Page 271 - Color Atlas of Biochemistry

262 Molecular genetics
PCR and protein expression
A. Polymerase chain reaction (PCR)
Thepolymerasechain reaction (PCR)is an
important procedure in genetic engineering
that allows any DNA segment to be replicated
(amplified) without the need for restriction
enzymes, vectors, or host cells (see p. 258).
However, thenucleotidesequenceof the seg-
ment has to be known. Two oligonucleotides
(primers) are needed, which each hybridize
with one of the strands at each end of the DNA
segment to be amplified; also needed are suf-
ficient quantities of the four deoxyribonucleo- two PCR amplificates (lanes 1 and 2) is shown
side triphosphates and a special heat-tolerant
DNA polymerase. The primers are produced
by chemical synthesis, and the polymerase is
obtained from thermostable bacteria.
First, the starter is heated to around 90 °C 800 bp for fragment 1 and 1800 bp for frag-
to separate the DNA double helix into single
strands (a; cf. p. 84). The mixture is then
cooled to allow hybridization of the primers
(b). Starting from the primers, complemen-
tary DNA strands are now synthesized in
both directions by the polymerase (c). This
cycle (cycle 1) is repeated 20–30 times with
thesamereaction mixture (cycle 2 and sub-
sequent cycles). The cyclic heating and cool-
ing are carried out by computer-controlled
thermostats.
After only the third cycle, double strands
start to form with a length equal to the dis-
tance between the two primers. The propor-
tion of these approximately doubles during
each cycle, until almost all of the newly syn-
thesized segments have the correct length.
B. DNA electrophoresis
The separation of DNA fragments by electro-
phoresis is technically simpler than protein
electrophoresis (see p. 78). The mobility of
molecules in an electrical field of a given
strength depends on the size and shape of
the molecules, as well as their charge. In con- clots (see p. 292), and the growth hormone
trast to proteins, in which all three factors
vary, the ratio of mass to charge in nucleic
acids is constant, as all of the nucleotide com-
ponents have similar masses and carry one
negative charge. When electrophoresis is car-
ried out in a wide-meshed support material
that does not separate according to size and
shape, the mobility of the molecules depends
Koolman, Color Atlas of Biochemistry, 2nd edition © 2005 Thieme
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on their mass alone. The supporting material
generally used in genetic engineering is a gel
of the polysaccharide agarose (see p. 40).
Agarosegelsare notverystableand arethere-
fore poured horizontally into a plastic cham-
ber in which they are used for separation
(top).
To make the separated fragments visible,
after running the procedure the gels are
placed in solutions of ethidium bromide.
This is an intercalator (see p. 254) that shows
strong fluorescence in UV light after binding
to DNA, although it barely fluoresces in an
aqueous solution. The result of separating
in the lower part of the illustration. Compar-
ing their distances with those of poly-
nucleotides of known lengths (lane 3; bp =
base pairs) yields lengths of approximately
ment 2. After staining, the bands can be cut
outof the geland theDNA canbeextracted
from them and used for further experiments.
C. Overexpression of proteins
To treat some diseases, proteins are needed
that occur in such small quantities in the or-
ganism that isolating them on a large scale
would not be economically feasible. Proteins
of this type can be obtained by overexpression
in bacteria or eukaryotic cells. To do this, the
corresponding gene is isolated from human
DNA and cloned into an expression plasmid
as described on p. 258. In addition to the gene
itself, the plasmid also has to contain DNA
segments that allow replication by the host
cell and transcription of the gene. After trans-
formation and replication of suitable host
cells, induction is used in a targeted fashion
to trigger ef cient transcription of the gene.
Translation of the mRNA formed in the host
cell then gives rise to large amounts of the
desired protein. Human insulin (see p. 76),
plasminogen activators for dissolving blood
somatotropin are among the proteins pro-
duced in this way.