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目的研究黄胸鼠不同地理区域种群的遗传差异,用磁珠富集法筛选其微卫星位点。方法将黄胸鼠基因组DNA用限制性内切酶消化后的小片段与接头连接,再用生物素标记的(AC)15重复序列探针与酶切片段杂交,应用链霉素亲和磁珠捕获300～600bp含有微卫星序列的DNA片段,通过载体转化到JM109感受态细胞中,构建富集微卫星序列的小片段插入文库,测序后设计引物并筛选。结果本次实验从约900个转化子中获得304个阳性克隆,对其中140个进行测序,并成功设计黄胸鼠微卫星引物13对。结论筛选出的13对微卫星引物多态性高,能稳定扩增出目标条带,可用于种群遗传研究。
Objective To study the genetic diversity of different geographical populations of Rattus flavipectus and to screen their microsatellite loci by magnetic bead enrichment. Methods The genomic DNA of Rattus flavipectus was ligated with the linker using the small fragment digested with restriction endonuclease and then hybridized with the biotin-labeled (AC) 15 repeat probe to the digested fragment. Streptavidin magnetic beads DNA fragments containing 300 ~ 600bp microsatellite sequences were captured and transformed into JM109 competent cells by vector. Small fragments enriched in microsatellite sequences were inserted into the library, and primers were designed and screened after sequencing. Results 304 positive clones were obtained from about 900 transformants in this experiment, of which 140 were sequenced and 13 pairs of microsatellite primers were successfully designed. Conclusion Thirteen pairs of microsatellite primers were screened for high polymorphism and could stably amplify the target bands and could be used for population genetic research.