Recombinant DNA Technology
Recombinant DNA technology is used to mass-produce proteins and genes inside bacteria. The basic procedure used in this form of genetics engineering is diagrammed in figure and described following. The procedure begins by isolating the gene that codes for protein to be mass-produced. The segment of DNA containing the gene is removed from its atural
location, usually a human cell, and attached to a piece of DNA that invades a bacterium. The attached gene is the donor gene and the DNA molecule that receives it the vector: Together, the donor gene and vector are called recombinant DNA; they are a combination of two kind of DNA. Bacteria are used in recombinant DNA technology because they are easy to grow in the laboratory, they reproduce rapidly, and their mechanisms for turning genes on and off are well understood. Moreover, bacteria readily accept pieces of DNA from one another almost piece of DNA placed inside a bacterium will be used to build proteins. Isolating the Gene Before a gene can be transferred from one cell to another, it has to be snipped out of its DNA molecule. The “scissors” of the genetic engineer are restriction enzymes, which occur naturally in bacteria where they shred the DNA molecules of viruse that infect these cells. Hundreds of restriction enzymes have been discovered; each kind cuts DNA molecules at a specific base sequence. These molecular scissors enable us to break and rejoin DNA molecules with ease. A typical restriction enzyme consists of two identical parts arranged in mirror images, like a left and right hand. The two parts recognize and cut mirrorimage
sequences of bases. If one part of the enzyme cuts one strand between the G and the A of the base sequence GAATTC, then the other part cuts the other strand between the G and the A of the mirror image sequence
The two cuts are made near each other, usually within a span of less than ten bases. After the enzyme has cut both strands of the DNA molecule, the bonds joining the bases in the tiny segment between the cuts break and the two strands come apart: Recombinant DNA technolgy. A donor gene is cut from its DNA molecule and spliced into plasmid from a bacterium. Together, they form a molecule of recombinant DNA, which invades a bacterium and replicates itself. The
bacterium reproduces to form many copies of itself and the recombinant DNA, including the donor gene, to synthesize proteins. The donor gene’s protein can be harvested in massive quantites. Restriction enzymes. Each restriction enzyme
cuts DNA at a particular sequence of bases. (a) Two restriction enzymes of the same kind cutting a DNA molecule. In the upper strand, they are cutting between a C and an A of the sequence GAAITC. In the lower strand they are cutting
between a C and an A of the sequence CITAAG. The cut strands separate and their base pairs come apart, leaving a short sequence of unpaired bases on the ends. The segment cut out of this molecule has the unpaired bases AATT on its upper strand and TTAA on its lower strand. (b) Two molecules of DNA cut with the same restriction enzyme have the same sequences of unpaired bases on their ends. They join when their unpaired bases form bonds according to the base pairing rules: A with T and G with C. Here a donor gene, cut from a longer molecule of DNA, joins a ring of DNA called a plasmid. (c) The two molecules, cut with the same restriction enzyme, become a single molecule of recombinant DNA.
A restriction enzyme cuts the DNA molecule wherever its particular sequence of bases appear; it breaks a molecule of
DNA into many fragments: A short stretch of unpaired bases remains at the end of each fragment AATT on one fragment and TTAA on the other fragment in the previous example. These ends are “sticky” their unpaired bases readily bond
with complementary bases (T with A; 0 with C) An enzyme, DNA ligase, then forms covalent bonds that join the strands of the two fragments. Two DNA molecules cut by the same restriction enzyme have the same unpaired bases at the ends of their fragments. As a consequence, the unpaired bases on a fragment from one of the molecules will join with complementary unpaired bases on a fragment from the other molecule , even when the two molecules are from different species of organisms.
(b) Plasmid: Bacteria contain small loops of DNA, called plasmids, in addition to their single large molecule of DNA. (a) Highly magnified photograph of plasmids. (b) In one of these bacteria, a plasmid has changed from a loop to a
thread and is moving into the other bacterium. Both bacteria are of the species Escherichia coli which normally lives within human intestines. Preparing a Vector Once a gene has been isolated, it is spliced into a special kind of DNA the vector for transport into a bacterium. The most commonly used vector in recombinant DNA technology is a bacterial plasmid. A plasmid is a tiny loop of DNA, carrying only a few genes , that occurs naturally within bacteria. Like a virus, a plasmid uses the cell’s ribosomes, RNA, and enzymes to synthesize its proteins and replicate itself. Unlike
a virus, its genes code for proteins that are useful, although not essential, to the survival and reproduction of the bacterium. A plasmid travels from one bacterium to another through a process called conjugation, the bacterial
equivalent of sex. Each bacterium readily accepts a foreign plasmid and synthesizes its proteins. Resistance of a bacterium to antibiotics, for example, is often controlled by genes within plasmids. This resistance is passed, via plasmids, from one species of bacterium to another, as described in the accompanying Wellness Report. Genetic engineers attach donor genes to plasmids for transport into bacteria. A plasmid is removed from a bacterium and cut by the same restriction enzyme that cut the donor gene out of its DNA molecule. The two pieces of DNA have complementary bases on their cut ends. When mixed together, the donor gene joins the plasmid to form a recombinant DNA molecule.
Mass-Producing the Protein: The recombinant DNA, consisting of donor gene and plasmid vector, readily invades a bacterium. There the recombinant DNA replicates itself and its proteins are synthesized. At the same time, the bacterium divides repeatedly to form many bacteria, with each bacterium holding copies of the recombinant DNA (including the donor gene). The formation of identical bacteria from one bacterium is know as cloning, and the formation of many copies of the donor gene from one copy is known as gene cloning. When the recombinant DNA molecules are not replicating, their genes are used to build proteins. Huge amounts of the donor gene’s protein are synthesized. This remarkable technique for synthesizing proteins is used to mass-produce a variety of medically important proteins,
some of which are listed in table.