Utilization of DNA Probes with Digoxigenin-Modified Nucleotides in Southern Hybridizations

Probes prepared with either digoxigenin- or biotin-modified nucleotides can be hybridized to Southern blots to detect target nucleic acid sequences. These methods offer an attractive alternative to “radioactively tagged” probes in terms of safety, cost, and efficiency. Most previous nonradioactive strategies utilized the detection of the modified base by the use of a coupled antibody- or avidin-alkaline phosphatase with subsequent exposure to Nitro Blue Tetrazolium (NBT). NBT is converted to an insoluble, colored compound at the site of hybridization. Replacement of this calorimetric reaction with a chemiluminescent process provides better sensitivity, as well as easier reusability of membranes. The development of compounds, such as adamantyl 1,2 dioxetane phosphate (AMPPD) (1 ) provides a convenient alternative to previous detection schemes, since this compound is destabilized by alkaline phosphatase, resulting in the production of light, which will expose a standard X-ray film. Probes produced by methods analogous to those used for NBT detection are also usable in this process. The membranes with the bound alkaline phosphatase are soaked in a dilute solution of AMPPD and then exposed to film at room temperature or 37�C instead of −70�C. Exposure times of 45 min to several hours are all that is necessary to detect single-copy sequences, even in genomic DNA preparations from organisms with very large genomes.