四川梅花鹿(CAG)_n微卫星文库构建

Microsatellite Enrichment by Magnetic Beads in Cervus nippon sinchuanicus

应用Dynal磁珠-生物素标记的微卫星探针与四川梅花鹿基因组酶切片段杂交,捕获200～750bp含有微卫星序列的DNA片段,连接pMD18-T载体,再转化到感受态细胞JM109中以构建文库。通过PCR方法从(CAG)n文库中筛选阳性克隆,从576个转化子中获得了234个阳性克隆,对其全部进行序列测定,其中73个含有微卫星序列。除获得的目的微卫星序列(CAG)n外,还观察到(AG)n、(AT)n的重复序列。本研究表明,经过优化的磁珠富集法能够稳定、高效地获得四川梅花鹿微卫星标记。

The microsatellite loci from Cervus nippon sinchuanicus genome was isolated by Streptavidin-coated Magnetic Beads Adsorption method. The length of 200 -750 bp DNA fractions containing microsatellite sequences were captured by oligonucleotide probes. The enriched DNA segments were ligated on pMD18-T and then transformed into E. coli JM109 competent cells. Then PCR was applied to screen positive clones from the libraries. All 234 positive clones were identified from 576 transformants. As a result, 73 sequences were acquired. Besides （CAG）n, （AG）nand （AT）, repeat sequences were also obtained. The resuhs indicated that the method was efficient to isolate microsatellite markers. The markers will be useful tools for further studies on C. n. sinchuanicus genetics and genomics.