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In molecular biology, DNA ligase is a particular type of ligase (EC 18.104.22.168) that can link together DNA strands that have double-strand breaks (a break in both complementary strands of DNA). The alternative, a single-strand break, is easily fixed by DNA polymerase using the complementary strand as a template but still requires DNA ligase to create the final phosphodiester bond to fully repair the DNA.
DNA ligase has applications in both DNA repair and DNA replication (see Mammalian ligases). In addition, DNA ligase has extensive use in molecular biology laboratories for Genetic recombination experiments (see Applications in molecular biology research).
Additional recommended knowledge
The mechanism of DNA ligase is to form covalent phosphodiester bonds between 3' hydroxyl ends of one nucleotide with the 5' phosphate end of another. ATP is required for the ligase reaction.
A pictorial example of how a ligase works (with sticky ends):
In mammals, there are four specific types of ligase.
Applications in molecular biology research
DNA ligases have become an indispensable tool in modern molecular biology research for generating recombinant DNA sequences. For example, DNA ligases are used with restriction enzymes to insert DNA fragments, often genes, into plasmids.
One vital, and often tricky, aspect to performing successful recombination experiments involving ligase is controlling the optimal temperature. Most experiments use T4 DNA Ligase (isolated from bacteriophage T4) which is most active at 25°C. However in order to perform successful ligations, the optimal enzyme temperature needs to be balanced with the melting temperature Tm (also the annealing temperature) of the DNA fragments being ligated.
If the ambient temperature exceeds Tm, homologous pairing of the sticky ends will not occur because the high temperature disrupts hydrogen bonding. The shorter the DNA fragments, the lower the Tm. Thus for sticky ends (overlaps) less than ten base pairs long, ligation experiments are performed at very low temperatures (~4-8°C) for a long period of time (often overnight).
The common commercially available DNA ligases were originally discovered in bacteriophage T4, E. coli or other bacteria.
|This article is licensed under the GNU Free Documentation License. It uses material from the Wikipedia article "DNA_ligase". A list of authors is available in Wikipedia.|