应用通用荧光引物筛选裸鼹鼠微卫星位点方法的建立  

Establishment of PCR Method Using Fluorescence Labeled Universal Primers for Screening Microsatellite Loci in Naked Mole Rat

作　　者：林丽芳[1] 李莉[2] 肖邦[1] 程继帅 杨文静[1] 丛薇[1] 赵善民[1] 汤球[1] 崔淑芳[1] 

机构地区：[1]第二军医大学实验动物中心,上海200433 [2]第二军医大学教学保障处,上海200433

出　　处：《实验动物与比较医学》2016年第1期76-80,共5页Laboratory Animal and Comparative Medicine

基　　金：上海市科技发展基金项目（12140900400）与国家科技支撑计划项目（2015BAI09B02）联合资助

摘　　要：目的 建立使用通用荧光引物分步PCR法筛选裸鼹鼠微卫星位点的方法.方法 在特异性引物F链的5＆#39;端连接通用引物,先以特异引物R链和加长引物F链进行PCR,再加入通用荧光引物进行第二步PCR,最后使用琼脂糖电泳和毛细管电泳检测扩增产物,同时以传统的荧光引物PCR为对照.结果 琼脂糖电泳结果显示,通用荧光引物PCR产生的目的片段清晰,引物特异性强,目的片段多态性与传统PCR产生的多态性一致,且片段大小均比传统PCR产生的目的片段大15 bp左右,与预期一致.毛细管电泳结果也显示,通用引物PCR结果检测的等位基因数与传统荧光引物PCR方法产生的数量完全一致,无杂带,扩增效率高,具有较强的可操作性.结论 使用通用荧光引物分步PCR法结果可应用于裸鼹鼠基因组大量微卫星位点的筛选.Objective Fluorescent labeled universal primers PCR in two-steps was established to screen microsatellite loci of naked mole rat. Methods A fusion of the forward primer and universal primers in 5＇ end was created. The first PCR was conducted with reverse primers and forward primer extended with universal primers. The second PCR was initiated after the adding of fluorescent labed universal primers. With conventional fluorescent labeled primers PCR as a control, PCR product was detected by agarose gel electrophoresis and capillary electrophoresis. Results Fluorescent labeled universal primer PCR had strong specificity and bands in agarose gel electrophoresis was sharp. Furthermore, the product had high consistent polymorphism with those in traditional PCR, with size being about 15 bp. They were in line with expectations. After analysis of capillary electrophoresis, it also showel equal number of allele with these two different method. As well, this fluorescent labeled universal primer PCR had high amplification efficiency and being specific, operational. Conclusion The fluorescent labed universal primers PCR in two-steps was applicable to screen a high-flux of microsatellite loci in naked mole rat.

关 键 词：裸鼹鼠 微卫星位点筛选 通用荧光引物 

分 类 号：Q95-33[生物学—动物学]