Investigation of the Effect of NdeI on DNA Fragments Destined for Ligation

Abstract

In molecular biology the ability to generate recombinant DNA is of great importance for studying gene function and gene expression. Of the numerous restriction endonucleases available for cutting DNA fragments, some show better ligation efficiency than others. For example, DNA cleavage using NdeI consistently ligates inefficiently compared to other common enzymes such as HindIII. The functional reason for this discrepancy was the focus of this investigation. During experimentation NdeI was found to be heat inactivated, furthermore, it was not reactivated upon incubation at non-denaturing temperatures. Therefore, it was hypothesized that NdeI may be complexing with the cleaved DNA and disrupting the DNA ligase interaction. This was investigated by generating fragments of similar size to NdeI cleaved fragments and comparing migration on an agarose gel. These experiments were plagued by inconsistent electric fields in the gel box, and a buffer induced salt effect. As a result, NdeI interaction with the DNA fragments could not be conclusively demonstrated. Other methods for structural determination must be experimented with in order to prove or disprove this hypothesis, such as end labeling techniques, X-ray crystallography or NMR spectroscopy.