Abstract
Molecular cloning is one of the most fundamental technologies in molecular biology, and has been critical for driving biotechnological advances. In this study, we have developed a novel method for standardized molecular cloning. The cloning technique known as “Nimble Cloning” uses the restriction enzyme, SfiI, in combination with the T5 exonuclease, to linearize the vector and generate 3′-overhangs simultaneously. Both PCR products and plasmids can be used for the cloning reaction in the Nimble Cloning system. The cloning system is highly efficient, suitable for gene expression in both prokaryotic and eukaryotic expression systems, and enables the reuse of DNA fragments or plasmid entry clones. Nimble Cloning is applicable for the cloning of single or multiple fragments, as well as multi-site cloning. Due also to its simplicity and versatility, the cloning method has great potential for the modular assembly of DNA constructs.
Highlights
Molecular cloning, which is one of the most fundamental procedures available for modern molecular biology research, has been critical for driving biotechnological advances
We have developed a novel method for standardized molecular cloning known as Nimble Cloning
The DNA fragment in the entry clone or the PCR product flanked by the adapters can be cloned into the circular destination vector with the Nimble Mix during the Nimble Cloning reaction and the transformation of E coli
Summary
Molecular cloning, which is one of the most fundamental procedures available for modern molecular biology research, has been critical for driving biotechnological advances. To the best of our knowledge, of the many molecular cloning protocols that have been developed, the following are the main techniques currently used for routine cloning: restriction digestion- and ligation-based cloning (Cohen et al, 1973), Gateway cloning (Hartley et al, 2000), Gibson assembly (Gibson et al, 2009), and Golden Gate cloning (Engler et al, 2008). Each of these methods have specific limitations (Liang et al, 2017). Gateway cloning has been widely used in many experimental systems, the following are the four main disadvantages with this method: (i) the associated two-step (BP and LR recombination reactions) cloning process is labor-intensive and time-consuming;
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