Development of an Efficient Genotyping Method to Detect Obese Mutation in the Mouse Leptin Gene for Use in SPF Barrier Facilities
Development of an Efficient Genotyping Method to Detect Obese Mutation in the Mouse Leptin Gene for Use in SPF Barrier Facilities
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DOI：
10.1292/jvms.12-0348
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发表时间：
2013-05-01
影响因子：
1.2
通讯作者：
Ikeda, Takuya
中科院分区：
文献类型：
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作者：
Ayabe, Hiroaki;Ikeda, Shogo;Ikeda, Takuya
我们建立了一种快速、有效的检测小鼠瘦素肥胖突变（Lep（ob））的四引物扩增突变系统-聚合酶链反应（tetra-primer ARMS-PCR）基因分型方法。在该方法中，将采集到γ射线灭菌的Flinders Technology Associates（FTA）滤纸上的全血用作PCR模板，无需DNA纯化步骤。单管PCR和电泳鉴定的Lep（ob）/Lep（ob）、Lep（ob）/+和Lep（ob）+/+3种基因型与PCR-RFLP检测结果完全一致。这种方法可以节省材料成本和操作时间，因为它不需要限制性内切酶消化，并且可以在大多数无特定病原体（SPF）屏障设施中建立。
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.