Investigation of the Ligation Efficiency of Digested λ Fragments of Different Sizes and Compatibility of Ends

Abstract

T4 Ligase is an enzyme derived from the T4 bacteriophage. Its remarkable ability to catalyze a ligation reaction by covalently joining two DNA molecules has allowed it to become a very useful tool in recombinant DNA technology. A previous study has indicated that not all! DNA fragments generated from a restriction digest ligate at the same efficiency. This finding led to an investigation to determine the effects of fragment size and fragment end compatibility on the ligation efficiency of digested! DNA fragments. In this study, Hind III, Nco I/ApaL I, and Acc I digests of! DNA were prepared to generate different DNA fragments for a time-course ligation experiment. Regardless of the enzymes used, it was found that larger fragments preferentially ligate over smaller fragments. Also, fragments with incompatible ends were used up more rapidly to form ligation products than those with compatible restriction ends. These results suggest that while size and end compatibility of fragments have some control over preferences for ligation, the structural characteristics and sequence variations of fragment ends may have a more profound effect on ligation efficiency.