The document provides information on RAPD (Random Amplified Polymorphic DNA) and RFLP (Restriction Fragment Length Polymorphism) molecular marker techniques. RAPD uses short random primers to amplify random DNA segments via PCR. RFLP involves digesting DNA with restriction enzymes, separating fragments by size, and detecting variants by probing fragmented DNA attached to membranes. Both techniques are used for applications like genetic diversity analysis, but RAPD requires less DNA and is quicker while RFLP has higher reproducibility and can detect allelic variants.

1. Presented By
BIKASH KUMAR SINGH
RAPD & RFLP
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2. •RAPDs are DNA fragments amplified by PCR using short
synthetic primers (generally 10 bp) of random sequence.
•RAPD analysis is a PCR based molecular marker
technique. Single short oligonucleotide primer is
arbitrarily selected to amplify a set of DNA segments
distributed randomly throughout the genome.
RAPD-Random Amplified Polymorphic DNA
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3. 1. The DNA of a selected species is isolated.
2. An excess of selected Decaoligonucleotide added.
3. This mixture is kept in a PCR equipment and is subjected to
repeated cycles of DNA denaturation-renaturation-DNA
replication.
4. During this process, the decaoligonucleotide will pair with the
homologous sequence present at different locations in the DNA.
RAPD involves following steps:-
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4. 5.DNA replication extend the decaoligonucleotide and copy the sequence
continuous with the sequence with which the selected oligonucleotide has
paired.
6.The repeated cycles of denaturation - renaturation-DNA replication will amplify
this sequence of DNA.
7. Amplification will takes place only of those regions of the genome that has the
sequence complementary to the decaoligonucleotide at their both ends.
8. After several cycles of amplification the DNA is subjected to gel electrophoresis.
9. The amplified DNA will form a distinct band. it is detected by ethidium bromide
staining and visible fluorescence's under U.V. light
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6. 6
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8. Template
DNA
Primers point away
from each other, so
amplification won’t
happen
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9. Template
DNA
Primers point in
the same
direction, so
amplification
won’t happen
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10. RAPD
Template
DNA
Primers too far
apart, so
amplification
won’t happen > 2,000 bases
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11. RAPD
Template
DNA
100 - 1,500 bases
Primers are just the
right distance apart, so
fragment is amplified
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12. Advantages
• It requires no DNA probes and sequence information for the design of
specific primers.
• It involves no blotting or hybridisation steps, hence, it is quick, simple and
efficient.
• It requires only small amounts of DNA (about 10 ng per reaction) and the
procedure can be automated.
• High number of fragments.
• Arbitrary primers are easily purchased.
• Unit costs per assay are low compared to other marker technologies.
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13. • The main advantage of RAPDs is that they are quick and easy to
assay. Because PCR is involved, only low quantities of template
DNA are required.
• Since random primers are commercially available, no sequence
data for primer construction are needed. Moreover, RAPDs have
a very high genomic abundance and are randomly distributed
throughout the genome.
Advantage
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14. Disadvantages
• Nearly all RAPD markers are dominant,
• PCR is an enzymatic reaction, therefore, the quality and concentration
of template DNA, concentrations of PCR components, and the PCR
cycling conditions may greatly influence the outcome.
• Mismatches between the primer and the template may result in the
total absence of PCR product as well as in a merely decreased amount
of the product.
• Lack of a prior knowledge on the identity of the amplification products.
• Problems with reproducibility.
• Problems of co-migration.
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15. Applications
• genetic diversity/polymorphism,
• germplasm characterization,
• genetic structure of populations,
• hybrid purity,
• genome mapping,
• developing genetic markers linked to a trait in question,
• population and evolutionary genetics,
• plant and animal breeding,
• animal-plant-microbe interactions,
• pesticide/herbicide resistance.
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16. •A restriction fragment length
polymorphism (RFLP) is a genetic
variant that can be examined by
cleaving the DNA into fragments
(restriction fragments) with a
restriction enzyme.
WHAT IS RFLP:
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18. • Isolating DNA is the first step for many DNA-based
technologies. DNA is found either in nuclear
chromosomes or in organelles (mitochondria and
chloroplasts).
• To extract DNA from its location, several laboratory
procedures are needed to break the cell wall and
nuclear membrane, and so appropriately separate
the DNA from other cell components.
• When doing so, care must be taken to ensure the
process does not damage the DNA molecule and that
it is recovered in the form of a long thread.
Isolating DNA
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19. • Extracted DNA is digested with specific, carefully chosen, restriction
enzymes.
• Each restriction enzyme, under appropriate conditions, will
recognize and cut DNA in a predictable way, resulting in a
reproducible set of DNA fragments (‘restriction fragments’) of
different lengths.
• The millions of restriction fragments produced are commonly
separated by electrophoresis on agarose gels. Because the
fragments would be seen as a continuous ‘smear’ if stained with
ethidium bromide, staining alone cannot detect the polymorphisms.
