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Blotting - Illustrative Examples

dna gene student probe

Blotting is perhaps best understood with illustrative examples. Suppose a student was studying a newly identified gene, X, from cows. The student then asks three basic questions as part of a research project: (1) Do pigs also have gene X on their chromosomes? (2) Do cows express gene X in their brain tissue? (3) Is the protein product of gene X found in the cow's blood plasma? Blotting experiments can answer all three of these questions.

Southern blots, such as this one, are useful in forensic examinations of DNA evidence.

A Southern (DNA) blot will answer the first question. The student obtains DNA from a pig, uses a restriction enzyme to cut the DNA into a large pool of fragments of different sizes, and then fractionates the DNA fragments using gel electrophoresis. The contents of the gel are then chemically treated so that the double-stranded DNA molecule "unzips" and exists in a single-stranded form, which is then blotted onto nitrocellulose paper. At this point, the student can take gene X (or a portion of the gene) from the cow, label it, make it single-stranded, and use it as a probe to analyze the pig's DNA. The labeled probe is then added to the nitrocellulose blotted with the pig DNA. If the pig's DNA also contains gene X, there should be a fragment on the nitrocellulose with a nucleotide sequence sufficiently complementary to the probe such that the probe will bind. In other words, the labeled probe will bind to any fragment from the blotted pig DNA that contains gene X, allowing the student to detect the presence of gene X in pigs.

To answer the second question, a Northern (RNA) blot would be used. The procedure is essentially the same as with the Southern blot, except that the student would isolate RNA from the cow's brain tissue and run it out on the gel. The same DNA probe described above would then be used to detect whether the RNA that represents gene X expression is present in the brain.

To answer the third question, the student would use a Western (protein) blot. This requires the use of an antibody that specifically reacts with the protein coded for by gene X. The student first obtains plasma from the cow and uses standard biochemical techniques to isolate the proteins for analysis. These proteins can then be run out on a gel and transferred to nitrocellulose. The proteins can then be probed with the labeled antibody. If the product of gene X is in the plasma, it will bind with the labeled antibody and can thus be detected.

Michael J. Bumbulis

Bibliography

Bloom, Mark V., Greg A. Freyer, and David A. Micklos. Laboratory DNA Science: An Introduction to Recombinant DNA Techniques and Methods of Genome Analysis. Menlo Park, CA: Addison-Wesley, 1996.

Russell, Peter. Genetics, 5th ed. Menlo Park, CA: Benjamin Cummings, 1998.

Watson, James D., et al. Recombinant DNA, 2nd ed. New York: Scientific American Books, 1992.

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