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CASE FILES: BIOCHEMISTRY
clearance and increasing the incidence of airway infections. In sweat glands,
defects in CFTR prevent the reabsorption of Cl- in the sweat gland duct, thus
increasing the concentration of NaCl in sweat. The presence of salty sweat in chil-
dren with CF gives rise to the Northern Europe adage: “Woe to that child which
when kissed on the forehead tastes salty. He is bewitched and soon must die.”
Gel electrophoresis is a method routinely used in biochemistry and molec-
ular biology to separate, identify, and purify peptides, proteins, and DNA
fragments based on their size. Electrophoresis is a method by which
charged molecules in a solution are separated in an electric field because
of their different mobilities. The mobility of an ion in the electric field is
dependent on the charge of the ionized molecule, the voltage gradient of the
electric field, and the frictional resistance of the supporting medium. The
supporting medium of choice for most protein and peptide analyses, as well as
small DNA fragments, is polyacrylamide gel. For large DNA fragments,
agarose gel gives the best results.
In general, DNA molecules have a high negative charge-to-mass ratio
owing to their sugar-phosphate backbone. Therefore, when an electric field
is applied, DNA will migrate through the agarose (or polyacrylamide) gel
toward the positive pole in the electrophoresis apparatus. Because of the high
charge-to-mass ratio, the relative electrophoretic mobility of DNA depends
primarily on the size of the DNA molecule and porosity of the gel matrix,
which can be varied by the concentration of agarose or polyacrylamide in the
gel. Small DNA strands will move fast, whereas large fragments will lag
behind smaller fragments as the DNA migrates through the gel. To estimate
the size of DNA in the samples, molecular weight markers (a mixture of DNA
fragments of known molecular size) are applied to one of the lanes in the gel.
Gel electrophoresis separates DNA molecules based on their frictional coeffi-
cient, which is dependent on the length and conformation of the fragment. For
most linear double-stranded DNA molecules, separation will be according to
their molecular weight. However, circular DNA molecules (e.g., from bacte-
ria or plasmids) can adopt a super coiled structure and therefore will pass
through the gel with less resistance than the rod shape of linear DNA.
Gel electrophoresis can be applied as a method for detection of genetic dis-
eases such as Cystic fibrosis (CF). Larger deletions in the cystic fibrosis
transmembrane conductance regulator (CFTR) gene will cause it to move
differently than the full-length wild type gene. In most cases, the DNA sample
of the patient is amplified by a polymerase chain reaction (PCR) using specific
primers. After the electrophoresis of the PCR samples the bands are visualized
under ultraviolet (UV) light using ethidium bromide staining.
Bands of molecules separated by electrophoresis can be detected in the gel
by various staining and destaining methods, such as the above mentioned
ethidium bromide. However, more specific information can be obtained by
application of blotting techniques. There are three different blotting tech-
niques based on whether DNA, RNA, or protein is being analyzed. However,
all blotting techniques have major steps in common. These include (1) the
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