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.