东方田鼠部分BAC文库的微卫星筛选  

Screening of microsatellite DNA sequence from the BAC library of Microtus fortis

作　　者：汤敏[1] 倪丽菊 高骏[4] 肖君华[1] 胡建华 高诚 

机构地区：[1]东华大学,上海201620 [2]上海市实验动物质量监督检验站,上海201203 [3]上海实验动物研究中心,上海201203 [4]上海市农业科学院畜牧兽医研究所,上海201106

出　　处：《中国实验动物学报》2012年第3期50-55,共6页Acta Laboratorium Animalis Scientia Sinica

基　　金：上海市科委基金项目(编号:09140900101)

摘　　要：目的从东方田鼠的部分BAC文库中筛选微卫星。方法应用非放射性的菌落杂交方法和磁珠富集法从东方田鼠的BAC文库中筛选高质量的微卫星标记。结果以地高辛标记的寡聚核苷酸(CA)20为探针,通过菌落杂交法从136个东方田鼠BAC克隆中筛选出杂交信号最强的20个阳性克隆。再将这20个阳性克隆分别通过链霉亲和素磁珠法构建亚克隆文库,从中选取400个经PCR鉴定为阳性的亚克隆进一步测序分析,共得到220个微卫星序列,阳性率55%。选取重复次数高,侧翼序列完整的微卫星序列设计74对引物,共有35对引物能扩增出清晰的条带,其中16对引物具有多态性。结论成功且高效地从阳性BAC克隆中筛选出微卫星序列,这些微卫星和阳性BAC克隆可用于后续的定位研究。Objective To obtain microsatellite DNA sequence of Microtus fortis from its BAC library.Methods To screen the microsatellite DNA sequence with nonradioactive analysis method—colony hybridization and enrichments by magnetic beads.Results Twenty positive clones with the strongest signals were screened out of 136 BAC clones through hybridization of digoxin-labeled oligonucleotide（CA）20.Then these positive BAC clones were devoted to construct the subclone libraries separately.220 microsatellite sequences were isolated from 400 positive subclones identified by PCR.According to those microsatellite sequences with more copy numbers and complete flanking sequence,74 pairs of primers were designed.As a result,35 pairs of microsatellite primers got clear bands,of which 16 pairs had polymorphism.Conclusions Microsatellite DNA sequences have been screened successfully and efficiently from BAC library of Microtus fortis.The sequences and positive BAC clones may contribute to further research of their location.

关 键 词：东方田鼠 BAC文库 微卫星 菌落杂交 链霉亲和素磁珠 

分 类 号：Q95-33[生物学—动物学]