title: Eprobe-mediated screening system for somatic mutations in the KRAS locus.
abstract: Activating mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) loci are largely predictive of resistance to epidermal growth factor receptor (EGFR) therapy in colorectal cancer (CRC). A highly sensitive detection system for the KRAS gene mutations is urgently needed; however, conventional methods have issues with feasibility and cost performance. Here, we describe a novel detection system using a fluorescence 'Eprobe' capable of detecting low level KRAS gene mutations, via real-time PCR, with high sensitivity and simple usability. We designed our Eprobes to be complementary to wild-type (WT) KRAS or to the commonly mutated codons 12 and 13. The WT Eprobe binds strongly to the WT DNA template and suppresses amplification by blocking annealing of the primer during PCR. Eprobe-PCR with WT Eprobe shows high sensitivity (0.05-0.1% of plasmid DNA, 1% of genomic DNA) for the KRAS mutation by enrichment of the mutant type (MT) amplicon. Assay performance was compared to Sanger sequencing using 92 CRC samples. Discrepancies were analyzed by mutation genotyping via Eprobe-PCR with full match Eprobes for 7 prevalent mutations and the next generation sequencing (NGS). Significantly, the Eprobe system had a higher sensitivity for detecting KRAS mutations in CRC patient samples; these mutations could not be identified by Sanger sequencing. Thus, the Eprobe approach provides for highly sensitive and convenient mutation detection and should be useful for diagnostic applications.
authors.affiliation: Departments of Thoracic and Visceral Organ Surgery, Gunma University Graduate School of Medicine, Maebashi, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Atsumi
authors.affiliation: Division of Genomic Technologies, RIKEN Center for Life Science Technologies, Yokohama, Kanagawa, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Hanami
authors.affiliation: Departments of Thoracic and Visceral Organ Surgery, Gunma University Graduate School of Medicine, Maebashi, Japan.
authors.forename: Yasuaki
authors.initials: Y
authors.lastname: Enokida
authors.affiliation: Departments of Thoracic and Visceral Organ Surgery, Gunma University Graduate School of Medicine, Maebashi, Japan.
authors.forename: Hiroomi
authors.initials: H
authors.lastname: Ogawa
authors.affiliation: Division of Genomic Technologies, RIKEN Center for Life Science Technologies, Yokohama, Kanagawa, Japan.
authors.forename: Diane
authors.initials: D
authors.lastname: Delobel
authors.affiliation: Division of Genomic Technologies, RIKEN Center for Life Science Technologies, Yokohama, Kanagawa, Japan.
authors.forename: Yasumasa
authors.initials: Y
authors.lastname: Mitani
authors.affiliation: Division of Genomic Technologies, RIKEN Center for Life Science Technologies, Yokohama, Kanagawa, Japan.
authors.forename: Yasumasa
authors.initials: Y
authors.lastname: Kimura
authors.affiliation: Division of Genomic Technologies, RIKEN Center for Life Science Technologies, Yokohama, Kanagawa, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Soma
authors.affiliation: Division of Genomic Technologies, RIKEN Center for Life Science Technologies, Yokohama, Kanagawa, Japan.
authors.forename: Michihira
authors.initials: M
authors.lastname: Tagami
authors.affiliation: Departments of Thoracic and Visceral Organ Surgery, Gunma University Graduate School of Medicine, Maebashi, Japan.
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Takase
authors.affiliation: Division of Genomic Technologies, RIKEN Center for Life Science Technologies, Yokohama, Kanagawa, Japan.
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Ichihara
authors.affiliation: Departments of Thoracic and Visceral Organ Surgery, Gunma University Graduate School of Medicine, Maebashi, Japan.
authors.forename: Izumi
authors.initials: I
authors.lastname: Takeyoshi
authors.affiliation: Division of Genomic Technologies, RIKEN Center for Life Science Technologies, Yokohama, Kanagawa, Japan.
authors.forename: Kengo
authors.initials: K
authors.lastname: Usui
authors.affiliation: RIKEN Preventive Medicine and Diagnosis Innovation Program, Yokohama, Kanagawa, Japan.
authors.forename: Yoshihide
authors.initials: Y
authors.lastname: Hayashizaki
authors.affiliation: Departments of Thoracic and Visceral Organ Surgery, Gunma University Graduate School of Medicine, Maebashi, Japan.
authors.forename: Kimihiro
authors.initials: K
authors.lastname: Shimizu
mesh_terms: D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D009154:Mutation; D009691:Nucleic Acid Denaturation / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins / Q000235:genetics*
chemical_list: C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins
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