10 - Analysis of the Dictyostelium discoideum Genome

Abstract

This chapter describes methods for studying the structure of the Dictyostelium genome, the distribution of gene families, and the function of genes. The chromosomes of Dictyostelium are too small and too morphologically similar to allow the assignment of DNA markers to them by in situ hybridization techniques. Milligram quantities of high-molecular weight DNA in solution can be obtained from 2 l of Dictyostelium cells grown in suspension. The change in the viscosity of the DNA provides a good measure of the progress of the digestion, and can be used to determine the exact time of digestion. It is better to slightly overdigest the DNA since any small partial digestion products will be purified away from large fragments later. The most robust methods for contig construction using Dictyostelium yeast artificial chromosome (YAC) clones are based on the use of random hybridization probes. Small groups of linked YACs can be identified easily by hybridization of the YAC set with single-copy probes. The isolation and restriction digestion of YAC DNA are also elaborated.