λ噬菌体red重组系统介导的靶向突变及其机理的研究生物化学与分子生物学专业论文.docxVIP

生璺睑型匡挝盔堂盟土堂焦造塞 生璺睑型匡挝盔堂盟土堂焦造塞 而与先导链序列一致的单链寡聚核苷酸打靶效率较低：3)Red重组技术的效率受DNA 分子转录水平的影响。DNA分子的转录能够增加Red重组技术的效率。4)12NA分子的 错配修复对Red重组技术有影响。当敲除掉错配修复系统之后，不但使总体的打靶效率 增高，而且能影响不同单链寡聚核苷酸的效率比值。 9 尘囤进塑医魁盔堂丛±堂垃途塞 Abstract The traditional cloning method in vitro is based 011 restriction endonuclease cleavage followed by DNA joining with DNA ligase．Although vital to molecplar biology and its applications， restriction endonuclease strategies for DNA engineering rely on the favorable disposition of cleavage sites，which imposes practical limitations． Recently，a novel homologous recombination method based on homologous recombination， has been developed in E．coil，DNA Recombineering is all exciting and powerf／．tl new approach towards engineering genetic material．It utilizes homologous recombination to ‘piece together’DNA moleeples，including both single-stranded oligonucleotides(SSOs)and double—stranded DNA fragments in all exquisitely precise manner．It may be used to create changes in DNA such弱deletions，insertions and point mutations，which are difficlalt or time- consuming to do using traditional molecglar biology techniques．In E coli，the most efficient recombination system that has been developed to—date is the Red-system．in which recombination is mediated by the Exo，Beta and Gam proteins，the system is dependent on the over—expression ofthe九phage genes exo,beta and gam Exo has 5’—÷3’exonuclease activity． that carl generate 3-extruding ends of dsDNA．Beta is a single—strand DNA—binding protein that can promotes its annealing with homologous sequence．Gam inhibits RecBCD nuclease from degrading linear DNA．Daiguan Yu and co-workers have placed these three genes under the tight control of a temperature sensitive A．-c1857 repressor to regglate their activities．At 320C when the repressor is active，the expressions of three genes are inhibited．However， when cells are shifted to 420C for I 5 minutes，the repressor is inactived and can’t bind X-pL promotor anymore，SO the genes are expressed from X-pL p