Preparation of genomic DNA from Dictyostelium discoideum for PCR analysis

The soil amoeba Dictyostelium discoideum is a practical genetic system in which targeted gene disruption by homologous recombination allows the study of specific gene functions (1–3). The efficiency of homologous recombination, however, can be less than 1%, and the identification of the desired clones requires the analysis of the genotype of many individual clones. However, the standard protocols to prepare genomic DNA for genotyping require a large number of cells (>10 7 ) and also necessitate manipulation of organic solvents and ethanol precipitation (4). Commercial kits are expensive and highly inef ficient for the isolation of D. discoideum genomic DNA. The easiest protocols known to us take at least a few hours for the preparation of genomic DNA. Genotyping of the nematode Caenorhabditis elegans, another genetic model, is very simple and is based on the use of a lysis protocol employing detergent and proteinase K (PK) (5–7). Here we adapted this method for the isolation of D. discoideum genomic DNA, which is amenable to PCR analysis. Our optimized protocol is the fol