DNA ligase is one of the most important, but least celebrated, enzymes used in modern biotechnology. This enzyme plays a very important role in DNA replication and repair by acting like a “glue” that can connect different DNA fragments to one another. Scientists take advantage of this molecular glue to combine DNA fragments to make an entirely new sequence. However, many people are merely using DNA ligase as a tool without knowing the story and mechanism behind it.
DNA ligase was first identified in E.coli in 1967. The ligase enzyme uses the energy from the hydrolysis of NAD to catalyze the formation of a phosphodiester bond between the 5’ phosphate (-PO4) of one DNA strand and the 3’hydroxyl (-OH) from the other strand. In short, this enzyme forms covalent bonds that permanently link two DNA strands. The DNA ligase from E.coli can only bond two DNA strands that are complementary (sticky) to each other, which means it cannot bring two blunt ends together. This severely limits the utility of E.coli DNA ligase in biotechnology experiments.
Fortunately, other types of DNA ligase are more flexible. The most commonly used DNA ligase in biotechnology experiments was isolated from bacteriophage T4 – thus it has become known as T4 DNA ligase. Unlike the enzyme found in E.coli, T4 DNA ligase uses energy from ATP. Moreover, T4 DNA ligase can ligate both sticky and blunt ends, and can function on DNA, RNA, or DNA/RNA hybrids.
Besides the DNA ligases found in prokaryotes and viruses, many kinds of DNA ligases have been identified in eukaryotes. In mammals, there are 4 kinds of DNA ligases that participate in important cellular processes. DNA ligase I works during DNA replication by stitching together the Okazaki fragments of the lagging strand. DNA ligase II is derived from DNA ligase III after a proteolytic process. DNA ligase III and DNA ligase IV work in eukaryotic DNA repair pathways. While DNA ligase III can repair DNA nicks during the base excision repair process, DNA ligase IV is required in the repair of double-strand breaks. Importantly, the activity of DNA ligase IV generates the variety of immunoglobulins and T-cell receptors during immune system development. As you can see, there is a specialized DNA ligase for every task within a cell.
DNA ligation is performed at temperatures between 4°C to 22°C, although the reaction reaches its highest efficiency at 16°C. The ligation should be allowed to process for 2 to 16 hrs. Because of its versatility, scientists prefer T4 DNA ligase for molecular cloning and DNA repair experiments. In a ligation reaction, you will need a reaction buffer (which contains the ATP), T4 ligase enzyme, and a mixture of the DNA fragments to be ligated. These components are combined and mixed in very small volumes, usually 20 µl or less. Edvotek ligation kits include a convenient freeze-dried T4 DNA ligase that arrives pre-mixed with the buffer and ATP. Students simply need to add TE or distilled water to reconstitute the enzyme before adding their DNA fragments.
Are you interested in exploring cloning techniques using ligation in your classroom? Check out our Kit #300 Blue/White Cloning of a DNA Fragment & Assay of ß-galactosidase or Kit #301 Construction and Cloning of a DNA Recombinantfor exciting experiments using T4 DNA ligase.