JoVE Science Education Database. Basic Methods in Cellular and Molecular Biology. DNA Ligation Reactions. Journal of Visualized Experiments, Cambridge, MA, doi: 10.3791/5069 (2014).

Overview

Ligation can be defined as the act of joining, and in biology the term refers to an enzymatic reaction that joins two biomolecules with a covalent bond. This video describes the application of DNA ligation in molecular biology research.

Summary
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 introduction to DNA ligation. The basic principle of ligation is described as well as a step-by-step procedure for setting up a generalized ligation reaction. Critical aspects of ligation reactions are discussed, such as how the length of a sticky end overhang affects the reaction temperature and how the ratio of DNA insert to vector should be tailored to prevent self-ligation. Molecular tools that assist with ligations like the Klenow Fragment and shrimp alkaline phosphatase (SAP) are mentioned, and applications , such as proximity ligations and the addition of linkers to fragments for sequencing are also presented.

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