磁珠富集法分离刀鲚微卫星标记.pdfVIP

生物技术通报 ·研究报告· BIOTECHNOLOGY BULLETIN 年第 期 2014 6 磁珠富集法分离刀鲚微卫星标记 邓平平  施永海  张根玉  张之文  张海明  陆根海  刘永士  （上海市水产研究所，上海 200433） 摘 要 ： 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 I 酶切， Mse 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 ） 与其杂交，磁珠富集 12 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。 关键词 ： 刀鲚 磁珠富集 微卫星 多态性 Isolation of Microsatellite in C. ectenes by Magnetic Beads Deng Pingping Shi Yonghai Zhang Genyu Zhang Zhiwen Zhang Haiming Lu Genhai Liu Yongshi （ ， 200433 ） Shanghai Fisheries Research Institute Shanghai Abstract:  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 ） , the 12 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