This study introduces a novel cost-effective technique for cloning of linear DNA plasmid inserts, aiming to address the associated expenses linked with popular in vitro DNA assembly methods. Specifically, we introduce ECOLI (Efficient Cloning Of Linear Inserts), a method utilizing a PCR product-based site-directed mutagenesis. In comparison to other established in vitro DNA assembly methods, our approach is without the need for costly synthesis or specialized kits for recombination or restriction sites. ECOLI offers a fast, efficient, and economical alternative for cloning inserts up to several hundred nucleotides into plasmid constructs, thus enhancing cloning accessibility and efficiency. This method can enhance molecular biology research, as we briefly demonstrated on the Dishevelled gene from the WNT signaling pathway.

Department of Experimental Biology Faculty of Science Masaryk University 625 00 Brno Czech Republic

Zobrazit více v PubMed

Cohen, S. N. DNA cloning: A personal view after 40 years. Proc. Natl. Acad. Sci. USA110(39), 15521–15529 (2013). 10.1073/pnas.1313397110 PubMed DOI PMC

McKinnell, R. G. & Di Berardino, M. A. The biology of cloning: History and rationale. BioScience49(11), 875–885 (1999).10.2307/1313647 DOI

Alberts, B. et al. Isolating, cloning, and sequencing DNA. In Molecular Biology of the Cell 4th edn (Garland Science, 2002).

Chong, Z. X., Yeap, S. K. & Ho, W. Y. Transfection types, methods and strategies: A technical review. PeerJ9, e11165 (2021). 10.7717/peerj.11165 PubMed DOI PMC

Kim, T. K. & Eberwine, J. H. Mammalian cell transfection: The present and the future. Anal. Bioanal. Chem.397(8), 3173–3178 (2010). 10.1007/s00216-010-3821-6 PubMed DOI PMC

Stepanenko, A. A. & Heng, H. H. Transient and stable vector transfection: Pitfalls, off-target effects, artifacts. Mutat. Res. Rev. Mutat. Res.773, 91–103 (2017). 10.1016/j.mrrev.2017.05.002 PubMed DOI

Kwon, H. et al. Emergence of synthetic mRNA: In vitro synthesis of mRNA and its applications in regenerative medicine. Biomaterials156, 172–193 (2018). 10.1016/j.biomaterials.2017.11.034 PubMed DOI

Sive, H. L., Grainger, R. M. & Harland, R. M. Microinjection of Xenopus oocytes. Cold Spring Harb. Protoc.2010(12), 5536 (2010).10.1101/pdb.prot5536 PubMed DOI

Lino, C. A. et al. Delivering CRISPR: A review of the challenges and approaches. Drug Deliv.25(1), 1234–1257 (2018). 10.1080/10717544.2018.1474964 PubMed DOI PMC

Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science337(6096), 816–821 (2012). 10.1126/science.1225829 PubMed DOI PMC

Fu, C. et al. Hot Fusion: An efficient method to clone multiple DNA fragments as well as inverted repeats without ligase. PLoS ONE9(12), e115318 (2014). 10.1371/journal.pone.0115318 PubMed DOI PMC

Wang, W., Zheng, G. & Lu, Y. Recent advances in strategies for the cloning of natural product biosynthetic gene clusters. Front. Bioeng. Biotechnol.9, 692797 (2021). 10.3389/fbioe.2021.692797 PubMed DOI PMC

Sharma, M. et al. Dishevelled: A masterful conductor of complex Wnt signals. Cell Signal.47, 52–64 (2018). 10.1016/j.cellsig.2018.03.004 PubMed DOI PMC

Gao, C. & Chen, Y. G. Dishevelled: The hub of Wnt signaling. Cell Signal.22(5), 717–727 (2010). 10.1016/j.cellsig.2009.11.021 PubMed DOI

Mlodzik, M. The dishevelled protein family: Still rather a mystery after over 20 years of molecular studies. Curr. Top. Dev. Biol.117, 75-91. 10.1016/bs.ctdb.2015.11.027 (2016). 10.1016/bs.ctdb.2015.11.027 PubMed DOI PMC

Paclikova, P. et al. Roles of individual human Dishevelled paralogs in the Wnt signalling pathways. Cell Signal.85, 110058 (2021). 10.1016/j.cellsig.2021.110058 PubMed DOI

Strakova, K. et al. Dishevelled enables casein kinase 1-mediated phosphorylation of Frizzled 6 required for cell membrane localization. J. Biol. Chem.293(48), 18477–18493 (2018). 10.1074/jbc.RA118.004656 PubMed DOI PMC

Bernatik, O. et al. Functional analysis of dishevelled-3 phosphorylation identifies distinct mechanisms driven by casein kinase 1ϵ and frizzled5. J. Biol. Chem.289(34), 23520–23533 (2014). 10.1074/jbc.M114.590638 PubMed DOI PMC

Korinek, V. et al. Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC-/- colon carcinoma. Science275(5307), 1784–1787 (1997). 10.1126/science.275.5307.1784 PubMed DOI

Quan, J. & Tian, J. Circular polymerase extension cloning for high-throughput cloning of complex and combinatorial DNA libraries. Nat. Protoc.6(2), 242–251 (2011). 10.1038/nprot.2010.181 PubMed DOI

Obradovic, J. et al. Optimization of PCR conditions for amplification of GC-Rich EGFR promoter sequence. J. Clin. Lab. Anal.27(6), 487–493 (2013). 10.1002/jcla.21632 PubMed DOI PMC

Bebenek, A. & Ziuzia-Graczyk, I. Fidelity of DNA replication-a matter of proofreading. Curr. Genet.64(5), 985–996 (2018). 10.1007/s00294-018-0820-1 PubMed DOI PMC