Received on January 28, 2004
Accepted on May 17, 2004

Molecular Diagnostics and Genetics

Three-Dimensional Microarray Compared with PCR-Single-Strand Conformation Polymorphism Analysis and DNA Sequencing for Mutation Analysis of K-ras Codons 12 and 13

¹ Department of Laboratory Medicine, Hamamatsu University School of Medicine, Hamamatsu, Japan.
² Department of Laboratory Medicine, Hamamatsu University School of Medicine, Hamamatsu, Japan and Genome Medical Business Division, OLYMPUS Corporation, Hachioji, Japan.
³ 1st Department of Pathology, Hamamatsu University School of Medicine, Hamamatsu, Japan.
⁴ Oncogene Research Unit/Cancer Prevention Unit, Tochigi Cancer Center Research Institute, Utsunomiya, Japan
⁵ Scientific Equipment Group, OLYMPUS America, Inc., New York, NY
⁶ Genome Medical Business Division, OLYMPUS Corporation, Hachioji, Japan.

^* To whom correspondence should be addressed. E-mail: mmaekawa{at}hama-med.ac.jp.

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%) 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 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.