1Department of Molecular and Environmental Biotechnology, Faculty of Biology and Biotechnology, University of Science, VNU-HCM
2Department Molecular and Environmental Biotechnology, Faculty of Biology and Biotechnology, University of Science, Ho Chi Minh City, Vietnam
3Vietnam National University, Ho Chi Minh City, Vietnam
4Department of Molecular and Environmental Biotechnology, Faculty of Biology and Biotechnology, University of Science, VNU-HCM
چکیده
Recombinant DNA technology has been playing the key role for a long time since its first beginning. DNA ligases have certainly contributed to the development of cloning techniques, as well as molecular study up to now. Despite being a prime cloning tool, DNA ligases still face some shortcomings which lead to their limit of use. Our study provided an improved method that simplified the basic restriction enzyme-based cloning (REC) by eliminating the ligation role, named recombinase-free cloning (RFC).This improved technique was designed with only one PCR reaction, one digestion reaction, and one temperature profile, which takes advantage of endogenous recombinase in E. coli host to create the target recombinant vector inside the cell. All purification steps were eliminated for effectively material- and time-saving. Five different clones were generated by RFC. This method showed relatively low efficiency yet successful at a range of 100% in every conducted trial with fragment sizes from 0.5-1.0 kbp. The RFC method could be completed within a day (about 9 hours), without the need of ligase or recombinase or purification steps, which significantly saved DNA components, materials as well as the time required. In conclusion, we expected to provide a more convenient cloning method, as well as enable faster generation of DNA clones, which would be well applied in the less equipped laboratories.
Vo-Nguyen H-V, Nguyen TT, Thi Vu HT, Thi Nguyen TT, Hoang QC, Tran TL. Recombinant human SCARB2 expressed in Escherichia coli and its potential in enterovirus 71 blockage. Iran J Sci Technol Trans A 2021;45:455-461.
March JB, J Clark. Enzymes by post—restriction enzyme stability. Nat Biotech 2000;18: 243.
Youngblom J. Extended stability of Taq DNA polymerase and T4 DNA ligase at various temperatures. BioTechniques 2003;34:264-268.
Li WY, Changjun L, Wu L, Wu JF, Yin XN, Deng KH, Zhang DY, Er M, MCT cloning: a seamless cloning strategy for inserting DNA fragments. Biotechnol Biotechnol Equipment 2018;32:1298-1305.
Aslanidis C, De Jong PJ. Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res 1990;18:6069-6074.
Van Den Ent F, Löwe J. RF cloning: a restriction-free method for inserting target genes into plasmids. J Biochmem Biophys Methods 2006;67:67-74.
Karimi M, D Inzé, A Depicker. GATEWAY vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 2002;7:193-195.
Zhu B, Cai G, Hall EO, Freeman GJ. In-Fusion™ assembly: seamless engineering of multidomain fusion proteins, modular vectors, and mutations. Biotechniques 2007;43:354-359.
Mathieu J, Alvarez E, Alvarez PJ. Recombination-assisted megaprimer (RAM) cloning. Methods X 2014;1:23-29.
Zhang Y, Werling U, Edelmann W. SLiCE: a novel bacterial cell extract-based DNA cloning method. Nucle Acids Res 2012;40:e55.
Motohashi K, A simple and efficient seamless DNA cloning method using SLiCE from Escherichia coli laboratory strains and its application to SLiP site-directed mutagenesis. BMC Biotech 2015;15:47.
Okegawa Y, Motohashi K. A simple and ultra-low cost homemade seamless ligation cloning extract (SLiCE) as an alternative to a commercially available seamless DNA cloning kit. Biochem Biophys Rep 2015;4:148-151.
Motohashi K. Evaluation of the efficiency and utility of recombinant enzyme-free seamless DNA cloning methods. Biochem Biophys Rep 2017;9:310-315.
Lovett ST, Hurley RL, Sutera VA, Aubuchon RH, Lebedeva MA. Crossing over between regions of limited homology in Escherichia coli: RecA-dependent and RecA-independent pathways. Genetics 2002;160:851-859.
Dutra BE, VA Sutera, ST Lovett. RecA-independent recombination is efficient but limited by exonucleases. Proc Natl Acad Sci USA 2007;104:216-221.
Jain K, Wood EA, Romero ZJ, Cox MM. RecA‐independent recombination: Dependence on the Escherichia coli RarA protein. Mol Microbiol 2021;115:1122-1137.
Jacobus AP, Gross J. Optimal cloning of PCR fragments by homologous recombination in Escherichia coli. PLoS One 2015;10:e0119221.
Wang Y, Liu Y, Chen J, Tang MJ, Zhang SL, Wei LN, Li CH, Wei DB. Restriction-ligation-free (RLF) cloning: a high-throughput cloning method by in vivo homologous recombination of PCR products. Genet Mol Res 2015;14:12306-12315.
Liang J, Liu Z, Low XZ, Ang EL, Zhao H. Twin-primer non-enzymatic DNA assembly: an efficient and accurate multi-part DNA assembly method. Nucleic acids research 2017; 45:e94.
Nguyen TT, Vo-Nguyen HV, Tran-Van H. Prokaryotic expression of chimeric GFP-hFc protein as a potential immune-based tool. Mol Biol Res Commun 2021;10:105-108.
Gellert M. Formation of covalent circles of lambda DNA by E. coli extracts. Proc Natl Acad Sci USA 1967;57:148-157.
