Page 267 - Color Atlas of Biochemistry

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258 Molecular genetics

DNA cloning B. DNA cloning
Most DNA segments—e. g., genes—occur in
The growth of molecular genetics since 1970
has mainly been based on the development very small quantities in the cell. To be able
to work with them experimentally, a large
and refinement of methods of analyzing and
manipulating DNA. Genetic engineering has number of identical copies (“clones”)first
have to be produced. The classic procedure
practical applications in many fields. For ex-
ample, it hasprovided new methodsof diag- for cloning DNA takes advantage of the ability
of bacteria to take up and replicate short,
nosing and treating diseases, and it is now
also possible to create targeted changes in circular DNA fragments known as plasmids.
The segment to be cloned is first cut out of
specific characteristics of organisms. Since bi- the original DNA using restriction endonu-
ological risks cannot be completely ruled out
with these procedures, it is particularly im- cleases (see above; for the sake of simplicity,
cleavage using EcoRI alone is shown here, but
portant to act responsibly when dealing with
genetic engineering. A short overview of im- in practice two different enzymes are usually
portant methods involved in genetic engi- used). As a vehicle (“vector”), a plasmid is
needed that has only one EcoRI cleavage site.
neering is provided here and on the following
pages. The plasmid rings are first opened by cleavage
with EcoRI and then mixed with the isolated
DNA fragments. Since the fragment and the
A. Restriction endonucleases vector have the same overhanging ends, some
of the molecules will hybridize in such a way
In many genetic engineering procedures, de-
fined DNA fragments have to be isolated and that the fragment is incorporated into the
vector DNA. When thecleavagesites are
then newly combined with other DNA seg- now closed again using DNA ligase, anewly
ments. For this purpose, enzymes are used
that can cut DNA and join it together again combined (“recombinant”) plasmid arises.
By pretreating a large number of host cells,
inside the cell. Of particular importance are
restriction endonucleases—a group of bacterial onecan cause someofthem to takeupthe
plasmid (a process known as transformation)
enzymes that cleavethe DNA doublestrandin
a sequence–specific way. The numerous re- and replicate it along with their own genome
strictionenzymes knownare named using when reproducing. To ensure that only host
bacteria that contain the plasmid replicate,
abbreviations based on the organism from
which they originate. The example used plasmids are used that give the host resistance
to a particular antibiotic. When the bacteria
here is EcoRI, a nuclease isolated from the are incubated in the presence of this antibi-
bacterium Escherichia coli.
Like many other restriction endonucleases, otic, only the cells containing the plasmid will
EcoRI cleaves DNA at thesiteofa palin- replicate. The plasmid is then isolated from
drome—i. e., a short segment of DNA in which these cells, cleaved with EcoRI again, and the
fragments are separated using agarose gel
both the strand and counter-strand have the electrophoresis (see p. 262). The desired frag-
same sequence (each read in the 5 3 direc-
tion). In this case, the sequence is 5 -GAATTC- ment can be identified using its size and then
extracted from the gel and used for further
3 . EcoRI, a homodimer, cleaves the phos-
phoric acid diester bonds in both strands be- experiments.
tween G and A. This results in the formation of
complementary overhanging or “sticky” ends
(AATT), which are held together by base pair-
ing. However, they are easily separated—e. g.
by heating. When the fragments are cooled,
the overhanging ends hybridize again in the
correct arrangement. The cleavage sites can
then be sealed again by a DNA ligase.

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