In Situ Hybridization to Polytene Chromosomes in Drosophila Using Digoxigenin-Labeled Probes

Abstract

INTRODUCTIONIn situ hybridization to polytene chromosomes frequently is used to find the chromosomal site of a cloned DNA sequence, as well as to investigate the distribution of families of repeated sequences in the chromosome set and to measure the amount of a repeated sequence at different sites. These different experimental goals are best accomplished by variations of the technique. Simple mapping of genes and even gene fragments of a few hundred base pairs is easily done using nonradioactive probes. The simplest and most successful probes are made by random primer labeling of gel-purified fragments of cloned DNA. Digoxigenin (DIG) is commonly used for nonradioactive labeling, as are biotin-labeled probes, and biotin and DIG probes can be used in conjunction in double labeling experiments. DIG probes are detected with an antibody coupled to an enzyme or to a fluorescent dye. This protocol describes a procedure for labeling gel-purified DNA fragments with DIG and for performing in situ hybridization using an antibody coupled to alkaline phosphatase.