利用微波-热效应进行限制性内切核酸酶反应的可行性验证

Digesting Plasmid DNA with Restriction Endonuclease(s) under the Condition of Microwave-heating

限制性内切酶催化的酶解反应是分子生物学实验中的常用技术.鉴于酶切反应时间长,有人提出采用微波释放的热效应催化限制性内切酶解反应,以期缩短酶解时间.但微波是否能代替传统酶切反应还需验证.本研究设计了一系列的酶切试验,验证微波酶切是否有效.结果表明:短时间(2 min)微波炉高火处理未导致DNA降解及内切酶失活.对于质粒的单酶切和部分双酶切,短时间(2 min)微波炉处理可替代水浴加热完成酶解反应.对于部分双酶切体系,微波炉处理酶切不完全,不能替代温水浴.使用微波酶解需严格控制时间,切勿时间过长.

During molecular biological experiments,restriction endonuclease-digestion was used frequently,but the digestion reaction normally needs to incubate the mixture in water bath for a long time.The microwave was thought to be able to catalyze restriction endonuclease-digestion and save the incubation time.In this study,we designed a series of experiments to verify whether the microwave really could catalyze the reaction.The results showed that DNA degradation and enzyme inactivation didn＇t happen when the restriction endonuclease-digestion reaction system was treated by microwave at high temperature for 2 min.For plasmid digestion with one or two restriction endonucleases,treatment by microwave for short time（2 min） could alternate water bath to complete digestion reaction.For some double digestion systems,microwave digestion is not complete and can＇t substitute for water bath.The digestion time had to control strictly when the microwave was used to catalyze the digestion.