Biotechnology Index Glossary
The term recombinant DNA encapsulates the concept of recombining fragments of DNA from different sources into a new, and hopefully useful DNA molecule. Joining linear DNA fragments together with covalent bonds is called ligation. More specifically, DNA ligation involves creating a phosphodiester bond between the 3' hydroxyl of one nucleotide and the 5' phosphate of another.
The enzyme used to ligate DNA fragments is T4 DNA ligase, which originates from the T4 bacteriophage. This enzyme will ligate DNA fragments having overhanging, cohesive ends that are annealed together, as in the EcoRI example below - this is equivalent to repairing "nicks" in duplex DNA. T4 DNA ligase will also ligate fragments with blunt ends, although higher concentrations of the enzyme are usually recommended for this purpose.
A ligation reaction requires three ingredients in addition to water:
- Two or more fragments of DNA that have either blunt or compatible cohesive ("sticky") ends.
- A buffer which contains ATP. The buffer is usually provided or prepared as a 10X concentrate which, after dilution, yields an ATP concentration of roughly 0.25 to 1 mM. Most restriction enzyme buffers will work if supplemented with ATP.
- T4 DNA ligase. A typical reaction for inserting a fragment into a plasmid vector (subcloning) would utilize about 0.01 (sticky ends) to 1 (blunt ends) units of ligase.
The optimal incubation temperature for T4 DNA ligase is 16C and when very high efficiency ligation is desired (e.g. making libraries) this temperature is recommended. However, ligase is active at a broad range of temperatures, and for routine purposes such as subcloning, convenience often dictates incubation time and temperature - ligations performed at 4C overnight or at room temperature for 30 minutes to a couple of hours usually work well.
The figure to the right depicts the effects of T4 DNA ligase. DNA fragments generated by digestion of a plasmid with two restriction enzymes were incubated with different amounts of ligase for varying periods of time, then electrophoresed in 1% agarose. Note that even after 5 minutes with ligase, essentially all the fragments have been ligated to one another, and shifted to higher molecular weights. Click on the image for details. This simple test is sometimes useful to check a tube of ligase suspected of being "dead".
Back to the index of Biology and Activity of Restriction Endonucleases
Last updated on October 20, 1999
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