关键词: 微卫星, 基因组文库, 筛选, 小熊猫

Abstract: 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 12% 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.

Key words: Genomic DNA library, Isolation, Microsatellite, Red panda(Ailurus