title: Novel Methodology for Rapid Detection of KRAS Mutation Using PNA-LNA Mediated Loop-Mediated Isothermal Amplification.
abstract: Detecting point mutation of human cancer cells quickly and accurately is gaining in importance for pathological diagnosis and choice of therapeutic approach. In the present study, we present novel methodology, peptide nucleic acid-locked nucleic acid mediated loop-mediated isothermal amplification (PNA-LNA mediated LAMP), for rapid detection of KRAS mutation using advantages of both artificial DNA and LAMP. PNA-LNA mediated LAMP reactions occurred under isothermal temperature conditions of with 4 primary primers set for the target regions on the KRAS gene, clamping PNA probe that was complimentary to the wild type sequence and LNA primers complementary to the mutated sequences. PNA-LNA mediated LAMP was applied for cDNA from 4 kinds of pancreatic carcinoma cell lines with or without KRAS point mutation. The amplified DNA products were verified by naked-eye as well as a real-time PCR equipment. By PNA-LNA mediated LAMP, amplification of wild type KRAS DNA was blocked by clamping PNA probe, whereas, mutant type KRAS DNA was significantly amplified within 50 min. Mutant alleles could be detected in samples which diluted until 0.1% of mutant-to-wild type ratio. On the other hand, mutant alleles could be reproducibly with a mutant-to-wild type ratio of 30% by direct sequencing and of 1% by PNA-clamping PCR. The limit of detection (LOD) of PNA-LNA mediated LAMP was much lower than the other conventional methods. Competition of LNA clamping primers complementary to two different subtypes (G12D and G12V) of mutant KRAS gene indicated different amplification time depend on subtypes of mutant cDNA. PNA-LNA mediated LAMP is a simple, rapid, specific and sensitive methodology for the detection of KRAS mutation.
authors.affiliation: Department of Human Pathology, Wakayama Medical University, Wakayama, Japan.|Second Department of Internal Medicine, Wakayama Medical University, Wakayama, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Itonaga
authors.affiliation: Department of Human Pathology, Wakayama Medical University, Wakayama, Japan.
authors.forename: Ibu
authors.initials: I
authors.lastname: Matsuzaki
authors.affiliation: Department of Human Pathology, Wakayama Medical University, Wakayama, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Warigaya
authors.affiliation: Department of Human Pathology, Wakayama Medical University, Wakayama, Japan.
authors.forename: Takaaki
authors.initials: T
authors.lastname: Tamura
authors.affiliation: Department of Human Pathology, Wakayama Medical University, Wakayama, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Shimizu
authors.affiliation: Department of Human Pathology, Wakayama Medical University, Wakayama, Japan.
authors.forename: Masakazu
authors.initials: M
authors.lastname: Fujimoto
authors.affiliation: Department of Human Pathology, Wakayama Medical University, Wakayama, Japan.
authors.forename: Fumiyoshi
authors.initials: F
authors.lastname: Kojima
authors.affiliation: Second Department of Internal Medicine, Wakayama Medical University, Wakayama, Japan.
authors.forename: Masao
authors.initials: M
authors.lastname: Ichinose
authors.affiliation: Department of Human Pathology, Wakayama Medical University, Wakayama, Japan.
authors.forename: Shin-Ichi
authors.initials: S
authors.lastname: Murata
mesh_terms: D045744:Cell Line, Tumor; D004252:DNA Mutational Analysis / Q000379:methods*; D011905:Genes, ras / Q000235:genetics*; D006801:Humans; D057230:Limit of Detection; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009841:Oligonucleotides / Q000378:metabolism*; D020135:Peptide Nucleic Acids / Q000378:metabolism*; D017354:Point Mutation; D016133:Polymerase Chain Reaction
chemical_list: D009841:Oligonucleotides; D020135:Peptide Nucleic Acids; C477371:locked nucleic acid