Agriculture Reference
In-Depth Information
separation of RNA and restriction fragments of DNA by gel electrophoresis in order
to detect specific sequences among the bands by Northern and Southern blotting in
an analogous way in which antibodies are used in Western blotting.
For a long time, since most hybridization was done with radioactive probes and
filter-bound nucleic acids, it was reserved for specialists. Because of the develop-
ment of non-radioactive methods it is now considered safer, less troublesome but
still rather time-consuming to perform. Many current commercial nucleic-based tests
employ the sandwich assay (Sylvanen
et al.,
1986), in which adjacent sections of the
target nucleic acid are hybridized by two probes. One is the capture probe which, by
hybridization, links the target to the solid support to which this probe is already
bound. The other probe is labelled with a reporter group, which may be a fluorescent
molecule, an enzyme or a radioactive atom that hybridizes with an adjacent section
of the target nucleic acid. As the reporter group must be readily distinguished even
in minute amounts, fluorescent molecules have many advantages. However, there
are some disadvantages since many other molecules in biological samples, including
several components of healthy plants, can fluoresce without being labelled. Serious
background fluorescence can often compete with the weaker signals from the probe,
thus limiting sensitivity to unsatisfactory levels.
Enzyme-labelled probes can be used to generate coloured molecules which are
then readily detected. In spite of this, thorough washing is imperative to remove
impurities derived from the hybridization reaction as they inhibit the enzyme. Probes
attached to biotin (a vitamin) bind to avidin (a protein) extremely tightly. As a result,
a more effective complex of many enzyme molecules can be produced by binding
enzymes first to biotin and then to avidin, around which they cluster. Digoxigenin
may be used in a similar way with anti-digoxigenin enzymes. There are several other
ways of enhancing detection systems, employing complexes of enzymes or
luminescent reactions. It is also possible to fasten more than one reporter group to
the labelled probe by making the probe very long or branched. In these ways, non-
radioactive methods are starting to attain a sensitivity equivalent to that expected
from radioactive labels. Improvement in the automation of hybridization is another
beneficial development. The procedures for hybridization developed for these 'cold'
non-radioactive probes are similar to those used for the 'hot' radioactive probes.
Probes of DNA are more widely used than those of RNA. In addition to DNA
viruses, DNA probes can be used to detect RNA viruses by reverse transcription to
produce a complementary DNA (cDNA) copy (Hull, 1986). Generally, probes
consist of a few kilobases (kb) of DNA but may be as short as 15-20 bp or as large
as 30 kb. Most often, the probes for a specific purpose are screened from randomly
cloned fragments of DNA. Even a genomic library covering the whole pathogen can
eventually be built up from such fragments. However, in most cases only a few
specific probes need to be selected as diagnostics. This selection process can be
simplified by subtractive hybridization or genomic subtraction. The non-specific
DNA that is common between the pathogen and another organism, such as its host,
is depleted while that specific to the pathogen only is enriched. This is done by
hybridizing labelled DNA from the target organism - the pathogen - with an excess
(100-200 times the amount) of DNA from the driver, which can be a related
organism or the host. The driver DNA mops up the target DNA common to it,
