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

Abstract

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 ϳ16%. The PamChip microarray could detect mutant alleles repre-

Genetic mutations of p53 and k-ras in gastric carcinoma patients from Hunan, China

“…The ability to detect small amounts of mutant DNA within a large amount of wild-type DNA is absolutely necessary for analyzing clinical DNA samples. In the present study, PCR-SSCP technique was applied as the combination of PCR-SSCP analysis and DNA sequencing provides the greatest sensitivity [24]. Also the PCR-SSCP is a simple and rapid method for the evaluation of p53 and k-ras gene mutations and it provides a more precise use of these genes as a prognostic marker in gastric cancer.…”

Rapid, Simple, and Accurate Detection of K-ras Mutations From Body Fluids Using Real-Time PCR and DNA Melting Curve Analysis

“…However, both SSCP and RLFP retain some problematic issues that must be elucidated, especially regarding the long TAT, labor-consumption, poor sensitivity, and difficulty of interpreting the band status. 2,6 For this reason, several kinds of different methodologies to analyze the amplicons are now being developed, such as microarray-based methods, 7,8 the capillary electrophoresis method, the DHPLC system, 9,10 and the MCA-based method. 14,17,18 Of these new technologies, promising methods based on the MCA platform are now being developed and some successful results have been reported as clinical use for genotyping single nucleotide polymorphism (SNP) and mutations 8,9,17 and for the identification of infected bacterial and viral pathogens.…”

“…2,6 For this reason, several kinds of different methodologies to analyze the amplicons are now being developed, such as microarray-based methods, 7,8 the capillary electrophoresis method, the DHPLC system, 9,10 and the MCA-based method. 14,17,18 Of these new technologies, promising methods based on the MCA platform are now being developed and some successful results have been reported as clinical use for genotyping single nucleotide polymorphism (SNP) and mutations 8,9,17 and for the identification of infected bacterial and viral pathogens. 18,19 We also tried to establish a rapid, simple, sensitive, and precise method that uses real-time PCR with an MCA platform for scanning K-ras alterations from body fluids with little tumor DNA.…”

“…Our real-time PCR/MCA method can give results within about 4 hours, improving the TAT, and saving about 8 hours compared with our current PCR-RFLP method. Similarly, the entire process of the methods of Maekawa, 8 Dabritz, 12 and Chen 13 can be completed within 1.0 to 3.5 hours, respectively. The detection sensitivity of our assay revealed a value of at least 10 -3 , which is equivalent or 1 order better than that of our current PCR-RFLP assay.…”

“…At the present, the single-strand conformational polymorphism (SSCP) 5 and RFLP 6,7 analyses have been used in hospital laboratories. Furthermore, experimental and promising methods [DNA chip analysis, 8 DHPLC analysis, 9,10 hetereoduplex analysis, 11 and DNA melting curve analysis (MCA) 10,[12][13][14] ] are in development.…”