Definitions

Ligates

DNA ligase

In molecular biology, DNA ligase is a special type of ligase that can link together two DNA strands that have single-strand breaks (a break in both complementary strands of DNA). The alternative, a double-strand break, is fixed by a different type of DNA ligase 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).

Ligase mechanism

The mechanism of DNA ligase is to form two covalent phosphodiester bonds between 3' hydroxyl ends of one nucleotide with the 5' phosphate end of another. ATP is not required for the ligase reaction.

A pictorial example of how a ligase works (with sticky ends):

Ligase will also work with blunt ends, although higher enzyme concentrations and different reaction conditions are required.

Mammalian ligases

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 and other bacteria.

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