Blotting Hybridization Techniques Allow Visualization of Specific Fragments

Visualization of a specific DNA or RNA fragment among the many thousands of "contaminating" molecules requires the convergence of a number of techniques, collectively termed blot transfer. Figure 40-5 illustrates the Southern (DNA), Northern (RNA), and Western (protein) blot transfer procedures. (The first is named for the person who devised the technique, and the other names began as laboratory jargon but are now accepted terms.) These procedures are useful in determining how many copies of a gene are in a given tissue or whether there are any gross alterations in a gene (deletions, insertions, or rearrangements). Occasionally, if a specific base is changed and a restriction site is altered, these procedures can detect a point mutation. The Northern and Western blot transfer techniques are used to size and quantitate specific RNA and protein molecules, respectively. A fourth hybridization technique, the Southwestern blot, examines protein^DNA interactions. Proteins are separated by electrophoresis, renatured, and analyzed for an interaction by hybridization with a specific labeled DNA probe.

Colony or plaque hybridization is the method by which specific clones are identified and purified. Bacteria are grown on colonies on an agar plate and overlaid with nitrocellulose filter paper. Cells from each colony stick to the filter and are permanently fixed thereto by heat, which with NaOH treatment also lyses the cells and denatures the DNA so that it will hybridize with the probe. A radioactive probe is added to the filter, and (after washing) the hybrid complex is localized by exposing the filter to x-ray film. By matching the spot on the autoradiograph to a colony, the latter can be picked from the plate. A similar strategy is used to identify fragments in phage libraries. Successive rounds of this procedure result in a clonal isolate (bacterial colony) or individual phage plaque.

All of the hybridization procedures discussed in this section depend on the specific base-pairing properties of complementary nucleic acid strands described above. Perfect matches hybridize readily and withstand high temperatures in the hybridization and washing reac-

Southern

Northern

Western

Southern

Northern

Western

Transfer to paper

Antibody* Add probe

Autoradiograph

Autoradiograph

Figure 40-5. The blot transfer procedure. In a Southern, or DNA, blot transfer, DNA isolated from a cell line or tissue is digested with one or more restriction enzymes. This mixture is pipetted into a well in an agarose or polyacrylamide gel and exposed to a direct electrical current. DNA, being negatively charged, migrates toward the anode; the smaller fragments move the most rapidly. After a suitable time, the DNA is denatured by exposure to mild alkali and transferred to nitrocellulose or nylon paper, in an exact replica of the pattern on the gel, by the blotting technique devised by Southern. The DNA is bound to the paper by exposure to heat, and the paper is then exposed to the labeled cDNA probe, which hybridizes to complementary fragments on the filter. After thorough washing, the paper is exposed to x-ray film, which is developed to reveal several specific bands corresponding to the DNA fragment that recognized the sequences in the cDNA probe. The RNA, or Northern, blot is conceptually similar. RNA is subjected to electrophoresis before blot transfer. This requires some different steps from those of DNA transfer, primarily to ensure that the RNA remains intact, and is generally somewhat more difficult. In the protein, or Western, blot, proteins are elec-trophoresed and transferred to nitrocellulose and then probed with a specific antibody or other probe molecule. (Asterisks signify labeling, either radioactive or fluorescent.)

tions. Specific complexes also form in the presence of low salt concentrations. Less than perfect matches do not tolerate these stringent conditions (ie, elevated temperatures and low salt concentrations); thus, hybridization either never occurs or is disrupted during the washing step. Gene families, in which there is some degree of homology, can be detected by varying the stringency of the hybridization and washing steps. Cross-species comparisons of a given gene can also be made using this approach. Hybridization conditions capable of detecting just a single base pair mismatch between probe and target have been devised.

Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

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