A NOVEL METHOD FOR PRODUCING PARTIAL RESTRICTION DIGESTION OF DNA FRAGMENTS BY PCR WITH 5-METHYL-CTP

Partial digestion of DNA fragments is a standard procedure for subclon ing analysis and for generating restriction maps. We have developed a novel method to generate a partial digestion for any DNA fragment that can be amplified by PCR. The method involves the incorporation of 5-m ethyl-dCTP into the PCR product to protect most of the restriction sit es. As a result, complete digestion of the modified PCR products with a 5-methyl-dCTP-sensitive enzyme will produce an array of restriction fragments equivalent to a partial restriction enzyme digestion reactio n done on unmethylated PCR products. This method reduces the time and material needed to produce partially-digested DNA fragments by traditi onal methods. Furthermore, using fluorescein-labeled primers in the re action, we were able to detect the fluorescein-labeled end fragments r esulting from the enzyme digestion using a fluorimager or anti-fluores cein-AP antibody and thus determine the restriction maps.