Biomedical Engineering Reference
In-Depth Information
CHAPTER 6
Cloning strategies
replicated; and (iv) a method for selecting recipient
cells that have acquired the recombinant (Fig. 6.1).
To simplify the description of such procedures, the
assumption is made that we know exactly what
we are cloning. This is indeed the case with simple
subcloning
strategies, where a defined restriction
fragment is isolated from one cloning vector and
inserted into another. However, we also need to
consider what happens in cases where the source
of donor DNA is very complex. We may wish, for
example, to isolate a single gene from the human
Introduction
In the early chapters of this topic, we discussed DNA
cutting and joining techniques, and introduced
the different types of vectors that are available for
cloning DNA molecules. Any cell-based cloning
procedure has four essential parts: (i) a method
for generating the DNA fragment for cloning; (ii) a
reaction that inserts that fragment into the chosen
cloning vector; (iii) a means for introducing that
recombinant vector into a host cell wherein it is
Restriction
endonuclease
digestion
Duplex
cDNA
synthesis
Direct
chemical
synthesis
Mechanical
shearing
DNA
fragments
PCR
Ligation
cohesive
termini
Blunt-end
ligation
(no linker)
Homopolymer
tailing
TA
cloning
Linker
molecules
Joining to
vector
Transfection
with recombinant
phage DNA
Transformation
with recombinant
plasmid DNA
In vitro
packaging into phage
coat: transduction with
recombinant phage or cosmid
Introduction
into host cell
Protein-protein
interactions
Protein-ligand
interactions
Functional
complementation
Gain of
function
Selection or
screening
Hybridization
PCR
Immunochemical
Fig. 6.1
Generalized overview of cloning strategies, with favoured routes shown by arrows. Note that in cell-based cloning
strategies, DNA fragments are initially generated and cloned in a non-specific manner, so that screening for the desired clone is
carried out at the end of the process. Conversely, when specific DNA fragments are obtained by PCR or direct chemical synthesis,
there is no need for subsequent screening.
