title: An oligonucleotide-tagged microarray for routine diagnostics of colon cancer by genotyping KRAS mutations.
abstract: Colorectal cancer (CRC) is one of the most prevalent types of cancer, causing significant morbidity and mortality worldwide. CRC is curable if diagnosed at an early stage. Mutations in the oncogene KRAS play a critical role in early development of CRC. Detection of activated KRAS is of diagnostic and therapeutic importance. In this study, KRAS gene fragments containing mutations in codon 12 were amplified by multiplex PCR using a 5'-Cy5-labeled reverse primer in combination with 3'-mutation-specific forward primers that were linked with four unique nucleotide-sequence tags at the 5'-end. The Cy5-labeled reverse primer was extended under PCR amplification to the 5'-end of the mutation-specific forward primers and thus included the complimentary sequence of the tag. PCR products were hybridized to tag-probes immobilized on various substrates and detected by a scanner. Our results indicate that all mutations at codon 12 of KRAS derived from cancer cells and clinical samples could be unambiguously detected. KRAS mutations were accurately detected when the mutant DNA was present only in 10% of the starting mixed materials including wild-type genomic DNA, which was isolated from either cancer cells or spiked fecal samples. The immobilized tag-probes were stable under multiple thermal cycling treatments, allowing re-use of the tag-microarray and further optimization to solid PCR. Our results demonstrated that a novel oligonucleotide-tagged microarray system has been developed which would be suitable to be used for detection of KRAS mutations and clinical diagnosis of CRC.
authors.affiliation: DTU Veterinary Laboratory of Applied Micro-Nanotechnology, Department of Poultry, Fish and Fur Animals, National Veterinary Institute, Technical University of Denmark, DK-8200, Aarhus N, Denmark.
authors.forename: Yuliang
authors.initials: Y
authors.lastname: Liu
authors.affiliation: DTU Nanotech. BioLabchip, Department of Micro and Nanotechnology, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark.
authors.forename: Haukur
authors.initials: H
authors.lastname: Gudnason
authors.affiliation: DTU Veterinary Laboratory of Applied Micro-Nanotechnology, Department of Poultry, Fish and Fur Animals, National Veterinary Institute, Technical University of Denmark, DK-8200, Aarhus N, Denmark.
authors.forename: Yi-Ping
authors.initials: YP
authors.lastname: Li
authors.affiliation: DTU Veterinary Laboratory of Applied Micro-Nanotechnology, Department of Poultry, Fish and Fur Animals, National Veterinary Institute, Technical University of Denmark, DK-8200, Aarhus N, Denmark.
authors.forename: Dang Duong
authors.initials: DD
authors.lastname: Bang
authors.affiliation: DTU Nanotech. BioLabchip, Department of Micro and Nanotechnology, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark.
authors.forename: Anders
authors.initials: A
authors.lastname: Wolff
mesh_terms: D015179:Colorectal Neoplasms / Q000175:diagnosis* / Q000235:genetics; D055088:Early Detection of Cancer / Q000379:methods*; D060005:Genotyping Techniques / Q000379:methods; D019073:HT29 Cells; D006801:Humans; D061986:MCF-7 Cells; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D012680:Sensitivity and Specificity; D018631:ras Proteins / Q000235:genetics*
chemical_list: C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins