102
U N I T 1
Cell and Tissue Function
Pharmaceutical Applications
Recombinant DNA technology has also made it possible
to produce proteins that have therapeutic properties. One
of the first products to be produced was human insu-
lin. Recombinant DNA corresponding to the A chain of
human insulinwas isolated and inserted into plasmids that
were in turn used to transform
Escherichia coli
. The bac-
teria then synthesized the insulin chain. A similar method
was used to obtain the B chains. The A and B chains were
then mixed and allowed to fold and form disulfide bonds,
producing active insulin molecules. Human growth hor-
mone has also been produced in
E. coli
. More complex
proteins are produced in mammalian cell culture using
recombinant DNA techniques. These include erythropoi-
etin, which is used to stimulate red blood cell production;
factor VIII, which is used to treat hemophilia; and tissue
plasminogen activator (tPA), which is frequently adminis-
tered after a heart attack to dissolve the thrombi that are
obstructing coronary blood flow.
DNA Fingerprinting
The technique of DNA fingerprinting is based in part on
those techniques used in recombinant DNA technology
and on those originally used in medical genetics to detect
slight variations in the genomes of different individuals.
Using restriction enzymes, DNA is cleaved at specific
regions (Fig. 5-13). The DNA fragments are separated
Chromosomal DNA
Digest with restriction
endonucleases
Separate fragments
by gel electrophoresis
Denature and transfer DNA
to nitrocellulose paper
Incubate with probe, wash
and perform autoradiography
to observe labeled DNA bands
Evidence
Victim
Suspect 1
Suspect 2
Radioactive
DNA probe
Evidence
Victim
Suspect 1
Suspect 2
DNA fragments
Gel
FIGURE 5-13.
Deoxyribonucleic acid (DNA) fingerprinting.
Restrictive enzymes are used to break chromosomal DNA into
fragments, which are then separated by gel electrophoresis,
denatured, and transferred to nitrocellulose paper; DNA
bands are labeled with a radioactive probe and observed
using autoradiography. (Modified from Smith C, Marks AD,
Lieberman M. Marks’ Basic Medical Biochemistry. 2nd ed.
Philadelphia, PA: Lippincott Williams &Wilkins; 2005:309.)
Vector DNA
Recombinant
DNA molecule
Chromosomal DNA
fragment for cloning
Cut DNA molecules with
restriction enzyme to
generate complementary
sequences on the vector
and the fragment
Join vector and
chromosomal
DNA fragment using
the enzyme DNA ligase
Introduce into bacterium
Recombinant
DNA molecule
Bacterial
chromosome
FIGURE 5-12.
Recombinant DNA technology. By fragmenting
DNA of any origin and inserting it in the DNA of rapidly
reproducing foreign cells, billions of copies of a single gene
can be produced in a short time.The DNA to be cloned is
inserted into a plasmid (a small, self-replicating circular
molecule of DNA) that is separate from chromosomal DNA.
When the recombinant plasmid is introduced into bacteria, the
newly inserted segment will be replicated along with the rest
of the plasmid. DNA, deoxyribonucleic acid. (From Office of
Biological and Environmental Research of the U.S. Department
of Energy Office of Science.
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