EVALUATION OF CAPILLARY ELECTROPHORESIS FOR ANALYSIS OF DNA RESTRICTION DIGESTS GENERATING VERY SMALL OLIGONUCLEOTIDE FRAGMENTS

Abstract

loning the products of the polymerase chain reaction (PCR) has become a common laboratory technique. In order to clone the PCR product into cloning vectors, the PCR product may need to be cut by restriction enzymes to generate sticky ends for subsequent ligat ions. To improve the ligation efficiency, it is often important to separate the digested products from undigested or partially digested DNA fragments. In addition, it is useful to monitor the restriction enzyme digestion for completion. However, many PCR products are small and restriction sites are engineered near the ends of the PCR products resulting in small differences between the native and digested DNA fragments. These differences cannot be readily detected or quantitated using conventional laboratory techniques of agarose or polyacrylamide gel electrophoresis. In this work, capillary gel electrophoresis (CGE) was investigated as an alternative approach to separate various small DNA fragments. PCR was used initially to generate a 327 bp DNA fragment encoding the 303 bp cyanovirin (CVN) gene. This PCR product was then digested with restriction enzymes to generate a 305 bp restriction fragment designed for ligation into an expression vector. Because it was difficult to visualize the size difference between the 305 bp restriction fragment and the 327 bp PCR product using conventional slab gel electrophoresis (SGE), CGE was tested to determine if this method would resolve the 305 bp and 327 bp DNA fragments. CGE was found to be an effective method capable of distinguishing between the 327 bp PCR product and the 305 bp restriction fragment. Aspects of sample preparation, mode of injection, separation chemistries and limits of sample detection (LOD)- using UV absorption and laser induced fluorescence (LIF) detection systems were addressed.