ArticleNucleic acids research2018
A single-molecule sequencing assay for the comprehensive profiling of T4 DNA ligase fidelity and bias during DNA end-joining.
Article in Nucleic acids research, 2018. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 23 papers.
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23 citing papers in PubMed, 51 citations in OpenAlex.
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- Nucleic acid joining enzymes: biological functions and synthetic applications beyond DNA.The Biochemical journal · 2025Review
- Golden Gate Cloning of Expression Plasmids for Synthetic Small RNAs in Bacteria.Methods in molecular biology (Clifton, N.J.) · 2025Article
- Advancements in Golden Gate Cloning: A Comprehensive Review.Methods in molecular biology (Clifton, N.J.) · 2025Review
- Selection of Fusion-Site Overhang Sets for High-Fidelity and High-Complexity Golden Gate Assembly.Methods in molecular biology (Clifton, N.J.) · 2025Article
- Use of a Golden Gate Plasmid Set Enabling Scarless MoClo-Compatible Transcription Unit Assembly.Methods in molecular biology (Clifton, N.J.) · 2025Article
- GGAssembler: Precise and economical design and synthesis of combinatorial mutation libraries.Protein science : a publication of the Protein Society · 2024Article
- POMBOX: A Fission Yeast Cloning Toolkit for Molecular and Synthetic Biology.ACS synthetic biology · 2024Article
- One-pot Golden Gate Assembly of an avian infectious bronchitis virus reverse genetics system.PloS one · 2024Article
- Article
- Advances in ligase-based nucleic acid amplification technology for detecting gene mutations: a review.Molecular and cellular biochemistry · 2023Review
- DIGGER-Bac: prediction of seed regions for high-fidelity construction of synthetic small RNAs in bacteria.Bioinformatics (Oxford, England) · 2023Article
- Profiling DNA Ligase Substrate Specificity with a Pacific Biosciences Single-Molecule Real-Time Sequencing Assay.Current protocols · 2023Article
- Human adipose tissue as a major reservoir of cytomegalovirus-reactive T cells.Frontiers in immunology · 2023Article
- An Easy-to-Use Plasmid Toolset for Efficient Generation and Benchmarking of Synthetic Small RNAs in Bacteria.ACS synthetic biology · 2022Article
- Mismatch discrimination and sequence bias during end-joining by DNA ligases.Nucleic acids research · 2022Article
- PacBio sequencing output increased through uniform and directional fivefold concatenation.Scientific reports · 2021Article
- Rapid and Efficient Synthetic Assembly of Multiplex Luciferase Reporter Plasmids for the Simultaneous Monitoring of Up to Six Cellular Signaling Pathways.Current protocols in molecular biology · 2020Article
Corrections and comments
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Authors and funding
9 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
DNA ligases are key enzymes in molecular and synthetic biology that catalyze the joining of breaks in duplex DNA and the end-joining of DNA fragments. Ligation fidelity (discrimination against the ligation of substrates containing mismatched base pairs) and bias (preferential ligation of particular sequences over others) have been well-studied in the context of nick ligation. However, almost no data exist for fidelity and bias in end-joining ligation contexts. In this study, we applied Pacific Biosciences Single-Molecule Real-Time sequencing technology to directly sequence the products of a highly multiplexed ligation reaction. This method has been used to profile the ligation of all three-base 5'-overhangs by T4 DNA ligase under typical ligation conditions in a single experiment. We report the relative frequency of all ligation products with or without mismatches, the position-dependent frequency of each mismatch, and the surprising observation that 5'-TNA overhangs ligate extremely inefficiently compared to all other Watson-Crick pairings. The method can easily be extended to profile other ligases, end-types (e.g. blunt ends and overhangs of different lengths), and the effect of adjacent sequence on the ligation results. Further, the method has the potential to provide new insights into the thermodynamics of annealing and the kinetics of end-joining reactions.
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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.