cells, because of their ease of growth and short doubling time. Gram-negative bacteria
such asE. colican be madecompetentfor the introduction of extraneous plasmid DNA
into cells (Section 6.3.1). The natural ability of bacteriophage to introduce DNA intoE.
colihas also been well exploited and results in 10–100-fold higher efficiency for the
introduction of recombinant DNA compared to transformation of competent bacteria
with plasmids. These well-established and traditional approaches are the reason why so
many cloning vectors have been developed forE. coli. The delivery of cloning vectors
into eukaryotic cells is, however, not as straightforward as that for the bacteriumE. coli.
It is possible to deliver recombinant molecules into animal cells bytransfection.
The efficiency of this process can be increased by first precipitating the DNA with
Ca^2 þor making the membrane permeable with divalent cations. High-molecular-
weight polymers such as DEAE-dextran or polyethylene glycol (PEG) may also be
used to maximise the uptake of DNA. The technique is rather inefficient although a
selectable marker that provides resistance to a toxic compound such as neomycin can
be used to monitor the success. Alternatively, DNA can be introduced into animal cells
byelectroporation. In this process the cells are subjected to pulses of a high-voltage
gradient, causing many of them to take up DNA from the surrounding solution. This
technique has proved to be useful with cells from a range of animal, plant and
microbial sources. More recently the technique oflipofectionhas been used as the
delivery method. The recombinant DNA is encapsulated by a core of lipid-coated
particles which fuse with the lipid membrane of cells and thus release the DNA into
the cell. Microinjection of DNA into cell nuclei of eggs or embryos has also been
performed successfully in many mammalian cells.
The ability to deliver recombinant molecules into plant cells is not without its prob-
lems. Generally the outer cell wall of the plant must be stripped, usually by enzymatic
digestion, to leave a protoplast. The cells are then able to take up recombinants from the
supernatant. The cell wall can be regenerated by providing appropriate media. In cases
where protoplasts have been generated transformation may also be achieved by electro-
poration. An even more dramatic transformation procedure involves propelling micro-
scopically small titanium or gold pellet microprojectiles coated with the recombinant
DNA molecule, into plant cells in intact tissues. Thisbiolistictechnique involves the
detonation of an explosive charge which is used to propel the microprojectiles into the
cells at a high velocity. The cells then appear to reseal themselves after the delivery of
the recombinant molecule. This is a particularly promising technique for use with plants
whose protoplasts will not regenerate whole plants.6.4 Hybridisation and gene probes
6.4.1 Cloned DNA probes
The increasing accumulation of DNA sequences in nucleic acid databases coupled
with the availability of custom synthesis of oligonucleotides has provided a relatively
straightforward means to design and produce gene probes and primers for PCR. Such223 6.4 Hybridisation and gene probes