ArticlePloS one2019
Mutant T4 DNA polymerase for easy cloning and mutagenesis.
Article in PloS one, 2019. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 6 papers.
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Who cites it
6 citing papers in PubMed, 20 citations in OpenAlex.
- Comparative evaluation of noncanonical amino acids as site-specific NMR probes for the complex of E. coli SSB with single-stranded DNA without isotope labelling.The FEBS journal · 2026Article
- Efficient genome editing with chimeric oligonucleotide-directed editing.Nature communications · 2026Article
- Site-selective generation of lanthanoid binding sites on proteins using 4-fluoro-2,6-dicyanopyridine.Magnetic resonance (Gottingen, Germany) · 2022Article
- Localising individual atoms of tryptophan side chains in the metallo-Magnetic resonance (Gottingen, Germany) · 2022Article
- Phosphoserine for the generation of lanthanide-binding sites on proteins for paramagnetic nuclear magnetic resonance spectroscopy.Magnetic resonance (Gottingen, Germany) · 2021Article
- Altered conformational sampling along an evolutionary trajectory changes the catalytic activity of an enzyme.Nature communications · 2020Article
Corrections and comments
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Authors and funding
2 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
The advent of high-fidelity DNA polymerases that can be used to linearize and amplify whole plasmids by PCR opened the door to greatly simplified cloning and mutagenesis protocols. Commercially available kits work well, but often have been optimized using undisclosed or proprietory components. Here we show that a mutant T4 DNA polymerase (Y320A) with attenuated 3'-exonuclease activity is uniquely suited to generate single-stranded DNA overhangs of uniform length in a more easily controllable manner than the wild-type enzyme, and this can be used to increase the yields of colonies containing correctly modified plasmids in cloning and mutagenesis experiments, which is particularly useful when E. coli cells are of relatively low competency. Standard protocols using the mutant T4 DNA polymerase are provided for the sequence and ligation independent cloning (SLIC) method and a modified QuikChange method, where the mutant enzyme enhances the yield of correctly mutated plasmid and further suppresses parental plasmid during digestion with DpnI. Single-stranded DNA overhangs generated by the mutant T4 DNA polymerase facilitate subsequent plasmid circularization, annealing and ligation in E. coli.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.