狗獾粪便DNA提取方法的初步探讨

在京杭大运河扬州段堤坝上采集狗獾的新鲜粪便,采用预处理-酚/氯仿抽提法、NaCl改良法、异硫氰酸胍裂解法、淀粉吸附法及QIAamp试剂盒法5种方法对粪便样品中狗獾DNA进行提取,探讨狗獾粪便样品中DNA提取方法和优化条件。结果表明,在5种提取方法中,淀粉吸附法的效果明显优于其它4种方法。采用粪便裂解液快速裂解细胞后,加入淀粉去除其中的大量PCR抑制物,然后用蛋白酶K裂解、酚/氯仿/异戊醇抽提,最后使用UNIQ-10柱纯化粪便DNA,对线粒体控制区和微卫星位点的PCR扩增反应及测序结果证实该方法的可行性。以上结果表明,通过该方法获得的粪便DNA能够用于更深入的分子遗传学等学科的研究。

A preliminary study on the DNA extraction method from faeces of badger (Meles meles)

The fresh feces of badger (Meles meles) were collected from the dam of Jing-Hang Canal near Yangzhou, and five DNA extraction methods including amylum-subsiding method, preprocess-organicsolvent extraction method, NaCl modified method, GuSCN-SiO2 method and QIAamp DNA Stool Mini Kit method were used to extract the DNA from them. The results showed that the highest amplification success rate was in the amylum-subsiding method. In this study, fecal sample was lysed rapidly with digested solution, then DNA was extracted using chloroform/phenol after incubation with proteinase K, following by purification to remove numerous potential PCR inhibitors. The mitochondrial DNA control region and 5 microsatellite loci were amplified. showing that the mitochondrial DNA control region sequenced successfully. Our results suggested that DNA extraction based on this method was reliable.