Interpretation Of The Southern Blot

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. 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. If nonra-dioactive, colorimetric probing techniques were used, the bands would be visible to the naked eye. Chemiluminescent and radioactive probes must be visualized by exposing the hybridized membrane to X-ray film, producing an autoradiograph. Figure 4 shows an autoradiograph generated by hybridization with a 32P-labeled radioactive probe. 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. 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. 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. Membranes that have been hybridized with radioactive or chemiluminescent probes can also be analyzed directly in a phosphorimager (Amersham Biosciences, Piscataway, NJ). This instrument m W m M W - 8 0 Kb xnF-rf

Fig. 4. Southern blot analysis of transforming growth factor (TGF-a). DNA was isolated from cultures of rat liver epithelial cells. Ten-microgram DNA smaples were digested with the restriction enzyme BamHl for 18 h at 37°C. The digested DNA was subjected to Southern blot analysis as described in Table 1. The 32P-labeled probe was generated by random primer extension, utilizing a 1.8-kb fragment of the rat TGF-a gene. The membrane was hybridized, washed, and exposed to X-ray film for 48 h. 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.

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. The scanning densitometer and phosphorimager enable the user to evaluate the positive signals on a blot with concrete numerical data instead of guesswork.

Proper interpretation of Southern blots necessitates the use of controls throughout the procedure. In order to compare the gene copy number between one sample and another, it is imperative that the same quantity of DNA be loaded onto the gel for all samples being compared. This can be assured by measuring the DNA concentration carefully prior to loading and by evaluating the relative concentrations of DNA on the elec-trophoresed gel by staining with ethidium bromide after electrophoretic separation. When analyzing patient samples that are suspected to contain abnormalities (gene deletions, rearrangements, or amplifications), one should include DNA that is known to be normal for comparison.

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Responses

  • mateusz douglas
    How would interpret results in southern blotting?
    4 years ago
  • MEDHANE
    How would u analyze or interpret results on southern blotting?
    3 years ago
  • Lucas Schmidt
    How to analyse southern blots results?
    2 years ago
  • mantissa
    How to read southern blot?
    10 months ago
  • sinit
    How to read a southern blot results?
    8 months ago
  • arianna
    How to Interpret data from southern blots?
    7 months ago
  • Negisti
    How to interpret southern blot results?
    5 months ago
  • liviano
    How to interpret southern blot test?
    5 months ago

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