ArticlePloS one2013
DNA fragments assembly based on nicking enzyme system.
Article in PloS one, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 11 papers.
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Who cites it
11 citing papers in PubMed.
- Post-assembly Plasmid Amplification for Increased Transformation Yields inChem & bio engineering · 2025Article
- Efficient assembly of long DNA fragments and multiple genes with improved nickase-based cloning and Cre/loxP recombination.Plant biotechnology journal · 2022Article
- Construction of a cDNA expression library in a binary vector using a nicking enzyme.Plant biotechnology (Tokyo, Japan) · 2020Article
- Nicking Endonuclease-Mediated Vector Construction Strategies for Plant Gene Functional Research.Plants (Basel, Switzerland) · 2020Article
- Scarless assembly of unphosphorylated DNA fragments with a simplified DATEL method.Bioengineered · 2017Article
- BioBrick-based 'Quick Gene Assembly'Synthetic biology (Oxford, England) · 2017Article
- Synthetic biology of microbes synthesizing polyhydroxyalkanoates (PHA).Synthetic and systems biotechnology · 2016Review
- Recent advances in DNA assembly technologies.FEMS yeast research · 2015Review
- Cloning Should Be Simple: Escherichia coli DH5α-Mediated Assembly of Multiple DNA Fragments with Short End Homologies.PloS one · 2015Article
- Recombinant expressed vector pET32a (+) S constructed by ligation independent cloning.Molecules (Basel, Switzerland) · 2014Article
- Emerging tools for synthetic genome design.Molecules and cells · 2013Review
Corrections and comments
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Authors and funding
5 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
A couple of DNA ligation-independent cloning (LIC) methods have been reported to meet various requirements in metabolic engineering and synthetic biology. The principle of LIC is the assembly of multiple overlapping DNA fragments by single-stranded (ss) DNA overlaps annealing. Here we present a method to generate single-stranded DNA overlaps based on Nicking Endonucleases (NEases) for LIC, the method was termed NE-LIC. Factors related to cloning efficiency were optimized in this study. This NE-LIC allows generating 3'-end or 5'-end ss DNA overlaps of various lengths for fragments assembly. We demonstrated that the 10 bp/15 bp overlaps had the highest DNA fragments assembling efficiency, while 5 bp/10 bp overlaps showed the highest efficiency when T4 DNA ligase was added. Its advantage over Sequence and Ligation Independent Cloning (SLIC) and Uracil-Specific Excision Reagent (USER) was obvious. The mechanism can be applied to many other LIC strategies. Finally, the NEases based LIC (NE-LIC) was successfully applied to assemble a pathway of six gene fragments responsible for synthesizing microbial poly-3-hydroxybutyrate (PHB).
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