ArticlePloS one2013
A practical comparison of ligation-independent cloning techniques.
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 45 papers.
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Who cites it
45 citing papers in PubMed, 86 citations in OpenAlex.
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- Restriction-ligation-independent production of a TVCV infectious clone and a TVCV-based gene expression vector.Heliyon · 2023Article
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- RyhB Paralogs Downregulate the Expressions of Multiple Survival-Associated Genes and Attenuate the Survival ofMicroorganisms · 2023Article
- A Sequence- and Ligation-Independent Cloning (SLIC) Procedure for the Insertion of Genes into a Plasmid Vector.Methods in molecular biology (Clifton, N.J.) · 2023Article
- The Non Catalytic Protein ERG28 has a Functional Role in Cholesterol Synthesis and is Coregulated Transcriptionally.Journal of lipid research · 2022Article
- Lambda-PCR for precise DNA assembly and modification.Biotechnology and bioengineering · 2022Article
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Corrections and comments
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Authors and funding
4 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
The precise assembly of specific DNA sequences is a critical technique in molecular biology. Traditional cloning techniques use restriction enzymes and ligation of DNA in vitro, which can be hampered by a lack of appropriate restriction-sites and inefficient enzymatic steps. A number of ligation-independent cloning techniques have been developed, including polymerase incomplete primer extension (PIPE) cloning, sequence and ligation-independent cloning (SLIC), and overlap extension cloning (OEC). These strategies rely on the generation of complementary overhangs by DNA polymerase, without requiring specific restriction sites or ligation, and achieve high efficiencies in a fraction of the time at low cost. Here, we outline and optimise these techniques and identify important factors to guide cloning project design, including avoiding PCR artefacts such as primer-dimers and vector plasmid background. Experiments made use of a common reporter vector and a set of modular primers to clone DNA fragments of increasing size. Overall, PIPE achieved cloning efficiencies of ∼95% with few manipulations, whereas SLIC provided a much higher number of transformants, but required additional steps. Our data suggest that for small inserts (<1.5 kb), OEC is a good option, requiring only two new primers, but performs poorly for larger inserts. These ligation-independent cloning approaches constitute an essential part of the researcher's molecular-tool kit.
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