Cellular gels. Purifying and mapping long DNA molecules

Abstract

Long DNA molecules of greater than 10(5) bp (0.1 Mbp) are easily broken by pipetting. Therefore, chromosomal DNA is generally isolated after embedding cells in a protective coat of agarose. The embedded DNA can then be cut into long pieces and fractionated on gels using pulsed fields, but these pieces are again easily broken if the resolved DNA molecules are recovered from the gels. We now describe a novel gel matrix, a 'cellular' gel, that permits the recovery of resolved fragments from gels in a form that enables facile manipulation without shear. This facilitates purification and restriction mapping of fragments of 0.1-1.0 Mbp. We illustrate the utility of the method by mapping chromosome III of baker's yeast, which has a length of approximately 0.36 Mbp. This method should facilitate purification and restriction mapping of yeast artificial chromosomes.