Stephen Parziale

This project was in conjunction with my undergraduate research at Emmanuel College. It consisted of conducting research in the Department of Biological Chemistry and Molecular Pharmacology, using gel technique to investigate the effects of fluorescently tagged DNA in gel digestion.

I use a variety of methods to study mechanisms of DNA cleavage using restriction endonucleases as a model system. In a single molecule technique, micron sized beads are tethered with single DNAs in a microfluidic flow cell and imaged using video microscopy. We have used this assay to study facilitated diffusion as well as the role of divalent cations. My team has characterized the salt dependence of both NdeI and EcoRI and found that both enzymes exhibit a peak in cleavage rates near 80 mM, an observation consistent with the theory of facilitated diffusion. We are currently studying how DNA length affects the reaction rates and are studying both 1000 bp DNAs and 200 bp DNAs. We are comparing our single molecule technique to ensemble assays which use quantitative analysis of gel electrophoresis. Preliminary data shows cleavage to be much slower in the gel assay as compared to the single molecule data, suggesting the two assays are limited by different steps. Our efforts are focused on improving this ensemble assay. We are also developing single-molecule fluorescence methods to observe the diffusion of EcoRI on DNA. In our technique, lambda phage DNA which has been linearized and biotinylated on both ends is extended and tethered to a surface. Using HiLo imaging, we have observed the diffusion and binding of catalytically inactive mutant EcoRI labeled with a quantum dot. We hope to use this method to directly observe DNA sliding by EcoRI. Our work is currently focused on optimizing our method used to track enzyme search activity.