The scanning densitometer and phosphorimager enable the user to evaluate the positive signals on a blot with concrete numerical data instead of guesswork. Provides a very accurate evaluation of the blot by measuring the amount of radioactive chemiluminescence emission corresponding to each band on the membrane, generating a value expressed in counts per minute. When rat liver epithelial cell DNA is digested with BamHl, the TGF-a gene is cut into an 8.0-kb fragment, which hybridizes to the radiolabeled probe and appears as a dark band in the 8.0-kb range on the autoradiograph. The membrane was hybridized, washed, and exposed to X-ray film for 48 h. The 32P-labeled probe was generated by random primer extension, utilizing a 1.8-kb fragment of the rat TGF-a gene. The digested DNA was subjected to Southern blot analysis as described in Table 1. Ten-microgram DNA smaples were digested with the restriction enzyme BamHl for 18 h at 37☌. DNA was isolated from cultures of rat liver epithelial cells. Southern blot analysis of transforming growth factor (TGF-a). Membranes that have been hybridized with radioactive or chemiluminescent probes can also be analyzed directly in a phosphorimager (Amersham Biosciences, Piscataway, NJ). For example, the relative density of a band produced by hybridization with a gene whose copy number has been amplified will be much higher than the relative density of the band corresponding to the normal gene copy number. The end result is a numerical value, which can be used to determine the relative number of target sequences present in one sample compared to another. The autora-diographs that are produced by Southern blotting are easily analyzed by a scanning densitometer-an instrument that provides a measure of the relative density of the bands that are present on the X-ray film. Figure 4 shows an autoradiograph generated by hybridization with a 32P-labeled radioactive probe. Chemiluminescent and radioactive probes must be visualized by exposing the hybridized membrane to X-ray film, producing an autoradiograph. If nonra-dioactive, colorimetric probing techniques were used, the bands would be visible to the naked eye. Positive signals on the membrane are created when a labeled nucleic acid probe binds to complementary target DNA sequences, producing a band or bands that can be visualized. Once the membrane that contains the target DNA sequences has been "probed" with a specific labeled probe, the results of the Southern analysis can be interpreted.
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