遗传 ›› 2016, Vol. 38 ›› Issue (2): 163-169.doi: 10.16288/j.yczz.15-428

• 技术与方法 • 上一篇    下一篇

耐热DNA连接酶介导的TLCR技术在简化DNA重组实验中的应用

周翔达,宋晓,怀聪,孙海燕,陈红岩,卢大儒   

  1. 复旦大学生命科学学院,遗传工程国家重点实验室,上海 200433
  • 收稿日期:2015-10-10 修回日期:2015-12-02 出版日期:2016-02-20 发布日期:2016-01-05
  • 通讯作者: 卢大儒,博士,教授,研究方向:遗传学。E-mail: drlu@fudan.edu.cn
  • 作者简介:周翔达,硕士研究生,专业方向:分子遗传学。E-mail: 11210700078@fudan.edu.cn
  • 基金资助:
    国家自然科学基金项目(编号:81170786, 81372235, 31571371)和上海市教委项目(编号:14ZZ008)资助[Supported by the National Natural Science Foundation of China (Nos; 81170786, 81372235, 31571371) and Shanghai Education Commission Project (No.14ZZ008)]

The applications of thermostable ligase chain reaction in facilitating DNA recombination

Xiangda Zhou, Xiao Song, Cong Huai, Haiyan Sun, Hongyan Chen, Daru Lu   

  1. State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China
  • Received:2015-10-10 Revised:2015-12-02 Online:2016-02-20 Published:2016-01-05

摘要: 传统的DNA重组方法Type Ⅱ型限制酶技术受到片段纯化的限制,无法做到复杂混合体系中DNA片段的特异性连接。为解决这个问题,本研究将耐热连接酶链式反应(Thermostable ligase chain reaction, TLCR)引入DNA片段的连接与捕获。该技术利用耐热型DNA连接酶的特性,在热循环反应中配合针对目的片段末端序列设计的单链寡核苷酸连接模板——Helper,实现目的片段的特异性连接和产物数量的指数性增长。两个质粒构建实验被用于验证TLCR技术的可行性和应用效果。首先利用TLCR技术从一个未经纯化的含有7种不同大小片段的混杂体系中特异性地将一段1.5 kb的片段捕获进载体,随机抽取的克隆样品经检验准确率达到80%,验证TLCR技术在复杂混合体系中特异性连接DNA片段的可行性和准确性。在另一个质粒构建实验中,TLCR技术从λ噬菌体基因组Hind消化物中将两段总长达27 kb的片段按顺序捕获进载体,随机抽取的克隆样品经检验同样达到了80%的准确率。结果表明,TLCR技术能够简化DNA重组实验的操作,并且适用于多片段和大片段的连接,可以为生物学研究提供便利。

关键词: DNA片段连接, 耐热型连接酶, Helper, DNA片段捕获, 复杂混合体系

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.

Key words: DNA ligation, thermostable ligase, Helper, DNA capture, chaotic system