Illustrative Examples

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.

nucleotide the building block of RNA or DNA

antibodies immune-system proteins that bind to foreign molecules hybridization (molecular) base-pairing among DNAs or RNAs of different origins

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

restriction enzyme an enzyme that cuts DNA at a particular sequence probe molecule used to locate another molecule complementary matching opposite, like hand and glove

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. see also Gel Electrophoresis; In situ Hybridization; Restriction Enzymes; Sequencing DNA.

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.

Breast Cancer

Breast cancer remains the most common cause of cancer among women in the United States, and it results in more deaths from cancer among women than any other type of cancer, except lung cancer. Over 40,000 women die from breast cancer in the United States each year. A long history of research, now coupled with the new information emerging from the field of molecular genetics, is beginning to explain the basic steps leading to breast cancer, and it will enable the development of novel treatment and prevention strategies.

Almost all breast cancers begin in the glandular structures in the breast that, during lactation, produce milk. These mammary glands are under the control of reproductive hormones that stimulate the monthly cycle of gland expansion and shrinkage, which is a feature of the regular menstrual cycle. Many of the factors associated with the development of breast cancer appear to have their effect through interaction with the hormonal stimulation of these glands.

The risk of developing breast cancer increases throughout a woman's lifetime, and the disease is relatively rare in very young women. The overall association of breast cancer incidence with increasing age may be explained by a model of breast cancer in which a progressive and cumulative series of genetic changes within the cells of the glands is necessary for the initiation of cancer. The longer a woman lives, the more opportunities there are for these genetic changes to accumulate and reach a stage where cells can become cancerous.

One of the most consistent epidemiological observations is the association of reproductive events with risk of breast cancer. Women who have

MALE BREAST CANCER

According to the National Cancer Institute, male breast cancer is most common among males between 60 and 70 years of age. Two of the major risk factors for men include: exposure to radiation, and having a family history of breast cancer (especially the BRCA2 gene). The survival rate for men with breast cancer almost equals that for women.

10 Ways To Fight Off Cancer

10 Ways To Fight Off Cancer

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