Ayabe Hiroaki, Ikeda Shogo, Maruyama Shigeri, Shioyama Seiji, Kikuchi Mayumi, Kawaguchi Ayako, Yamada Tetsuo, Ikeda Takuya
Production Department, Plasma Team, Charles River Laboratories Japan, Inc., 795 Shimofurusawa, Atsugi-shi, Kanagawa 243-0214, Japan.
J Vet Med Sci. 2013;75(5):633-8. doi: 10.1292/jvms.12-0348. Epub 2012 Dec 10.
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.
我们开发了一种快速高效的基因分型方法，即利用四引物扩增阻滞突变系统-聚合酶链反应（四引物ARMS-PCR）检测小鼠瘦素肥胖突变（Lep(ob)）。在该方法中，采集到经γ射线灭菌的弗林德斯技术协会（FTA）滤纸上的全血用作PCR模板，无需进行DNA纯化步骤。通过单管PCR和电泳区分的三种基因型（Lep(ob)/Lep(ob)、Lep(ob)/+和+/+）与通过PCR-限制性片段长度多态性（PCR-RFLP）确定的基因型完全一致。该方法可节省材料成本和操作时间，因为它不需要限制性酶切，并且可以在大多数无特定病原体（SPF）屏障设施中进行。
