ArticlePloS one2016
Rapid and Robust PCR-Based All-Recombinant Cloning Methodology.
Article in PloS one, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 7 papers.
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Who cites it
7 citing papers in PubMed, 11 citations in OpenAlex.
- Linking multipoint folding and stability with functional regulation in the mitochondrial transmembrane β-barrel Sam50.Nature communications · 2026Article
- Elucidating the role of mycobacteriophage D29-encoded Gp36 in DNA binding and phage gene expression regulation.Nucleic acids research · 2025Article
- A positive SelectionFrontiers in bioengineering and biotechnology · 2021Article
- Article
- Salvaging the Thermodynamic Destabilization of Interface Histidine in Transmembrane β-Barrels.Biochemistry · 2018Article
- The Goldilocks Approach: A Review of Employing Design of Experiments in Prokaryotic Recombinant Protein Production.Bioengineering (Basel, Switzerland) · 2018Review
- Article
Corrections and comments
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Authors and funding
3 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
We report here a PCR-based cloning methodology that requires no post-PCR modifications such as restriction digestion and phosphorylation of the amplified DNA. The advantage of the present method is that it yields only recombinant clones thus eliminating the need for screening. Two DNA amplification reactions by PCR are performed wherein the first reaction amplifies the gene of interest from a source template, and the second reaction fuses it with the designed expression vector fragments. These vector fragments carry the essential elements that are required for the fusion product selection. The entire process can be completed in less than 8 hours. Furthermore, ligation of the amplified DNA by a DNA ligase is not required before transformation, although the procedure yields more number of colonies upon transformation if ligation is carried out. As a proof-of-concept, we show the cloning and expression of GFP, adh, and rho genes. Using GFP production as an example, we further demonstrate that the E. coli T7 express strain can directly be used in our methodology for the protein expression immediately after PCR. The expressed protein is without or with 6xHistidine tag at either terminus, depending upon the chosen vector fragments. We believe that our method will find tremendous use in molecular and structural biology.
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Registered trials
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