title: [The applications of thermostable ligase chain reaction in facilitating DNA recombination].
abstract: The traditional Type Ⅱ 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 assembly 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 feasibility and accuracy of using TLCR to specific assembly of DNA fragments in a complicated mixed system. In the other experiment, TLCR was used to capture two fragments (total length was 27 kb) from Hind Ⅲ digestion of Lambda genome into vector by order. The results also showed an accuracy of around 80%. As demonstrated in the results, TLCR can simplify the process of DNA recombination experiments and is suitable for the assembly of multiple and large DNA fragments. This technique can provide convenience to biological experiments.
authors.affiliation: State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China.
authors.forename: Xiang-da
authors.initials: XD
authors.lastname: Zhou
authors.affiliation: State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Song
authors.affiliation: State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China.
authors.forename: Cong
authors.initials: C
authors.lastname: Huai
authors.affiliation: State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China.
authors.forename: Hai-yan
authors.initials: HY
authors.lastname: Sun
authors.affiliation: State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China.
authors.forename: Hong-yan
authors.initials: HY
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China.
authors.forename: Da-ru
authors.initials: DR
authors.lastname: Lu
mesh_terms: D004247:DNA / Q000235:genetics* / Q000378:metabolism*; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000378:metabolism*; D004795:Enzyme Stability; D006358:Hot Temperature; D022521:Ligase Chain Reaction / Q000379:methods*; D008957:Models, Genetic; D016133:Polymerase Chain Reaction / Q000379:methods; D011995:Recombination, Genetic; D015203:Reproducibility of Results
chemical_list: D004247:DNA; D011088:DNA Ligases; D000072481:DNA Ligase ATP