Western blotting



South-Western blotting



in other organisms such as yeast or Drosophila. Many scientists use Southern blotting to identify the gene in a different organism. Southern blotting is a technique in which Using probes to detect DNA one DNA sample is hybridized to another DNA sample. Suppose we have a large DNA

sequences by hybridization molecule, such as the yeast chromosome, and we wish to locate the particular gene can be carried out on a whose sequence is similar to the human gene of interest. First the target or yeast DNA

membrane and is then referred is cut with a restriction enzyme, and the fragments are separated by gel electrophore-t° as "bl°tting". sis. The double-stranded fragments are melted into single-stranded fragments by soaking the gel in alkaline denaturing solution such as sodium hydroxide. Then the DNA fragments are transferred to a nylon membrane. Finally, the membrane is dipped in a solution of labeled DNA probe molecules, in this example, a radioactively labeled piece of the human gene (Fig. 21.32). The probe binds only to those fragments with sequences similar enough to base pair. When the probe hybridizes to the corresponding DNA, the filter will be "hot" or radioactive in that area, and if a piece of photographic film is placed over the filter, a black spot corresponding to the hybrid molecule will appear. Southern blotting only refers to hybridization of DNA to DNA.

Although Southern blotting was actually named after its inventor, Edward Southern, it set a geographical trend for naming other types of hybridization techniques (Table 21.01). Northern blotting refers to hybridization that uses RNA as the target molecule and DNA as a probe. For example, DNA probes may be used to locate messenger RNA molecules that correspond to the same gene. The mixture of RNA is run on the gel and transferred to the filter. The filter is then probed just as above.

Western blotting does not even involve nucleic acid hybridization. Proteins are separated on a gel, transferred to a membrane and detected by antibodies or other methods. Since Western blotting applies to proteins it is described more fully in Ch.

Northern blotting Hybridization technique in which a DNA probe binds to an RNA target molecule

Southern blotting A method to detect single stranded DNA that has been transferred to nylon paper by using a probe that binds DNA Western blotting Detection technique in which a probe, usually an antibody, binds to a protein target molecule

Fluorescence in Situ Hybridization (FISH) 595

Rat Cow Dog Human Rat Cow Dog Human

Rat Cow Dog Human Rat Cow Dog Human

Probe was non-coding; Probe is coding dna;


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