[Basic studies on mutation analysis of K-ras codon 12 by use of three-dimensional microarray system].
A next-generation DNA microarray system, FD10 has been developed. It is based around the PamChip, a custom-made microarray, which consists of a solid three-dimensional structure that facilitated the incorporation of probe molecules. We applied this microarray system on a detection of K-ras mutation at codon 12 in some cancer cell lines. The PCR products amplified by use of FITC labeled primers were applied onto probe-absorbed microarray. After hybridization, the signal was imaged by CCD camera and analyzed by the exclusive software. We confirmed the microarray results by PCR-SSCP and sequencing analyses. Ten, two and three out of 15 cell lines were homozygous for wild type allele, heterozygous for wild and mutant allele, and homozygous for mutant alleles, respectively. Signals hybridized with antisense probes were stronger than those with sense probes, without PSN1 cell line. The system had a good reproducibility. Essentially, the microarray results were consistent with PCR-SSCP and sequencing results. In conclusion, the FD10 microarray system was easy to operate and short to get results. It might be useful for a focused array applicable for specific purposes. The K-ras mutation detection system worked well and will be applied to clinical specimens soon.
Department of Laboratory Medicine, Hamamatsu University School of Medicine, Hamamatsu 431-3192.
Masato
M
Maekawa
Terumi
T
Taniguchi
Chika
C
Tatebayashi
Toshinobu
T
Horii
Akihiro
A
Takeshita
Haruhiko
H
Sugimura
Kokichi
K
Sugano
Hiroyuki
H
Yonekawa
Tomonori
T
Nagaoka
Takashi
T
Kanno
D003062:Codon / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods; D004273:DNA, Neoplasm; D011905:Genes, ras / Q000235:genetics*; D006801:Humans; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015203:Reproducibility of Results; D014407:Tumor Cells, Cultured
D003062:Codon; D004273:DNA, Neoplasm