Immobilization of the restriction enzymesHaeIII andHindIII on porous silica particles via a glutaraldehyde linkage for the micro-digestion of dsDNA with analysis by capillary electrophoresis

Abstract

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 minute quantities of DNA with minimal reagent consumption. In this study, the restriction enzymes, HaeIII, PstI, and HindIII, were successfully immobilized via glutaraldehyde linkages to porous silica micro-particles. Studies were carried out to examine the impact of immobilization on enzymatic activity. Digestions of ϕX174-RF DNA phage and SV40 viral DNA were performed with the immobilized enzymes by placing the silica particles in solution with the target DNA with digestion times of 120 min and 240 min respectively. The digests were analyzed off-line using capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection. Timed studies were performed to establish optimal conditions for complete digestion. Digests utilizing immobilized HaeIII and HindIII were similar in composition to homogeneous, free solution digests. PstI showed no evidence of activity upon immobilization. The immobilized restriction enzymes could also be used for multiple rounds of digestion; however, longer incubation times were required for successive runs probably due to partial denaturation of the restriction enzyme. Digests 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 weight cut-off filter. Using this approach, digestion times were comparable to solution digests as previously mentioned; however, enzyme reuse and reaction product isolation was facilitated.