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        Analysis of DNA Restriction Enzyme Digests by Two-Dimensional Electrophoresis in Agarose Gels

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        Two-dimensional (2D ) electrophoretic fractionation of digested DNA has been useful in analyzing and comparing the genomic structure of viruses and prokaryotic organisms (1 4 ). For eukaryotes, it has been used to examine the genomic distribution of multicopy gene families (5 ), to determine the methylation status of individual members of such families (6 8 ), and to analyze the physical organization of complex genetic loci (9 ,10 ). The basic principle is straightforward (Fig. 1 ): High-mol-wt DNA is digested in solution with a restriction enzyme of choice and the digest electrophoresed in the first dimension. The sample lane is cut as a longitudinal strip from the gel, equilibrated with appropriate buffer, and incubated with a second restriction enzyme. The strip is then embedded in a second gel and electrophoresed in a direction perpendicular to the first. Although simple in concept, the procedure involves many manipulations: thus the chances of accident are high in the beginning. Close attention to detail and a stoic attitude toward sudden failure are useful in persevering to a good series of high resolution gels. We describe first the basic 2D procedure as practiced in our laboratory (7 ,8 ) and additionally comment on the literature with regard to reported technical descriptions and applications (see Notes ).
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