耐热DNA连接酶介导的TLCR技术在简化DNA重组.PDFVIP

Hereditas (Beijing) 2016 2 , 38(2): 163169 DNA TLCR DNA 200433 DNA Type DNA (Thermostable ligase chain reaction, TLCR) DNA DNA ——Helper TLCR TLCR 7 1.5 kb 80% TLCR DNA TLCR λ Hind 27 kb 80%TLCR DNA DNA Helper DNA The applications of thermostable ligase chain reaction in facilitat- ing DNA recombination Xiangda Zhou, Xiao Song, Cong Huai, Haiyan Sun, Hongyan Chen, Daru Lu State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China Abstract: The traditional Type restriction enzyme-based method is restricted by the purification steps, and therefore, cannot be applied to specific DNA assembly in chaotic system. To solve this problem, Thermostable Ligase Chain Reaction (TLCR) was introduced in the process of DNA assembly and capture. This technique combines the feature of thermostable DNA ligase and sequence specific oligo ligation template, “Helper”, to achieve specific as- sembly of target fragments and exponential increase of products in multiple thermocyclings. Two plasmid construction experiments were carried out in order to test the feasibility and practical performance of TLCR. One was that, TLCR was used to specifically capture a 1.5 kb fragment into vector from an unpurified chaotic system which contained 7 different sizes of fragments. The results showed that the capturing accuracy was around 80%, which proved the feasi- : 20151010; : 20151202 : (14ZZ008)[Supported by the National Natural Science Foundation of China (Nos. and Shanghai Education Commission Project (No.14ZZ008)] : E-mail: 11210700078@ : E-mail: drlu@ DOI: 10.16288/j.yczz.15-428 : 2016/1/5 16:42:31 URL: /kcms/detail/11.1913.R1642.004.html 164 Hereditas (Beiji