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Polymorphic DNA)
RAPD (Random Amplified Polymorphic DNA)
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Genomic DNA
PCR
Buffer
Arbitrary Primer
The RAPD protocol usually uses a 10 bp arbitrary primer at
constant low annealing temperature (generally 34 – 37 °C).
RAPD primers can be purchased as sets or individually from
different sources, such as the University of British Colombia
(http://www.michaelsmith.ubc.ca/services/
NAPS/Primer_Sets) and the Operon Biotechnologies
(http://www.operon.com).
Two basic criteria indicated by Williams et al. (1990) must be
met: a minimum of 40% GC content (50 - 80%) GC content
is generally used) and the absence of palindromic sequence
Because G-C bond consists of three hydrogen bridges and
the A-T bond of only two, a primer-DNA hybrid with less than
50% GC will probably not withstand the 72 oC temperature at
which DNA elongation takes place by DNA polymerase.
The resulting PCR products are generally resolved on
1.5-2.0% agarose gels and stained with ethidium
bromide (EtBr); polyacrylamide gels in combination
with either AgNO3 staining (e.g., Huff et al., 1993; Vejl,
1997;Hollingsworth et al., 1998)
PCR
360 bp
Electrophoresis
A B C
260 bp
520bp
520 bp 360 bp
A B C 260 bp
PCR product occurs when:
The primers anneal in a particular orientation
(such that they point towards each other)
The primers anneal within a reasonable
distance of one another (150 -3000 bp)
The number of amplification products is related to the
number and orientation of the genome sequences which are
complementary to the primer
1 2 3
4 5 6
Product 1 Product 2
The nature of RAPD
polymorphism
a) nucleotide substitution within target sites may affect
the annealing process - either no fragment is detected
1 3
4 5 6
No product
Product 2
or detected fragment is of increased size
1 3
4 5 6
Product 1 Product 2
b) insertion or deletion of a small fragment of DNA - the
amplified fragments are changed in size
Insertion 3
1 2
Deletion
4 5 6
DNA template
PCR reaction
Product 1 Product 2
c) insertion of a large piece of DNA between the primer -
binding sites may exceed the capacity of PCR - no fragment
is detected
5 6
DNA template PCR reaction
No product Product 2
A schematic picture of an agarose gel
Monomorphic bands
Polymorphic bands