ArticleScientific reports2018
Efficient strategy for introducing large and multiple changes in plasmid DNA.
Article in Scientific reports, 2018. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 16 papers.
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Who cites it
16 citing papers in PubMed.
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- The N-Terminal Region of Cucumber Mosaic Virus 2a Protein Is Involved in the Systemic Infection inPlants (Basel, Switzerland) · 2024Article
- Protein thermal sensing regulates physiological amyloid aggregation.Nature communications · 2024Article
- In silico and in vitro models reveal the molecular mechanisms of hypocontractility caused byFrontiers in physiology · 2024Article
- Accelerated Long-Fragment Circular PCR for Genetic Manipulation of Plant Viruses in Unveiling Functional Genomics.Viruses · 2023Article
- An efficient and cost-effective method for directed mutagenesis at multiple dispersed sites-a case study with Omicron Spike DNA.Biology methods & protocols · 2023Article
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- OverFlap PCR: A reliable approach for generating plasmid DNA libraries containing random sequences without a template bias.PloS one · 2022Article
- A two-step PCR assembly for construction of gene variants across large mutational distances.Biology methods & protocols · 2021Article
- Synthesis of libraries and multi-site mutagenesis using a PCR-derived, dU-containing template.Synthetic biology (Oxford, England) · 2021Article
- A blueprint for academic laboratories to produce SARS-CoV-2 quantitative RT-PCR test kits.The Journal of biological chemistry · 2020Article
- A blueprint for academic labs to produce SARS-CoV-2 RT-qPCR test kits.medRxiv : the preprint server for health sciences · 2020Article
- Differentially Marked IncP-1β R751 Plasmids for Cloning via Recombineering and Conjugation.Polish journal of microbiology · 2019Article
- Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins.Journal of visualized experiments : JoVE · 2019Article
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8 authors.
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No grant is acknowledged in the PubMed record.
Abstract
While the QuikChange site-directed mutagenesis method and its later modifications are extremely useful and simple, they suffer from several drawbacks. Here, we propose a new method, named LFEAP mutagenesis (Ligation of Fragment Ends After PCR) for creating various mutations in plasmid by leveraging three existing concepts: inverse PCR, single primer PCR, and sticky-end assembly. The first inverse PCR on the target plasmid yielded linearized DNA fragments with mutagenic ends, and a second single primer PCR resulted in complementary single-stranded DNA fragments with the addition of overhangs at the 5' end of each strand. The resulting single strands were then annealed to produce double-stranded DNA with free 5' single-stranded DNA tails. These products with compatible sticky ends were efficiently assembled into a circular, mutagenized plasmid. With this strategy, multiple simultaneous changes (up to 15) and mutations in large plasmids (up to 50 kb) were achieved with high efficiency and fidelity. LFEAP mutagenesis is a versatile method that offers significant advantages for introducing large and multiple changes in plasmid DNA.
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