用PCR法快速筛选中国对虾含微卫星的重组阳性克隆

Rapid screening recombinant positive clones containing microsatellites of Penaeus chinensis with PCR

运用PCR法在中国对虾的小片段基因组文库中快速筛选含有微卫星序列的重组阳性克隆。PCR中使用的引物为载体pGEM - 3zf(+)上多克隆位点两侧的T7/Sp6启动子引物和根据微卫星核心重复序列设计的STR引物 :STR1:5’ -ATATATATATATAT - 3’ ;STR2 :5’ -TCTCTCTCTCTCTC - 3’。直接使用含有重组克隆的菌液 ,在PCR反应前增加一段裂解细菌的高温。筛选出来的含有微卫星序列的重组阳性克隆经DNA测序验证。结果证明 ,采用PCR方法用菌液快速筛选含有微卫星序列的重组阳性克隆完全可行 ,而且具有简便、快速、高效。

Recombinant positive clones containing microsatellite sequences of Chinese shrimp,Peneaus chinensis were obtained through rapid screening small-size fractionated genomic libraries with PCR technique. The primers used in PCR were T7/Sp6 promotor primers located at both sides of multiple clone site of pGEM-3zf(+)and STR primers:STR1:5'-ATATATATATATAT-3' and STR2:5'-TCTCTCTCTCTCTC-3' designed according to core repeats motifs of microsatellites. The bacterial suspensions were directly used in PCR, before which a short time period of high temperature (95℃) was kept to break bacteria. The recombinant positive clones containing microsatellite squences were obtained by screening and were verified after sequencing. The results show that PCR technique combined with bacterial suspension for rapid screening recombinant positive clones containing microsatellite squences is feasible and this method also has advantages of concenience,quickness, high efficiency and reliable outcome.