5’端与3’端间同源长度对质粒构建的影响

Length Effect of Homologous 5'-and 3'-end on Plasmid Construction

5’端/3’端间同源长度对分子内同源重组质粒构建的影响还不明确.本研究以与GFO对应的五种反向引物CR1、CR2、CR3、CR4、CR5,分别扩增5’端/3’端间同源长度有10、20、30、40、50 bp的5.9 kb线性质粒pET20b-C2-GH10-C2作为模式DNA,经重组酶Exnase Ⅱ 37℃体外重组30 min,42℃热激诱导转化BL21(DE3)感受态细胞,比较转化率、质粒位置正确率、接口正确率.结果显示,同源长度30 bp DNA重组后转化子正确质粒最多,正确接口转化率3.4个/ng DNA,其次是同源长度20 bp DNA.电泳检测显示DNA重组后产生nick质粒,是转化子的来源,胞内酶修饰nick可产生基因-载体接口异常.

Length effect of homologous 5’-and 3’-end is not clear on plasmid construction in intra-molecularrecombination. GFO forward primer and reverse primers CR1,CR2,CR3,CR4,and CR5 were used to amplify fivelinearized p ET20 b-C2-GH10-C2 DNAs containing 10,20,30,40,and 50 bp length of homologous 5’-and 3’-ends,respectively. The five DNAs were recombined in vitro with Exnase Ⅱ at 37 ℃ for 30 min,and transformed BL21（DE3）competent cells using 42 ℃ heat-shock method. After comparing transformation efficiency,plasmid position andDNA sequence,the 30 bp length DNA created the most accurate plasmids,and transformation efficiency was 3.4 transformants per ng,while the 20 bp length DNA created the second. Electrophoresis showed that nicked plasmidwas product of DNA recombination,which created transformants. Nick modification by host cell enzymes led to irregularend-joined junction of inserts and vectors.