Basic techniques - Principles of Gene Manipulation - page 9

recombinant molecules has also been termed genetic
engineering or gene manipulation because of the po-
tential for creating novel genetic combinations by
biochemical means. The process has also been termed
molecular cloning or gene cloning because a line of
genetically identical organisms, all of which contain
the composite molecule, can be propagated and grown
in bulk, hence amplifying the composite molecule
and any gene product whose synthesis it directs .
Although conceptually very simple, cloning of
a fragment of foreign, or passenger , or target DNA
in a vector demands that the following can be
accomplished.
• The vector DNA must be purified and cut open.
• The passenger DNA must be inserted into the
vector molecule to create the artificial recombinant.
DNA joining reactions must therefore be performed.
Methods for cutting and joining DNA molecules are
now so sophisticated that they warrant a chapter of
their own (Chapter 3).
• The cutting and joining reactions must be read-
ily monitored. This is achieved by the use of gel
electrophoresis.
• Finally, the artificial recombinant must be trans-
formed into E . coli or another host cell. Further details
on the use of gel electrophoresis and transformation
of E . coli are given in the next section. As we have
noted, the necessary techniques became available at
about the same time and quickly led to many cloning
experiments, the first of which were reported in
1972 ( Jackson et al . 1972, Lobban & Kaiser 1973).

kb pairs

21.226

7.421
5.804
5.643
4.878

3.530

-

+

Fig. 2.1 Electrophoresis of DNA in agarose gels. The direction
of migration is indicated by the arrow. DNA bands have been
visualized by soaking the gel in a solution of ethidium bromide
(see Fig. 2.3), which complexes with DNA by intercalating
between stacked base-pairs, and photographing the orange
fluorescence which results upon ultraviolet irradiation.

electrophoresis is not well understood (Holmes
& Stellwagen 1990). The migration of the DNA
molecules through the pores of the matrix must play
an important role in molecular-weight separations
since the electrophoretic mobility of DNA in free
solution is independent of molecular weight. An
agarose gel is a complex network of polymeric
molecules whose average pore size depends on the
buffer composition and the type and concentration
of agarose used. DNA movement through the gel
was originally thought to resemble the motion of a
snake (reptation). However, real-time fluorescence
microscopy of stained molecules undergoing elec-
trophoresis has revealed more subtle dynamics
(Schwartz & Koval 1989, Smith et al . 1989). DNA
molecules display elastic behaviour by stretching in
the direction of the applied field and then contract-
ing into dense balls. The larger the pore size of the

Agarose gel electrophoresis

The progress of the first experiments on cutting and
joining of DNA molecules was monitored by velocity
sedimentation in sucrose gradients. However, this
has been entirely superseded by gel electrophoresis.
Gel electrophoresis is not only used as an analytical
method, it is routinely used preparatively for the
purification of specific DNA fragments. The gel is
composed of polyacrylamide or agarose. Agarose is
convenient for separating DNA fragments ranging
in size from a few hundred base pairs to about 20 kb
(Fig. 2.1). Polyacrylamide is preferred for smaller
DNA fragments.
The mechanism responsible for the separation
of DNA molecules by molecular weight during gel