大熊猫和小熊猫粪便DNA提取的简易方法  

A simple protocol for DNA extraction from faeces of the giant panda and lesser panda

作　　者：张保卫[1] 魏辅文[1] 李明[1] 吕晓平[1] 

机构地区：[1]中国科学院动物研究所,北京100080

出　　处：《动物学报》2004年第3期452-458,共7页ACTA ZOOLOGICA SINICA

基　　金：国家自然科学基金重点项目 (No .3 0 2 3 0 0 80 ) ;国家杰出青年基金项目 (No .3 0 12 5 0 0 6) ;中国科学院知识创新工程项目 (KSCX2 1 0 3 )

摘　　要：采集了大熊猫和小熊猫的新鲜粪便样品 ,使用 1 0 0 %乙醇保存。通过重复离心富集研究动物的肠道脱落细胞 ,并使用乙醇和双蒸水洗涤以除去抑制物。用 1 %的SDS快速裂解细胞 ,离心除去残渣后 ,向裂解液中加入蛋白酶进行消化。消化结束后使用等体积的酚 /氯仿抽提 ,乙醇沉淀DNA。用双蒸水溶解粪便DNA后 ,使用PCR产物纯化试剂盒对粪便DNA进行纯化。电泳检测结果显示 ,从乙醇保存的大、小熊猫粪便样品中抽提到高质量的粪便DNA。对线粒体控制区、细胞色素b基因、 1 2SrRNA基因的PCR扩增反应以及测序结果也证实了样品保存方法和DNA抽提方法可靠而高效。此方法使用实验室内常用的分子生物学试剂 ,不仅克服了分子粪便学研究中常见的抑制物粪便DNA微量降解严重等障碍 ,与商业化的粪便抽提试剂盒 (QIAampDNAStoolMiniKit,Qiagen)相比还是一种经济的试验方法 (抽提反应成本为试剂盒的 1 / 5 )。文中还对粪便DNA内细菌基因组等背景DNA可能对分子粪便学试验结果的影响进行了探讨。在基于PCR技术的遗传学研究中 ,对于植食性动物而言 ,粪便内的背景DNA对目标动物DNA片断的扩增和序列测定未见影响 ;但对于肉食性动物 ,则必须考虑被捕食者基因组对试验可能产生的影响 。We developed a new method to extract DNA from faecal samples in this study. Fresh faecal samples of the giant panda Ailuropoda melanoleuca and lesser panda Ailurus fulgens were collected and stored in 100% ethanol. About 200-300 mg faecal samples were collected in a microcentrifuge tube through repeated centrifugation, then washed by ethanol and sterile water. Faecal sample was lysed rapidly with 1% SDS and centrifuged quickly, then transferred the supernatant to a new tube. DNA was extracted using Chloroform/Phenol after incubation with Proteinase K, following by precipitation and purification. Mitochondrial DNA control region, cytochrome b and 12 S rRNA gene were amplified and sequenced successfully from faecal DNA, suggesting that the methods of sample storage and DNA extraction were reliable and efficient. This method not only overcame previous barriers to molecular scatology, such as inhibitors, low quality and degradation of DNA, but also was more economical than commercial kits (e.g. QIAamp DNA Stool Mini Kit,Qiagen). We monitored some background DNA in faecal DNA, and discussed potential risks from background DNA contamination, such as genomes of bacteria, virus, parasites and prey. No negative effect was observed during amplification and sequencing of target DNA, which showed that the background DNA had no effect on genetic study of target animals.

关 键 词：大熊猫 小熊猫 粪便 DNA PCR技术 抽提方法 

分 类 号：Q781[生物学—分子生物学]