Published online before print October 2, 2008

This Article Full Text Full Text (PDF) Supplemental Material All Versions of this Article: jmoldx.2008.080024v1 10/6/520    most recent Purchase Article View Shopping Cart Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Tatsumi, K. Articles by Hayashizaki, Y. Search for Related Content PubMed PubMed Citation Articles by Tatsumi, K. Articles by Hayashizaki, Y.

Journal of Molecular Diagnostics 2008, Vol. 10, No. 6
Copyright © 2008 American Society for Investigative Pathology & Association for Molecular Pathology
DOI: 10.2353/jmoldx.2008.080024

Rapid Screening Assay for KRAS Mutations by the Modified Smart Amplification Process

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 and Yoshihide Hayashizaki^*

From the Genome Exploration Research Group (Genome Network Project Core Group), * RIKEN Genomic Sciences Center, RIKEN Yokohama Institute; the Department of Gastroenterological Surgery, Graduate School of Medicine, Yokohama City University; and DNAFORM, Inc., Kanagawa, Japan

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.

This article has been cited by other articles:

				T. Araki, K. Shimizu, K. Nakamura, T. Nakamura, Y. Mitani, K. Obayashi, Y. Fujita, S. Kakegawa, Y. Miyamae, K. Kaira, et al. Usefulness of Peptide Nucleic Acid (PNA)-Clamp Smart Amplification Process Version 2 (SmartAmp2) for Clinical Diagnosis of KRAS Codon12 Mutations in Lung Adenocarcinoma: Comparison of PNA-Clamp SmartAmp2 and PCR-Related Methods J. Mol. Diagn., January 1, 2010; 12(1): 118 - 124. [Abstract] [Full Text] [PDF]