Document Detail


Rapid screening assay for KRAS mutations by the modified smart amplification process.
MedLine Citation:
PMID:  18832461     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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:
Kenji Tatsumi; Yasumasa Mitani; Jun Watanabe; Hideki Takakura; Kanako Hoshi; Yuki Kawai; Takeshi Kikuchi; Yasushi Kogo; Atsuko Oguchi-Katayama; Yasuhiro Tomaru; Hajime Kanamori; Masaru Baba; Takefumi Ishidao; Kengo Usui; Masayoshi Itoh; Paul E Cizdziel; Alexander Lezhava; Michio Ueda; Yasushi Ichikawa; Itaru Endo; Shinji Togo; Hiroshi Shimada; Yoshihide Hayashizaki
Publication Detail:
Type:  Evaluation Studies; Journal Article     Date:  2008-10-02
Journal Detail:
Title:  The Journal of molecular diagnostics : JMD     Volume:  10     ISSN:  1525-1578     ISO Abbreviation:  J Mol Diagn     Publication Date:  2008 Nov 
Date Detail:
Created Date:  2008-10-16     Completed Date:  2008-12-09     Revised Date:  2013-06-05    
Medline Journal Info:
Nlm Unique ID:  100893612     Medline TA:  J Mol Diagn     Country:  United States    
Other Details:
Languages:  eng     Pagination:  520-6     Citation Subset:  IM    
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
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MeSH Terms
Descriptor/Qualifier:
Aged
Aged, 80 and over
Alleles
DNA Mutational Analysis / instrumentation,  methods*
Female
Humans
Male
Middle Aged
Nucleic Acid Amplification Techniques / instrumentation,  methods*
Point Mutation*
Proto-Oncogene Proteins / genetics*
Sensitivity and Specificity
Sequence Analysis, DNA / instrumentation,  methods*
ras Proteins / genetics*
Chemical
Reg. No./Substance:
0/KRAS protein, human; 0/Proto-Oncogene Proteins; EC 3.6.5.2/ras Proteins
Comments/Corrections

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