title: Development of an efficient genotyping method to detect obese mutation in the mouse leptin gene for use in SPF barrier facilities.
abstract: We have developed a rapid and efficient genotyping method for detection of the mouse leptin obese mutation (Lep(ob)) using tetra-primer amplification refractory mutation system-polymerase chain reaction (tetra-primer ARMS-PCR). In this method, whole blood collected onto gamma-ray sterilized Flinders Technology Associates (FTA) filter paper is used as PCR template without a DNA purification step. Three genotypes (Lep(ob)/Lep(ob), Lep(ob)/+ and +/+) differentiated by single-tube PCR and electrophoresis were perfectly consistent with those determined by PCR-restriction fragment length polymorphism (PCR-RFLP). This method can save material costs and operation time, because it does not require restriction enzyme digestion and could be set up in most specific pathogen-free (SPF) barrier facilities.
authors.affiliation: Production Department, Plasma Team, Charles River Laboratories Japan, Inc., 795 Shimofurusawa, Atsugi-shi, Kanagawa 243-0214, Japan. hiroaki.ayabe@crl.com
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Ayabe
authors.forename: Shogo
authors.initials: S
authors.lastname: Ikeda
authors.forename: Shigeri
authors.initials: S
authors.lastname: Maruyama
authors.forename: Seiji
authors.initials: S
authors.lastname: Shioyama
authors.forename: Mayumi
authors.initials: M
authors.lastname: Kikuchi
authors.forename: Ayako
authors.initials: A
authors.lastname: Kawaguchi
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Yamada
authors.forename: Takuya
authors.initials: T
authors.lastname: Ikeda
mesh_terms: D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D060005:Genotyping Techniques / Q000379:methods*; D020738:Leptin / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D009765:Obesity / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D013047:Specific Pathogen-Free Organisms / Q000235:genetics
chemical_list: D017931:DNA Primers; D020738:Leptin