Rapid screening assay for KRAS mutations by the modified smart amplification process.
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.
Tatsumi
Kenji
K
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
Mitani
Yasumasa
Y
Watanabe
Jun
J
Takakura
Hideki
H
Hoshi
Kanako
K
Kawai
Yuki
Y
Kikuchi
Takeshi
T
Kogo
Yasushi
Y
Oguchi-Katayama
Atsuko
A
Tomaru
Yasuhiro
Y
Kanamori
Hajime
H
Baba
Masaru
M
Ishidao
Takefumi
T
Usui
Kengo
K
Itoh
Masayoshi
M
Cizdziel
Paul E
PE
Lezhava
Alexander
A
Ueda
Michio
M
Ichikawa
Yasushi
Y
Endo
Itaru
I
Togo
Shinji
S
Shimada
Hiroshi
H
Hayashizaki
Yoshihide
Y
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*
C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins