Agriculture Reference
In-Depth Information
probes gives a wide range of possibilities for exposing
variation in the DNA sequences.
RFLP's are highly reproducible; they show codom-
inance in their expression and are reliably specific.
However, they are relatively time consuming, not easy
to automate, require fairly large amounts of 'clean' DNA
and, not inevitably, tend to use radioactive probes for
best results.
of the PCR conditions or ingredients and the markers
show dominance
More reliable methods that have been developed are:
Amplified Fragment Length Polymorphism (AFLP) and
this is not only more repeatable but also gives much
higher frequency of bands; and inter-simple sequence
repeats (ISSR or anchored micro-satellites). However,
the details of these are beyond our present remit.
PCR methods - site targeted techniques - single
locus systems
Rather than using arbitrary primers, it is possible to
specifically design primers to be used in PCR. There are
a number of possibilities to design primers but one such
approach is Sequence Tagged Micro-satellites (STMS).
Micro-satellites are simple sequence repeats which are
found around the genome and are generally quite vari-
able in exact base pair composition. If one pictures
these at different places in the genome, the DNA 'flank-
ing' these regions will be different depending on where
they are (i.e. the site at which they are found will be
unique). So you can 'fish' for these with simple repeats,
then sequence the bands and design primers with the
main part being simple repeats but the ends being other
unique 'tags'. This allows the production of much more
robust markers to be generated but with the advantage
of the PCR technology.
PCR methods - arbitrarily primed
techniques - multi-locus systems
The most commonly used approach is Randomly
Amplified Polymorphic DNA (RAPD). The technique
basically involves using a single 'arbitrary' primer in a
PCR reaction. The primer is basically just a short stretch
of DNA. The basic ingredient of the PCR reaction is
DNA polymerase, an enzyme that enables the copying
of a duplicate molecule of DNA from a DNA template,
and is commonly Ta q polymerase a thermally stable
DNA polymerase. The primer anneals to the comple-
mentary sequences in the DNA we are investigating
and 'primes' the polymerase amplification. So the events
which occur are:
Isolate the DNA from the organism of interest
Put in thermal cycler with the primer and polymerase
Denature the double-stranded DNA by heating
Anneal primers to initiate extension of sites flanking
region by cooling
Uses of molecular markers
Primer extension - synthesis of DNA strands comple-
mentary to the region between the flanking primers
with Ta q polymerase
Molecular markers can therefore be used to:
Repeat the three cycles, above, basically doubling the
specific region determined by the primer on each
cycle - so quickly enriching the mixture to be almost
purely pieces of this one region of DNA - the basis
of PCR
Identify cultivars (DNA finger printing), to differen-
tiate one cultivar from another (perhaps one already
released), or to be able to prove proprietary owner-
ship of specific cultivars. If you have a modest set of
markers it is possible to produce a 'DNA finger-print'
which is unique (or nearly so) and so be potentially
used to identify that particular genotype. Similarly
using the same principal it is possible to identify DNA
that is not supposed to be there and so can be used to
ensure that a particular cultivar is pure and free from
contaminants. A further possibility is afford by the
potential to assess how diverse genotypes are at the
DNA level and hence assess their level of difference
(genetic distance) if used as parents (so e.g. parents of
hybrid cultivars).
The products are separated on agarose gel, commonly,
in the presence of ethidium bromide and visualized
under ultraviolet light
The advantages of RAPDs are that it requires only
small amounts of DNA, it requires modest equipment
(thermal cycler and electrophoresis equipment); and
no prior knowledge of the gene or DNA sequence is
required. It is fast and relatively inexpensive. However,
the results can be variable depending on slight changes
 
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