一种筛选微卫星位点的改进方法及其在小熊猫微卫星基因文库构建上的应用

An Improved Method for Isolation of Microsatellite DNA and Application In Red Panda(Ailurus fulgens)

微卫星基因位点是在各个遗传学领域被广泛使用的分子标记.而对于首次研究的物种,必须筛选出可以应用的微卫星位点.对于分离微卫星位点来说由于只有大约0.5%～2%的重组体含有微卫星位点,所以标准的基因组文库需要至少产生5 000个重组体.传统的筛选方法就是通过检测大量重组体来定位这些少量的位点.近几年来出现了一些新的筛选方法,采用'富集'微卫星序列的技术将包含微卫星位点的重组体比例提高了10～100倍.这样就降低了筛选工作的时间和劳动强度,大大提高了筛选微卫星位点的效率.本文在结合了几种筛选微卫星位点技术路线的基础上,采用生物素标记探针杂交、富集和PCR筛选技术,对原有技术路线进行了改进.主要实验步骤为:1)基因组DNA的提取和消化;2)生物素标记探针富集;3)克隆建库;4)'PCR'筛选;5)引物设计和多态性检测.新的方法在小熊猫微卫星基因文库的构建中取得成功,获得了10个具有多态性的(CA)n重复序列微卫星位点.

Microsatellite loci have become one of the most popular molecular markers used in many genetics fields. However, it is crucial that polymorphic microsatellite loci need to be selected or isolated for the studied species at first. The task of microsatellite isolation can be quite involving in terms of effort and time because it traditionally consists of screening genomic libraries with appropriate probes. The number of positive clones that can be obtained by means of this traditional method usually ranges from 14% to less than 0.04%. Several new protocols, based on the ＂enrichment＂ protocol, have appeared in the last few years, which can increase the number of positive clone, but the high cost and complexity counteract their applicability. Thus, we proposed an economical and easy protocol, which was a combination and amelioration of different methods, based on ＂biotin ligated primer＂, ＂enrichment＂ and ＂PCR Screening＂ protocols. The main step involves： 1 ） Isolation and Digestion of the Genomic DNA, 2） Enrichment using the ＂biotin ligated primer＂, 3） Clone and Lib construction, 4） ＂PCR Screening＂, and 5） Primer design and Variability test. Using this new method, we have isolated and characterized 10 polymorphic （CA）n microsatellite loci in red panda （ Ailurus fulgens ）, and these loci of high variability and stability will be useful in population genetic studies of red panda.