[Objective] To establish an efficient, convenient and economical method for site-directed mutagenesis. [Method] The target mutation was introduced into primers designed by DNAMAN5.0 software. Through overlap extension...[Objective] To establish an efficient, convenient and economical method for site-directed mutagenesis. [Method] The target mutation was introduced into primers designed by DNAMAN5.0 software. Through overlap extension PCR for twice obtained the mutation gene which of the full length of the recombinant Human Tissue type plasminogen activator （Reteplase）. The mutation gene cloned it into pEASY- blunt simple cloning vector for sequencing. [Result] The sequencing results showed that three site mutations were fully consistent with the expected results （10~ site had been added a base-pair of A, C had been changed into G at 137~ site, G had been changed into A at 686~ site）.Three site mutations were introduced by using overlap extension PCR on one-step. The overall rate of obtaining the mutant sites was 100%. Site-directed mutagenesis will clone the recombinant Human Tissue type plas- minogen activator and laid the basis for the functional study. [Conclusion] Site-directed mutagenesis was successfully implemented based on the overlap extension PCR which is an efficient, convenient and economical DNA-directed mutagenesis method.展开更多
目的：建立同时测定全血中杀鼠灵、杀鼠醚、敌鼠、氯敌鼠、溴敌隆、氟鼠灵、溴鼠灵7种杀鼠剂的高效液相色谱方法。方法：全血样品经乙酸乙酯提取后,以甲醇-0.3%乙酸铵水溶液（82：18,v/v）为流动相,经ODS-3色谱柱（250 mm×4.6 mm i...目的：建立同时测定全血中杀鼠灵、杀鼠醚、敌鼠、氯敌鼠、溴敌隆、氟鼠灵、溴鼠灵7种杀鼠剂的高效液相色谱方法。方法：全血样品经乙酸乙酯提取后,以甲醇-0.3%乙酸铵水溶液（82：18,v/v）为流动相,经ODS-3色谱柱（250 mm×4.6 mm i.d,5μm）分离,在波长310 nm处检测。结果：分离测定过程在15 min内完成,各杀鼠剂在线性范围内,浓度与峰面积呈良好的线性关系（r=0.999）;两个浓度水平的样品加标回收率分别为91.2%-108.5%和79.2%-93.2%（杀鼠醚分别为65.8%和53.9%）;方法检出限（0.1-0.2）μg/ml;相对标准偏差（RSD）均小于3.0%。结论：方法快速、简便、准确,适用于中毒病人的中毒诊断检测。展开更多
基金Supported by National Natural Science Foundation of China(31160032)~~
文摘[Objective] To establish an efficient, convenient and economical method for site-directed mutagenesis. [Method] The target mutation was introduced into primers designed by DNAMAN5.0 software. Through overlap extension PCR for twice obtained the mutation gene which of the full length of the recombinant Human Tissue type plasminogen activator （Reteplase）. The mutation gene cloned it into pEASY- blunt simple cloning vector for sequencing. [Result] The sequencing results showed that three site mutations were fully consistent with the expected results （10~ site had been added a base-pair of A, C had been changed into G at 137~ site, G had been changed into A at 686~ site）.Three site mutations were introduced by using overlap extension PCR on one-step. The overall rate of obtaining the mutant sites was 100%. Site-directed mutagenesis will clone the recombinant Human Tissue type plas- minogen activator and laid the basis for the functional study. [Conclusion] Site-directed mutagenesis was successfully implemented based on the overlap extension PCR which is an efficient, convenient and economical DNA-directed mutagenesis method.
