Yu Fang, Li Xia, Wang Fei, Liu Yang, Zhai Chao, Li Wenqiang, Ma Lixin, Chen Wanping
State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Province Key Laboratory of industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan, China.
School of Pharmacy, Qingdao University, Qingdao, China.
Front Bioeng Biotechnol. 2023 Aug 11;11:1167534. doi: 10.3389/fbioe.2023.1167534. eCollection 2023.
Molecular cloning is used in a wide variety of biological and medical research. Here, we developed a rapid and efficient DNA-assembling method for routine laboratory work. We discovered that the cleavage speed of T5 exonuclease is approximately 3 nt/min at 0°C and hence developed a T5 exonuclease-mediated low-temperature sequence- and ligation-independent cloning method (TLTC). Two homologous regions of 15 bp-25 bp compatible with the ends of the vector backbones were introduced into the inserts through PCR. Approximately 120 fmol of inserts and linear vectors was mixed at a molar ratio of approximately 3:1 and treated with 0.5 U of T5 exonuclease at 0°C for 5 min. Then, the mixture was transformed into to generate recombinant plasmids. Single segment and multi-segments can be assembled efficiently using TLTC. For single segment, the overall cloning efficiency is above 95%. Moreover, extra nucleotides in the vectors can be removed during TLTC. In conclusion, an extremely simple and fast DNA cloning/assembling method was established in the present study. This method facilitates routine DNA cloning and synthesis of DNA fragments.
分子克隆被广泛应用于各种生物学和医学研究中。在此，我们开发了一种适用于常规实验室工作的快速高效的DNA组装方法。我们发现T5核酸外切酶在0°C时的切割速度约为3个核苷酸/分钟，因此开发了一种T5核酸外切酶介导的低温序列和连接无关克隆方法（TLTC）。通过PCR将与载体骨架末端兼容的两个15 bp - 25 bp的同源区域引入到插入片段中。将约120 fmol的插入片段和线性载体以约3:1的摩尔比混合，并在0°C下用0.5 U的T5核酸外切酶处理5分钟。然后，将混合物转化以产生重组质粒。使用TLTC可以高效组装单片段和多片段。对于单片段，总体克隆效率高于95%。此外，在TLTC过程中可以去除载体中的额外核苷酸。总之，本研究建立了一种极其简单快速的DNA克隆/组装方法。该方法有助于常规DNA克隆和DNA片段的合成。
