Fragmentation and labeling of probe DNA for whole-mount FISH in Drosophila

Good probes for whole-mount fluorescent in situ hybridization (FISH) must meet two criteria: The DNA fragments must be very small and they must be highly labeled. This article describes an effective labeling scheme that involves fragmenting the probe DNA and then adding a mixture of labeled and unlabeled nucleotides to the 3' ends using the enzyme terminal deoxynucleotidyl transferase (TdT). This method can be used to label a variety of DNA probes, regardless of their initial size (e.g., plasmid, cosmid, or P1 clones, polymerase chain reaction [PCR] products, or total genomic DNA). Short oligonucleotides may also be labeled in this way without digestion, because their small size allows them to diffuse through thick tissues. A potential advantage of end-labeling is that the modified nucleotides are not incorporated into the complementary probe sequence itself and may thus interfere less with hybridization. The free 3' tail may also make haptens more accessible to detection reagents.