• Hybridisation must therefore be used to detect specific fragments.
Restriction digestion and gel electrophoresis
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20. • DNA transfer is called ‘Southern blotting’, after E.M. Southern
(1975), who invented the technique.
• In this method, the gel is first denatured in a basic solution
and placed in a tray. A porous nylon or nitrocellulose
membrane is laid over the gel, and the whole weighted
down.
• All the DNA restriction fragments in the gel transfer as single
strands by capillary action to the membrane. All fragments
retain the same pattern on the membrane as on the gel.
DNA transfer by Southern blotting
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21. • The membrane with the target DNA is incubated with the DNA probe.
Incubation conditions are such that if strands on the membrane are
complementary to those of the probe, hybridisation will occur and
labeled duplexes formed.
The DNA probe is a single-stranded molecule, conveniently labeled, using any
standard method (e.g. a radioisotope or digoxygenin), and hybridized with the target
DNA, which is stuck to the membrane.
• Where conditions are highly stringent, hybridisation with distantly
related or non-homologous DNA does not happen.
• Thus, the DNA probe picks up sequences that are complementary and
'ideally‘ homologous to itself among the thousands or millions of
undetected fragments that migrate through the gel.
• Desired fragments may be detected after simultaneous exposure of the
hybridized membrane and a photographic film.
DNA hybridisation: The procedure
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22. RFLP technology in pictures
After agarose has been
poured into the gel
mould, combs are
immediately inserted to
form wells and left until
the gel hardens. The
combs are then removed
and the gel placed in an
electrophoresis chamber.
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23. Samples of
digested DNA,
with bromophenol
blue dye added,
are loaded into
the wells
with a pipette.
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24. After electrophoresis,
the gel is treated with
NaCl to break the
DNA double helix
bonds and make it
single-stranded. This
allows later
hybridisation with a
single-stranded DNA
probe.
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25. The blotting tray is
first prepared by
saturating sponges
with NaOH. Safety
glasses and gloves
are required, and a
laboratory coat
recommended.
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26. Absorbent paper is placed on top of the
sponges to prevent direct contact with
the gel.
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27. Bubbles between the absorbent paper and sponges are removed by rolling
a pipette or a glass rod across the paper. This ensures a complete transfer
of the solution all through
the gel.
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28. The treated agarose
gel is placed on top of
the absorbent paper.
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29. Membrane is cut
into the appropriate
size.
The membrane is
placed on top of the
gel, then covered
with a piece of
absorbent paper.
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30. The entire set-up is topped
with a weight (here, a
bottle of water standing on
a piece of glass) to
promote good transfer.
After some hours the
transfer is complete, the
blotting paper is taken
away, and the membrane
stored until hybridisation
with the probe.
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31. The process of hybridisation begins. A DNA is boiled
to denature it to single strands.
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32. The labeled probe is
added to the container
with the hybridisation
solution and membrane,
and incubated overnight
in an oven. The
following day, the
membrane is removed
from the hybridisation
set up, and washed with
the appropriate
stringency solution.
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33. The membrane is then
blotted dry and put into
a cassette for holding X-
ray film.
The cassette is wrapped,
or sealed with tape, and
stored in a freezer until
the film is
sufficiently exposed,
usually 1 to 4 days.
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34. This is an RFLP autoradiogram.
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35. RFLPs can be used in many different settings to
accomplish different objectives.
RFLPs can be used in paternity cases or criminal cases to
determine the source of a DNA sample. (i.e. it has forensic
applications).
RFLPs can be used determine the disease status of an
individual. (e.g. it can be used in the detection of mutations
particularly known mutations).
In human population genetics, geographical isolates and
comparison of genetical makeup of related species.
Applications of RFLP:
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36. RFLPs can be used in criminal cases
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37. • Highly robust methodology with good transferability
between laboratories.
• No sequence information required.
• highly recommended for phylogenetic analysis between
related species.
• Well suited for constructing genetic linkage maps.
• Simplicity—given the availability of suitable probes, the
technique can readily be applied to any plant.
Advantages of RFLPs
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38. • Large amounts of DNA required.
• Automation not possible.
• Low levels of polymorphism in some species.
• Time consuming, especially with single-copy probes
• Costly.
• Moderately demanding technically.
Disadvantages of RFLPs
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39. D
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F
F
R
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N
C
E
R
F
L
P
&
R
A
P
D
MOLECULAR MARKER RFLP & RAPD
RFLP RAPD
1-3 loci detected.  1-10 loci detected
Can detect allelic variants. Cannot detect allelic variants
Technique comparatively more
reliable.
Technique comparatively less
reliable.
Large quantity of purified DNA
required i.e. 2-10µg.
Quantity of DNA required for
analysis is small 10-50µg.
Different species specific probes are
required.
Same primers with arbitrary sequence
can be used for different species.
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