ArticleScientific reports2021
A high-efficiency method for site-directed mutagenesis of large plasmids based on large DNA fragment amplification and recombinational ligation.
Article in Scientific reports, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 13 papers.
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13 citing papers in PubMed.
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- NAD+ capping of sibD transcripts in E. coli is mediated by its minimal promoter and enhanced by ppGpp.Nucleic acids research · 2026Article
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- Interplay of YEATS2 and GCDH regulates histone crotonylation and drives EMT in head and neck cancer.eLife · 2025Article
- Brief guide to gene cloning.Molecules and cells · 2025Review
- P3a site-specific and cassette mutagenesis for seamless protein, RNA and plasmid engineering.Genes & cancer · 2025Article
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- The disruption of NEAT1-miR-125b-5p-SLC1A5 cascade defines the oncogenicity and differential immune profile in head and neck squamous cell carcinoma.Cell death discovery · 2024Article
- Improving the enzymatic activity and stability of N-carbamoyl hydrolase using deep learning approach.Microbial cell factories · 2024Article
- Site-Directed Mutagenesis Protocol to Determine the Role of Amino Acid Residues in Polycomb Group (PcG) Protein Function.Methods in molecular biology (Clifton, N.J.) · 2023Article
- Highly efficient and simple SSPER and rrPCR approaches for the accurate site-directed mutagenesis of large and small plasmids.New biotechnology · 2022Article
- A strategy for in-house production of a positive selection cloning vector from the commercial pJET1.2/blunt cloning vector at minimal cost.3 Biotech · 2022Article
- A simplified Gibson assembly method for site directed mutagenesis by re-use of standard, and entirely complementary, mutagenesis primers.BMC biotechnology · 2022Article
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12 authors.
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Abstract
Site-directed mutagenesis for large plasmids is a difficult task that cannot easily be solved by the conventional methods used in many laboratories. In this study, we developed an effective method for Site-directed Mutagenesis for Large Plasmids (SMLP) based on a PCR technique. The SMLP method combines several effective approaches, including a high-efficiency DNA polymerase for the large DNA amplification, two independent PCR reactions and a fast recombinational ligation. Using this method, we have achieved a variety of mutants for the filamin A gene (7.9 kb) cloned in the pcDNA (5.4 kb) or the pLV-U6-CMV-EGFP (9.4 kb) plasmids, indicating that this method can be applied to site-directed mutagenesis for the plasmids up to 17.3 kb. We show that the SMLP method has a greater advantage than the conventional methods tested in this study, and this method can be applied to substitution, deletion, and insertion mutations for both large and small plasmids as well as the assembly of three fragments from PCR reactions. Altogether, the SMLP method is simple, effective, and beneficial to the laboratories that require completing the mutagenesis of large plasmids.
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