Enzymatic Ligation
Enzymatic Ligation - Related Videos
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DNA Ligation Reactions
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In molecular biology, ligation refers to the joining of two DNA fragments through the formation of a phosphodiester bond. An enzyme known as a ligase catalyzes the ligation reaction. In the cell, ligases repair single and double strand breaks that occur during DNA replication. In the laboratory, DNA ligase is used during molecular cloning to join DNA fragments of inserts with vectors – carrier DNA molecules that will replicate target fragments in host organisms. This video provides an...
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Use of Enzymatic Biosensors to Quantify Endogenous ATP or H2O2 in the Kidney
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Enzymatic microelectrode biosensors enable real-time measurements of extracellular cell signaling in biologically-relevant concentrations. The following protocols extend the applications of biosensors to the ex vivo and in vivo detection of ATP and H2O2 in the kidney.
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Cecal Ligation Puncture Procedure
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The mouse model of cecal ligation and puncture as a valuable tool for the study of human sepsis.
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Light-driven Enzymatic Decarboxylation
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We describe a protocol for the light-catalyzed generation of hydrogen peroxide — a cofactor for oxidative transformations.
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Modified Technique for Coronary Artery Ligation in Mice
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The surgical procedure used to induce experimental myocardial infarction in mice begins with left thoracotomy between the third and the fourth ribs in order to visualize the anterior surface of the heart and left lung. The left coronary artery is ligated, the chest is closed and the mouse is allowed to recover spontaneously.
FAQs
Ligases first activate one substrate, commonly with ATP or another energy-rich cofactor. This activation supplies the energetic input needed to transfer the activated substrate to a second molecule and form a new covalent bond. The mechanism links energy use to bond formation, allowing biological polymers to be joined under the mild conditions associated with biochemical systems.
Substrate specificity allows a ligase to recognize particular molecular partners and join them selectively rather than promoting indiscriminate bond formation. This selectivity is especially valuable when assembling or modifying biological polymers, because the intended products can be produced with fewer unwanted connections. In biotechnology, specificity supports controlled DNA assembly and library construction.
DNA ligase seals adjacent DNA ends by forming a phosphodiester bond within the sugar-phosphate backbone. By restoring continuity between neighboring ends, the reaction supports DNA repair and replication. The same chemical capability also underlies laboratory strategies in which DNA fragments are joined to create designed constructs for molecular cloning or other assembly workflows.
Enzymatic ligation is valuable because it combines high molecular specificity with mild reaction conditions. These features help preserve the biological context of the molecules being manipulated while directing bond formation toward selected substrates. As a result, ligases are useful not only for studying natural processes such as DNA maintenance but also for practical biotechnology and synthetic biology.
A general DNA-ligation workflow brings together the DNA ends or molecules intended for joining with an appropriate ligase and the reaction’s energy source, such as ATP when required. The enzyme activates one substrate and transfers it to the second, producing a covalent connection. The resulting joined DNA can support cloning, assembly, or library construction.
Researchers choose this approach when they need to join DNA molecules in a controlled and biologically compatible manner. DNA ligase can seal adjacent ends by restoring the backbone’s phosphodiester linkage, making it useful for constructing recombinant DNA arrangements. Its specificity and mild conditions help support molecular cloning without changing the broader role of the DNA molecules involved.
In library construction, enzymatic ligation provides a way to connect DNA components into a collection of assembled molecules. The enzyme’s substrate selectivity helps direct joining events, while cofactor-driven activation supplies the energy for covalent bond formation. This combination makes ligation relevant when many DNA constructs must be assembled as part of a broader biotechnology workflow.