Immobilization of the restriction enzymes HaeIII and HindIII on porous silica particles via a glutaraldehyde linkage for the micro-digestion of dsDNAwith analysis by capillary electrophoresis

Solid-phase DNA restriction digest reactors have been developed consisting of silica particles modified with a covalently tethered restriction enzyme. This solid-phase restriction reactor enables digestion and separation of m inute quantities of DNA with minimal reagent consumption. In this study, th e restriction enzymes, HaeIII, Psd, and HindIII, were successfully immobili zed via glutaraldehyde linkages to porous silica micro-particles. Studies w ere carried out to examine the impact of immobilization on enzymatic activi ty. Digestions of phi X174-RF DNA phage and SV40 viral DNA were performed w ith the immobilized enzymes by placing the silica particles in solution wit h the target DNA with digestion times of 120 min and 240 min respectively. The digests were analyzed off-line using capillary electrophoresis (CE) wit h laser-induced fluorescence (LIF) detection. Timed studies were performed to establish optimal conditions for complete digestion. Digests utilizing i mmobilized HaeIII and HindIII were similar in composition to homogeneous, f ree solution digests. PstI showed no evidence of activity upon immobilizati on. The immobilized restriction enzymes could also be used for multiple rou nds of digestion; however, longer incubation times were required for succes sive runs probably due to partial denaturation of the restriction enzyme. D igests also were prepared and isolated by use of a simple micro-spin column consisting of a layer of immobilized enzyme-coated silica on a molecular w eight cut-off filter. Using this approach, digestion times were comparable to solution digests as previously mentioned; however, enzyme reuse and reac tion product isolation was facilitated.