title: Rapid screening assay for KRAS mutations by the modified smart amplification process.
abstract: Previously, the smart amplification process version 2 (SMAP-2) was developed to detect mutations from tissue and in crude cell lysates and has been used for rapid diagnosis of specific somatic mutations with single-nucleotide precision. The purpose of this study was to develop a rapid and practical method to detect cancer and metastasis in specimens using the SMAP-2 assay. We developed modified SMAP-2 assays that enabled detection of any change in a single codon using a single assay. Rapid SMAP-2 screening assays are suitable for routine clinical identification of critical amino acid substitutions such as codon 12 mutations in KRAS. Primers bracketing the first two nucleotides of KRAS codon 12 were designed so that all possible alleles would be amplified by the SMAP-2 assay. In combination with the peptide nucleic acid (PNA) with exact homology to the wild-type allele, our assay amplified all mutant alleles except for the wild-type sequence. With this new assay design (termed PNA-clamp SMAP-2), we could detect KRAS mutations within 60 minutes, including sample preparation. We compared results from PNA-clamp SMAP-2 assay, polymerase chain reaction-restriction fragment length polymorphism, and direct sequencing of clinical samples from pancreatic cancer patients and demonstrated perfect concordance. The PNA-clamp SMAP-2 method is a rapid, simple, and highly sensitive detection assay for cancer mutations.
authors.affiliation: Genome Exploration Research Group (Genome Network Project Core Group), RIKEN Genomic Sciences Center, RIKEN Yokohama Institute, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, Japan. landy-tk@bd5.so-net.ne.jp
authors.forename: Kenji
authors.initials: K
authors.lastname: Tatsumi
authors.forename: Yasumasa
authors.initials: Y
authors.lastname: Mitani
authors.forename: Jun
authors.initials: J
authors.lastname: Watanabe
authors.forename: Hideki
authors.initials: H
authors.lastname: Takakura
authors.forename: Kanako
authors.initials: K
authors.lastname: Hoshi
authors.forename: Yuki
authors.initials: Y
authors.lastname: Kawai
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kikuchi
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Kogo
authors.forename: Atsuko
authors.initials: A
authors.lastname: Oguchi-Katayama
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Tomaru
authors.forename: Hajime
authors.initials: H
authors.lastname: Kanamori
authors.forename: Masaru
authors.initials: M
authors.lastname: Baba
authors.forename: Takefumi
authors.initials: T
authors.lastname: Ishidao
authors.forename: Kengo
authors.initials: K
authors.lastname: Usui
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Itoh
authors.forename: Paul E
authors.initials: PE
authors.lastname: Cizdziel
authors.forename: Alexander
authors.initials: A
authors.lastname: Lezhava
authors.forename: Michio
authors.initials: M
authors.lastname: Ueda
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Ichikawa
authors.forename: Itaru
authors.initials: I
authors.lastname: Endo
authors.forename: Shinji
authors.initials: S
authors.lastname: Togo
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Shimada
authors.forename: Yoshihide
authors.initials: Y
authors.lastname: Hayashizaki
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D000483:Alleles; D004252:DNA Mutational Analysis / Q000295:instrumentation / Q000379:methods*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation / Q000379:methods*; D017354:Point Mutation; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000295:instrumentation / Q000379:methods*; D018631:ras Proteins / Q000235:genetics*
chemical_list: C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins