Recombinant Dna Technology
12 Temmuz 2007
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 natural
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
CTTAAC:
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.
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