连接方法对重组效率影响的比较研究

目的]为探索一种适于连接PCR产物和不同粘端的新方法。方法]分别采用冻融法、割胶法、试剂盒法和牙签法连接PCR产物与质粒DNA以及酶切产物与质粒DNA,比较不同连接方法对重组效率的影响。结果]采用冻融法和割胶法连接PCR产物与pMD18-T,其连接效率分别为94.00%和93.00%,略高于试剂盒法和牙签法。采用冻融法和割胶法连接酶切产物的重组效率分别为86.67%和88.00%,略高于试剂盒法和牙签法。牙签法简化了连接前DNA相关操作,具有较高的连接效率,完全能够满足普通连接反应,特别是粘端连接反应要求。结论]割胶法具有很高的重组效率和转化效率,可以满足普通连接反应、文库构建对连接效率和转化效率的要求,具有一定的应用前景。

Comparative Study on the Effects of Connection Methods on the Recombination Efficiency

Objective] The research aimed to discuss a new method for connecting PCR products and different cohesive ends. Method] PCR products and plasmid DNA, enzyme digestion products and plasmid DNA were connected by using freeze-thawing method, cutting gel method, reagent kit method and toothpick method to compare the effects of different connection methods on the recombination efficiency. Result] When PCR products were connected with pMD18-T by using freeze-thawing method and cutting gel method, the recombination efficiency were 94.00% and 93.00%, slightly higher than that by reagent kit method and toothpick method. The recombination efficiency of connecting enzyme digestion products by freeze-thawing method and cutting gel method were 86.67% and 88.00% resp.,which were slightly higher than that by reagent kit method and toothpick method. The toothpick method simplified related DNA operation before connection and had higher connection efficiency, which could completely meet the demands of common connection reactions, especially for the connection reaction of cohesive ends. Conclusion] Cutting gel method had higher recombination efficiency and transformation efficiency and could meet the demands of common connection reactions and the demands of library construction to the recombination efficiency and transformation efficiency, which had certain application foreground.