In Situ Hybridization to Polytene Chromosomes in Drosophila Using Tritium-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. Quantitation of sequences at different chromosomal sites, for example, is best done using radioactive probes where the signal is measured directly from the bound probe, rather than after the one or two subsequent steps (e.g., antibody or streptavidin binding, enzymatic color production, etc.) that are required to detect nonradioactive probes. Tritium (3H) is by far the best radioisotope for in situ hybridization because of the very low energy of the beta particle that is emitted. These beta particles travel <1 µm through autoradiographic emulsion, giving a precise localization for the probe. In addition, 3H probes can reliably detect sequences on polytene chromosomes significantly shorter than those detected by nonradioactive probes. The simplest and most successful probes are made by random primer labeling of gel-purified fragments of cloned DNA. The DNA can be labeled while still in the gel slice or it can be purified with a minicolumn. This protocol describes a procedure for 3H labeling of DNA in agarose and for performing in situ hybridization using the resulting radiolabeled probe.