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DNA Ligation

Ligation of DNA is a critical step in many modern molecular biology workflows. The sealing of nicks between adjacent residues of a single-strand break on a double-strand substrate and the joining of double-strand breaks are enzymatically catalyzed by DNA ligases. The formation of a phosphodiester bond between the 3' hydroxyl and 5' phosphate of adjacent DNA residues proceeds in three steps: Initially, the ligase is self-adenylated by reaction with free ATP. Next, the adenyl group is transferred to the 5'-phosphorylated end of the "donor" strand. Lastly, the formation of the phosphodiester bond proceeds after reaction of the adenylated donor end with the adjacent 3' hydroxyl acceptor and the release of AMP. In living organisms, DNA ligases are essential enzymes with critical roles in DNA replication and repair. In the lab, DNA ligation is performed for both cloning and non-cloning applications.

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DNA Ligation includes these areas of focus:
Non-Cloning Ligation
Cloning Ligation
Protocols for DNA Ligation
Application Notes DNA Ligation
    Publications related to DNA Ligation
  1. Anton, B.P., Morgan, R.D., Ezraty, B., Manta, B., Barras, F., Berkmen, M. 2019. Complete genome sequence of Escherichia coli BE104, an MC4100 drivative lacking the methionine reductive pathway Microbiol Resour Announc. 8 (29), PubMedID: 31296691, DOI: 10.1128/MRA.00721-19
  2. Potapov, V., Ong, J.L., Kucera, R.B., Langhorst, B.W., Bilotti, K., Pryor, J.M., Cantor, E.J., Canton, B., Knight, T.F., Evans, T.C., Lohman, G.J.S. 2018. Comprehensive profiling of four base overhang ligation fidelity by T4 DNA ligase and application to DNA assembly ACS Synthetic Biology. 7 (11), PubMedID: 30335370, DOI: 10.1021/acssynbio.8b00333
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Videos

  1. DNA Ligase

    DNA Ligase Fidelity: When does it matter?

    High fidelity polymerases are everywhere—but why would you need a high fidelity ligase? And what do we even mean by “fidelity” when we’re talking about ligation? In this webinar, NEB Scientist and ligase expert Greg Lohman discusses mismatch ligation by DNA ligases and the molecular diagnostics applications that depend on the use of high-fidelity DNA ligases like NEB’s HiFi Taq DNA Ligase to detect single base differences in DNA.

  2. DNA Ligase

    DNA Ligation

    Ligation, the process of joining DNA fragments with a DNA ligase, proceeds in three steps. Learn more about the function of ligation with our quick tutorial animation.

  3. BestRatios_thumb

    What molar ratios should I use for DNA Ligation?

    The optimal reactant ratio is contingent upon the downstream application.

  4. WhyUsePEG_thumb

    Why do I need to add PEG to my DNA ligation?

    Polyethylene glycol (PEG) is an important reagent in ligation reactions, find out why.

  5. BestReactionTimes_thumb

    What are the best conditions for DNA ligation?

    Find out how the downstream application dictates the best reaction conditions for ligation.

  6. DifficultLigation_thumb

    Are some ligations more difficult than others?

    Ligation of blunt ends and single-base overhangs require optimized reaction conditions.

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