GENE ANALYSIS: DNA
June 1st, 2008 | by admin |SOUTHERN BLOTTING
One of the most useful techniques for analyzing a gene at the level of genomic DNA is Southern blotting, named for its originator, E.M. Southern.18 In general, it allows one to determine whether specific nucleotide sequences in a cloned probe are present in a sample of genomic DNA. The presence of these sequences
usually means that the gene itself is present in the genomic DNA diagrams the technique. Purified genomic DNA is digested with a specific restriction endonuclease, which, as described above, will produce an array of differently sized DNA fragments.
Electrophoresis through an agarose gel then separates these fragments according to size. (Since the phosphate groups in DNA make the molecules negatively charged, they will migrate toward the anode in an
electric field. The semiporous agarose will allow molecules of DNA to pass with varying degrees of ease, at a rate inversely proportional to their size. At any time after electrophoresis begins, small molecules will be closer to the anode than large molecules). The agarose gel is usually cast in the form of a flat rectangle a few millimeters thick. The final goal of Southern blotting is to identify specific fragments of cut DNA using nucleic acid hybridization. Because the agarose gel used in electrophoresis is thick and the DNA fragments can move within it, DNA in the gel is not in a suitable form for further analysis. The DNA fragments must be transferred to a solid support to which they are irreversibly bound in order to carry out nucleic acid hybridization studies. Thus, after electrophoresis, a paper-thin membrane microfilter (made of nitrocellulose or nylon) is placed over the flat portion of the gel. Liquid is then forced through the agarose gel in a direction perpendicular to the direction in which the DNA moved during electrophoresis. As the liquid perfuses the gel, it carries DNA fragments with it, depositing them on the membrane filter, to which
the DNA sticks. After transfer, the DNA fragments are arrayed by size on the solid support.
At this point, a fragment of cloned DNA (the probe) is radiolabeled by using any of a variety of techniques. The membrane containing the transferred DNA is then soaked in a solution containing the radiolabeled probe. If there are any sequences in the genomic DNA that are complementary to those in the probe, the probe will hybridize to those sequences on the filter. The unbound probe can be washed away, and the remaining specifically hybridized probe can be visualized by exposing the filter to x-ray film.
What results from these studies is a pattern of one or more bands on x-ray film. Each band corresponds to a restriction endonucleasegenerated DNA fragment containing nucleotide sequences complementary to those in the radioactive probe. For any particular gene probe, the size (i.e., length) of the band it identifies will be the same from individual to individual (although see below for a discussion of RFLPs, an important exception). Therefore, if a gene has undergone a structural rearrangement, as, for example, when the c-abl oncogene is translocated from chromosome 9 to 22, the pattern may change. Suppose, for example, that the c-abl probe ordinarily recognizes a 2,000- base EcoRI fragment in normal genomic DNA. If the translocation break point in a CML patient occurs within that fragment, part of the c-abl gene and one of its EcoRI sites will move to chromosome 22. Southern blot analysis of the patient’s DNA may now detect either a larger fragment than normal, if the recipient chromosome has an EcoRI site farther away than the old EcoRI site, or a smaller fragment, if it has an EcoRI site closer than the old one. Southern blotting is thus a sensitive technique for detecting large structural rearrangements in the genome, such as those that are occasionally associated with malignancy.
Since the amount of the radiolabeled probe that hybridizes to a Southern blot is proportional to the number of copies of the specific gene present in the target DNA, this technique can be used quantitatively. For example, in an analysis of primary breast cancer tissue, Southern blotting was used to determine that 30% of these samples contained multiple copies of c-neu oncogene DNA, that is, the gene was amplified.
