DNA Ligase,Recombinant DNA,DNA Fragments,E coli Ligase,Molecular Biology,Ligase Reaction

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	DNA Ligase - Recombinant DNA experiments require the joining of two different DNA segments or fragments in vitro. The cohesive ends generated by some RE will anneal (join) themselves by forming hydrogen bonds. But the segments annealed thus are weak and do not withstand experimental conditions. To get a stable joining, the DNA should be joined by using an enzyme called ligase. DNA ligase joins the DNA molecule covalently by catalysing the formation of phosphodiester bonds between adjacent nucleotides.
DNA ligase isolated from E. coli and. T 4 bacteriophage is widely used in molecular biology experiments. These ligases more or less catalyse the reaction in the same way, and differ only in requirements of cofactor. T4 ligase requires ATP as cofactor and E coli ligase requires NADP as cofactor. The cofactor is first split (A TP à AMP + 2Pi) and then AMP binds to the enzyme to form the enzyme-AMP complex. This complex then binds to the nick or break (with 5' -PO4 and 3' -OH) and makes a covalent bond in the phosphodiester chain. The ligase reaction is carried out at 4°C for better results.