Genetic Manipulation - Environmental Biotechnology: Theory and Application - page 220

which changes from colourless to yellow on hydrolysis in much the same way as
the blue/white screening described above for the cloning vector, pGEM ® .Other
reporter genes produce enzymes which can cause the emission of light such as
the luciferase isolated from fireflies, or whose activity is easy and quick to assay
like the bacterial -glucuronidase (GUS) which is probably the most frequently

used reporter genes in transgenic plants. Reporter genes can only be a guide to
the process of transcription and translation occurring in the cell and it has been
acknowledged for some time that care must be exercised to avoid misinterpreting
data (Pessi, Blumer and Haas, 2002).
As with selector genes, the reporter genes serve no useful purpose once the
cloning procedure has been successfully accomplished to produce the finished
product. In the early days of this technology, these genes would normally be
left in situ to avoid the extra work of removing them which might also upset the
structure of the recombinant genome thus diminishing the quality of the carefully
engineered organism. There is, however, an argument to remove all genes which
were necessary for construction purposes but which no longer serve a useful
purpose, to reduce perceived potential risk of unwittingly increasing the spread of
genes throughout the environment. These concerns are addressed in Chapter 10.

β

Analysis of Recombinants

The design of the plasmid was such that insertion of 'foreign' DNA allows for
a colour test, or causes a change in antibiotic sensitivity, either to resistance
(positive selection) or sensitivity (negative selection). This constitutes the first
step in screening. The second stage is usually to probe for the desired gene
using molecules which will recognise it and to which is attached some sort of
tag, usually radioactive or one able to produce a colour change. The next stage
is normally to analyse the DNA isolated from possible recombinants, firstly by
checking the size of the molecule or pieces thereof, or by sequencing the DNA.
This is the most informative approach but used to be very laborious. With the
current and ever developing automated protocols, DNA sequencing has become
a standard part of recombinant analysis procedure. However, if a large number
of samples are to be analysed it is usually quicker and cheaper to scan them by
a procedure described as a Southern blot, after Ed Southern, the scientist who
designed the technique.
In this procedure, the DNA is spread out by electrophoresis on a gel which
is then probed by a piece of DNA complementary to the sequence of interest.
If a band shows up on autoradiography then the probe has found a mate and
the required sequences are present, at least in part. DNA sequencing is then
required to confirm exactly what has occurred during the cloning procedure but
the advantage is that only the samples which are very likely to contain the
required insert are sequenced, thus saving time and expense.
From this technique, the Northern blot has developed, which is much the same
idea except that the material spread out on the gel is RNA rather than DNA, and