15 cell lines and 80 of 81 clinical cancer specimens (98.8%; 95% confidence interval, 96.4-100%) were genotyped accurately with the microarray, a rate better than that of direct DNA sequencing (38.9%) or SSCP (93.8%). Only one clinical specimen was misdiagnosed as homozygous for the wild-type allele. Densitometric analysis of SSCP bands indicated that the content of the mutant allele in the specimen was approximately 16%. The PamChip microarray could detect mutant alleles representing more than 25% of the SSCP band proportions. Therefore, the limit for detection of mutant alleles by the PamChip microarray was estimated to be 16-25% of the total DNA. CONCLUSIONS: The PamChip microarray is a novel three-dimensional microarray system and can be used to analyze K-ras mutations quickly and accurately. The mutation detection rate was nearly 100% and was similar to that of PCR-SSCP together with sequencing analysis, but the microarray analysis was faster and easier.
BACKGROUND: We developed a rapid, precise, and accurate microarray-based method that uses a three-dimensional platform for detection of mutations. METHODS: We used the PamChip microarray to detect mutations in codons 12 and 13 of K-ras in 15 cell lines and 81 gastric or colorectal cancer tissues. Fluorescein isothiocyanate-labeled PCR products were analyzed with the microarray. We confirmed the microarray results with PCR-single-strand conformation polymorphism (SSCP) analysis and DNA sequencing. RESULTS: We could correctly identify wild-type, heterozygous, and homozygous mutant genotypes with the PamChip microarray in <3.5 h. The array data were consistent with those of PCR-SSCP analysis and DNA sequencing. All 15 cell lines and 80 of 81 clinical cancer specimens (98.8%; 95% confidence interval, 96.4-100%) were genotyped accurately with the microarray, a rate better than that of direct DNA sequencing (38.9%) or SSCP (93.8%). Only one clinical specimen was misdiagnosed as homozygous for the wild-type allele. Densitometric analysis of SSCP bands indicated that the content of the mutant allele in the specimen was approximately 16%. The PamChip microarray could detect mutant alleles representing more than 25% of the SSCP band proportions. Therefore, the limit for detection of mutant alleles by the PamChip microarray was estimated to be 16-25% of the total DNA. CONCLUSIONS: The PamChip microarray is a novel three-dimensional microarray system and can be used to analyze K-ras mutations quickly and accurately. The mutation detection rate was nearly 100% and was similar to that of PCR-SSCP together with sequencing analysis, but the microarray analysis was faster and easier.