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. Because of their short generation times and high breeding efficiency, rodents are extremely useful for researching disorders [2,12]. Point mutation-carrying strains, such as mice with an obese mutation in the leptin gene (Lep ob ) [16] and rats with a fatty mutation in the leptin receptor gene (Lepr fa ), [13] are included in representative rodent disease models [6]. For authentic research and for maintaining strains, it is important to verify the existence of point mutations.Tetra-primer amplification refractory mutation systempolymerase chain reaction (tetra-primer ARMS-PCR) employs 2 primer pairs to amplify, respectively, the 2 different alleles of a single nucleotide polymorphism (SNP) in a single PCR [15]. Moreover, the primers can be designed to amplify fragments of differing sizes for each allele [15]. Therefore, tetra-primer ARMS-PCR does not require restriction enzyme digestion and allows genotyping of a point mutation solely by inspection of PCR products with agarose gel electrophoresis. Combining Flinders Technology Associates (FTA) filter paper and appropriate PCR buffer allows for direct PCR amplification of DNA from unpurified blood on FTA filter paper [7]. To use FTA filter paper in our specific pathogenfree (SPF) barrier facility, we have so far used ethylene oxide gas (EOG) sterilization. However, ethylene oxide is a directly acting alkylating agent which is associated with an increase in chromosomal aberrations and sister chromatid exchange [1,5]. On the other hand, radiation leaves no toxic residues on treated medical items [10]. Here, we describe a rapid and efficient point mutation genotyping of the mouse leptin gene using tetra-primer ARMS-PCR and gamma-ray sterilized FTA filter paper in SPF barrier facilities.All procedures involving the use of mice were approved by the animal welfare committee of CHARLES RIVER LABORATORIES JAPAN. FTA filter paper (GE Healthcare Japan, Tokyo, Japan) was sterilized with EOG or with gamma-ray irradiation and carried into our in-house vivarium. Future breeders of the SPF B6.Cg-Lep ob /J strain (CHARLES RIVER LABORATORIES JAPAN, Yokohama, Japan), housed in the in-house vivarium, were used when weaning age. A small drop of whole blood, exuded by cutting the mice ...