Porth's Essentials of Pathophysiology, 4e

102

Cell and Tissue Function

U N I T 1

Cut DNA molecules with restriction enzyme to generate complementary sequences on the vector and the fragment

Chromosomal DNA

Chromosomal DNA fragment for cloning

Vector DNA

Digest with restriction endonucleases

DNA fragments

Join vector and chromosomal DNA fragment using the enzyme DNA ligase

Separate fragments by gel electrophoresis

Recombinant DNA molecule

Introduce into bacterium

Suspect 1

Suspect 2

Victim

Evidence

Recombinant DNA molecule

Bacterial chromosome

Gel

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 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. http://science.energy.gov/ber/)

Denature and transfer DNA to nitrocellulose paper

Incubate with probe, wash and perform autoradiography to observe labeled DNA bands

Radioactive DNA probe

Suspect 1

Suspect 2

Victim

Evidence

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.)