Nucleic Acids Research

A simple agaroae-gel apparatus has been developed that allows the separation of DNA molecules in the size range from 50 kb to well over 750 kb, the largest 3ize for which size standards were available. The apparatus is based on the recent discovery that large DNA molecules are readily fractionated on agarose gels if they are alternately subjected to two approximately orthogonal electric fields. The switching time, which wa3 on the order of 20-50 sec in our experiments, can be adjusted to optinize fractionation in a given size range. The resolution of the technique is sufficient to allow the fractionation of a sample of 3elf-ligated \ DNA into a ladder of approximately 15 bands, spaced at 50 kb intervals. We have applied the technique to the fractionation of yeast DNA into 11 distinct bands, several of which have been shown by DNA-DNA hybridization to hybridize uniquely to different chromosome-specific hybridization probes. In this paper, we describe the design of the apparatus, the electrophoretic protocol, and the sample-handling procedures that we have employed. INTRODUCTION Our knowledge of the structural and functional organization of chronosonal DNA has long been limited by the difficulty of fractionating mixtures of large DNA molecules or even assaying heterogeneous DNA sajnples for the presence of such molecules. Routine preparative and analytical techniques for manipulating DNA are only effective for molecules of approximately 100 kb or less, while the average amount of DNA per chromosome Is on the order of 1000 kb In lower eukaryotes such as yeast and over 100,000 kb in higher plants and animals. Nonetheless, numerous efforts have been made to characterize chromosome-sized DNA molecules in organisms such as yeast and Drosophlla, primarily by electron microscopy, velocity sedimentation, and measurements of visco-elasticity. Although these techniques have met with some success—for example, in providing evidence that the chromosomes of lower eukaryotes probably contain a single linear molecule of duplex DNA (1,2)—they have not led to any detailed knowledge of the composition of the highly heterogeneous samples of large DNA molecules that are produced by the gentle lysis of cells © IRL Pren Limited, Oxford, England. 5647 Nucleic Acids Research or nuclei. The single chromosoae-sized DNA molecule that has been well characterized was isolated fro* a nutant yeast strain that harbors a ring cnromosoie containing about half of the genetic material present on a wild-type copy of chromosome III. Thi3 lolecule could be purified aa a 190 kb supercoiled circle and shown to be free of proteaseor RNase-3ensitlve