磁珠富集法分离刀鲚微卫星标记

Isolation of Microsatellite in C.ectenes by Magnetic Beads

利用磁珠富集法分离微卫星序列,以开发长江刀鲚微卫星分子标记。将长江刀鲚基因组DNA经限制内切酶Mse I酶切,回收400-1 000 bp片段,安装接头,构建长江刀鲚全基因组PCR文库。用生物素标记的微卫星探针(CA)12与其杂交,磁珠富集含有微卫星序列的DNA片段。将洗脱所得片段进行PCR扩增,然后进行克隆。经过菌落PCR检验后挑选出118个阳性克隆进行测序,其中97条含有微卫星序列。用设计合成59对微卫星引物对30尾养殖长江刀鲚进行引物的多态性筛选,得到9对多态性引物。

It was a study to develop microsatellite markers with the method of microsatellite enrichment by magnetic beads. Genomic DNA of C. ectenes was digested with restriction enzyme Mse I. The fragments of 400-1 000 bp were recycled and ligated with relevant adaptors, then the“complete genome PCR library”was constructed. The PCR products were hybridized by a biotin-labeled Oligo probe（CA）12, the microsatellites were enriched with magnetic beads. The eluted fragments from magnetic beads were amplified and cloned into pMD18-T vector. 118 positive clones were screened with PCR method from 170 clones. After sequenced, 97 microsatellite sequences were found, 59 pairs of SSR primers were designed, and 9 pairs of primers were polymorphism within 30 cultured Chang Jiang C. ectenes.