ArticleAnalytical biochemistry2013
A plasmid-based lacZα gene assay for DNA polymerase fidelity measurement.
Article in Analytical biochemistry, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 15 papers.
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Who cites it
15 citing papers in PubMed, 20 citations in OpenAlex.
- The A, B, C, D's of replicative DNA polymerase fidelity: utilizing high-throughput single-molecule sequencing to understand the molecular basis for DNA polymerase accuracy.Nucleic acids research · 2025Article
- Precise and Accurate DNA-3'/5-Ends Polishing withInternational journal of molecular sciences · 2024Article
- Human DNA polymerase ε is a source of C>T mutations at CpG dinucleotides.Nature genetics · 2024Article
- Chimeric Phi29 DNA polymerase with helix-hairpin-helix motifs shows enhanced salt tolerance and replication performance.Microbial biotechnology · 2021Article
- Construction of a highly error-prone DNA polymerase for developing organelle mutation systems.Nucleic acids research · 2020Article
- Human DNA polymerase delta requires an iron-sulfur cluster for high-fidelity DNA synthesis.Life science alliance · 2019Article
- High-resolution mapping of DNA polymerase fidelity using nucleotide imbalances and next-generation sequencing.Nucleic acids research · 2018Article
- Identification and characterization of a heterotrimeric archaeal DNA polymerase holoenzyme.Nature communications · 2017Article
- Archaeoglobus Fulgidus DNA Polymerase D: A Zinc-Binding Protein Inhibited by Hypoxanthine and Uracil.Journal of molecular biology · 2016Article
- Alternative divalent cations (Zn²⁺, Co²⁺, and Mn²⁺) are not mutagenic at conditions optimal for HIV-1 reverse transcriptase activity.BMC biochemistry · 2015Article
- Human PrimPol is a highly error-prone polymerase regulated by single-stranded DNA binding proteins.Nucleic acids research · 2015Article
- An archaeal family-B DNA polymerase variant able to replicate past DNA damage: occurrence of replicative and translesion synthesis polymerases within the B family.Nucleic acids research · 2014Article
- Human immunodeficiency virus reverse transcriptase displays dramatically higher fidelity under physiological magnesium conditions in vitro.Journal of virology · 2014Article
- DNA polymerase hybrids derived from the family-B enzymes of Pyrococcus furiosus and Thermococcus kodakarensis: improving performance in the polymerase chain reaction.Frontiers in microbiology · 2014Article
- Unwinding of primer-templates by archaeal family-B DNA polymerases in response to template-strand uracil.Nucleic acids research · 2013Article
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Authors and funding
3 authors at 2 institutions in 1 country.
Funding
Abstract
A significantly improved DNA polymerase fidelity assay, based on a gapped plasmid containing the lacZα reporter gene in a single-stranded region, is described. Nicking at two sites flanking lacZα, and removing the excised strand by thermocycling in the presence of complementary competitor DNA, is used to generate the gap. Simple methods are presented for preparing the single-stranded competitor. The gapped plasmid can be purified, in high amounts and in a very pure state, using benzoylated-naphthoylated DEAE-cellulose, resulting in a low background mutation frequency (~1 × 10(-4)). Two key parameters, the number of detectable sites and the expression frequency, necessary for measuring polymerase error rates have been determined. DNA polymerase fidelity is measured by gap filling in vitro, followed by transformation into Escherichia coli and scoring of blue/white colonies and converting the ratio to error rate. Several DNA polymerases have been used to fully validate this straightforward and highly sensitive system.
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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.