CLINICAL CASES
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isolation of the biomolecules of interest (DNA, RNA, or protein); (2) sepa-
ration of the mixture using gel electrophoresis; and (3) subsequent transfer
of the sample molecules from the gel to a nitrocellulose or nylon mem-
brane. The final identification step involves incubation of the membrane
blot with probes that bind specifically to the molecule of interest. DNA is
analyzed using Southern blotting, named after E. M. Southern, who first
described this procedure in 1975. Based on this initial terminology, Northern
and Western blotting were given as names referring to RNA and protein-
blotting transfer, respectively. The major difference between the procedures
involves the probes used for detection and identification of the molecules of
interest. Southern and Northern blots take advantage of the ability of com-
plementary nucleic acid strands to hybridize.
In a Southern blot, after the DNA fragments have been transferred from the
electrophoretic gel to the membrane, they are allowed to interact with a single-
stranded DNA oligonucleotide that contains the sequence of nucleic acids in
the gene of interest. The oligonucleotide probe is usually radioactively
labeled, which allows the DNA band to be identified following exposure to
x-ray film. Northern blots are used to monitor the expression of a gene of
interest by determining the amount of mRNA present. Then the mRNA is
isolated, separated by electrophoresis, and, after transfer to a membrane, is
allowed to interact with a radioactive DNA polynucleotide that has a
sequence of the gene of interest. Visualization is by exposure to x-ray film.
In the case of a Western blot of a protein mixture, after the proteins have been
transferred to the membrane, the membrane is probed with an antibody that
binds specifically to the target protein/antigen. The antibody may be
radioactive or coupled to a fluorescent chromophore to allow easy detection.
The ability to sequence DNA and analyze genes at the nucleotide level has
become a powerful tool in many areas of analysis and research. The Sanger
dideoxynucleotide method of DNA sequencing was first developed in 1975
by Frederick Sanger, after whom the technique was named. The strategy used
in this method is to create four sets of labeled fragments, corresponding to
the four deoxyribonucleotides. It involves the enzymatic duplication of a
DNA strand that is complementary to the strand of interest. In the Sanger
method, 2',3'-dideoxyribonucleotide triphosphates (ddNTPs) are used in
addition to deoxyribonucleotide triphosphates (dNTPs). Since the ddNTPs
lack the 3'-OH group, they will terminate DNA chain elongation because
they cannot form a phosphodiester bond with the next dNTP. Each of the four
sequencing reaction tubes (A, C, G, and T) would then contain a single-
stranded DNA template; a primer sequence; DNA polymerase to initiate syn-
thesis where primer is hybridized to the template; mixture of the four
deoxyribonucleotide triphosphates (dATP, dTTP, dCTP, and dGTP) to extend the
DNA strand; one labeled dNTP (using a radioactive element or dye); and one
ddNTP, which terminates the growing chain wherever it is incorporated. Tube A
would have ddATP, tube C ddCTP, and so forth. Since the concentration of
the ddNTP is low (approximately 1 percent of dNTP), the chain will terminate
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