Biomedical Engineering Reference
In-Depth Information
Enzymatic fragmentation of the DNA
Host cell
1
1
(a)
2
(b)
(c)
1
2
3
2
+
3
3
Vectors
rDNA molecules
(d)
(e)
1
1
3
3
2
2
Agar plate containing 3
colonies,each is a clone of
one of the host cells above
Colony 3 contains the
cloned gene of interest
2
n
Figure 3.12
A basic overview of the DNA cloning process. Refer to text for specifi c details
genes, often including one or more genes whose product renders the plasmid-containing cell
resistant to specifi c antibiotic(s). One plasmid often used in cloning experiments with
E. coli
is
pUC18 (Figure 3.13). Bacteriophage ('phage') are viruses capable of infecting and replicating
inside bacteria. Bacteriophage
DNA is approximately 48 500 bp in length. Another vector
type sometimes used are the bacterial artifi cial chromosomes (BACs), which are effectively
very large plasmids used to clone very large stretches of DNA (usually DNA fragments above
100 000 bp).
Integration of the DNA fragments into the chosen vector is undertaken by 'opening up' the cir-
cular vector via treatment with the same RE as used to generate the DNA fragments for cloning,
followed by co-incubation of the cleaved vector and the fragments under conditions that promote
the annealing of complementary sticky ends. Some vectors may simply recircularize to reform
their original structure, but pretreatment of the vector in various ways can prevent this from hap-
pening. Most of the recircularized plasmids will have incorporated a fragment of DNA to be
cloned. The plasmids are then incubated with another enzyme, a DNA ligase, which catalyses
the formation of phosphodiester bonds in the DNA backbone and thus will seal or 'ligate' the
plasmid.
The next stage of the cloning process entails the introduction of the engineered vector into
E. coli
cells. This can be achieved by a number of different means. One approach (called transfor-
mation) involves co-incubation of the plasmids and cells in a solution of calcium chloride, initially
at 0
λ
C, with subsequent increase in temperature to 42
C. This temperature shock facilitates entry
of plasmids into some cells.
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