RAPD markers are decamer DNA fragments. RAPD is a type of PCR reaction. as the name suggest it is a fast method when compared to the traditional PCR medthod.

1. RAPD
Random Amplified polymorphic DNA.
Presented by: Abhi Giri
M.sc-II (Oceanography)
Sem – III / Paper – II
Guided by: Mrs S. P. Jayani
(M.sc, DMLT)
Head of Department

2. Contents • A brief History of RAPD.
• An Introduction to RAPD.
• HOW? It works.
• Differences between genomes using RAPD.
• Interpreting RAPD banding patterns.
• Procedure / Protocol.
• Advantages.
• Disadvantages.
• Summary.
• Conclusions.
• References.

3. Markers
 Markers :- Any trait that can be identified with confidence &
relative case and can be followed in a mapping population is
called as marker.
 Genetic marker :- Genetic marker is a specific location on a
chromosome that is defined by a naked eye polymorphism as
differences in electrophoretic mobility of specific proteins, or as
differences in specific DNA sequence.
 There are three types of markers namely:-
• MORPHOLOGICAL MARKER
• BIOCHEMICAL MARKER
 MOLECULAR MARKER

4. History
• Shortly after Kary Mullis invented PCR it was realized that
short primer would bind several locations in a genome &
thus could multiple fragments.
• Williams et.al (1990) developed RAPD a technique used
very short 10 base primers to generate random fragments
from template DNAs
• RAPD fragments can be separated & used as genetic
markers or a kind of DNA fingerprint.
• Techniques related to RAPD include:
• DNAAmplification Fingerprinting (DAF) - Caetano-Anolles
et al. (1991) uses very short (eight nucleotide long) primers
• Arbitrary Primed PCR (AP-PCR) - Welsh and McClelland
(1990) uses longer primers, but lowers primer annealing
stringency to get priming at many sites

5. INTRODUCTION
• RAPD markers are decamer DNA fragments.
• RAPD is a type of PCR reaction, Segments amplified are
Random.
• No knowledge of DNA sequence required. Hence a
popular method.
• In recent years, RAPD is used to Characterize, & Trace,
the phylogeny of diverse plant & animal species.
• Identical 10-mer primer will or will not amplify a
segment of DNA, depending on positions that are
complementary to the primer sequence.

6. How it Works ?
• The principle is that, a single, short oligonucleotide primer,
which binds to many different loci, is used to amplify random
sequences from a complex DNA template.
• This means that the amplified fragment generated by PCR
depends on the length and size of both the primer and the target
genome.
• These amplified products (of up to 3.0 kb) are usually
separated on agarose gels (1.5-2.0%) and visualised by
ethidium bromide staining.

7. • Nucleotide variation between different sets of template
DNAs will result in the presence or absence of bands
because of changes in the priming sites.
• Recently, sequence characterised amplified regions
(SCARs) analysis of RAPD polymorphisms showed that
one cause of RAPD polymorphisms is chromosomal
rearrangements such as insertions/deletions.
• In order for PCR to occur:
1) the primers must anneal in a particular orientation
(such that they point towards each other) and,
2) they must anneal within a reasonable distance of one
another.

8. FINDING DIFFERENCES BETWEEN GENOMES
USING RAPD ANALYSIS
• Consider the Figure 2 (genome A). If
another DNA template (genome B) was
obtained from a different (yet related)
source, there would probably be some
differences in the DNA sequence of the
two templates. Suppose there was a
change in sequence at primer annealing
site #2
Figure 2 (genome A)
Figure 3 (genome B)

9. • Genome A and B can represent genomic DNA
from two individuals in the same species or
possibly from two different species.
• Certain portions of genomic DNA tend to
much conserved (very little variation) while
other portions tend to vary greatly among
individuals within a species or among different
species.
• The trick in RAPD PCR analysis is to:
1. Find those sequences which have just
enough variation to allow us to detect
differences among the organisms that we are
studying.
2. find the right PCR primers which
will allow us to detect sequence differences.
(genomes A and B) on a agarose gel

10. INTERPRETING RAPD BANDING PATTERNS
• Each gel is analysed by scoring the present (1) or absent (0)
polymorphic bands in individual lanes. The scoring can be done
based on the banding profiles which is clear and transparent (Fig. 5a)
otherwise the scoring is very difficult (Fig. 5b).
(Fig. 5a) (Fig. 5b)

11. • Criteria for selecting scoring bands:
1) reproducibility—need to repeat experiments.
2) thickness 3) size and,
4) expected segregation observed in a mapping
population.
• DNA polymorphism among individuals can be due to:
1) mismatches at the primer site.
2) appearance of a new primer site and,
3) length of the amplified region between primer sites.
• The NTSYS-pc software ver. 2.02 is used to estimate
genetic similarities with the Jaccard’s coefficient.

12. Components of a PCR and RAPD Reactions
RAPD
1. Buffer (containing Mg++) - usually
high Mg++ concentrations are used
lowering annealing stringency
2. Template DNA
3. 1 short primer (10 bases) not
known to anneal to any specific
part of the template DNA
4. dNTPs
5. Taq DNA Polymerase (or another
thermally stable DNA polymerase)
PCR
1. Buffer (containing Mg++)
2. Template DNA
3. 2 Primers that flank the
fragment of DNA to be
amplified
4. dNTPs
5. Taq DNA Polymerase (or
another thermally stable
DNA polymerase)

13. Procedure RAPD involves following steps:-
• 1.The DNA of a selected species is isolated.
-quality influences the outcome of the PCR
-high molecular weight.
-No impure template
-less amount of RNA
• 2. An excess of selected decaoligonucleotide added.
-10 base primer for PCR, only 1 primer per reaction
- Short primer bind randomly on the chromosome.

14. • 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.
• 5.DNA replication extend the decaoligonucleotide and
copy the sequence continuous with the sequence with
which the selected oligonucleotide has paired.

15. Procedurecont.
• 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.

16. • 8. After several cycles of amplification the DNA is
subjected to gel electrophoresis.
Initial denaturation at 94°C for 10 min.
Denaturation at 94°C for 1 min.
Annealing at 37-45°C for 1 min.
Extension at 72°C for 1 min.
Final extension at 72°C for 10 min.
Cooling at 4°C.
• 9.The amplified DNA will form a distinct band. it
is detected by ethidium bromide staining and
visible fluorescence's under U.V. light

17. PCR Melting
94 oC
Melting
94 oC
Annealing
Primers
50 oC
Extension
72 oC
Temperature
100
0
50
T i m e
5’3’
3’5’
3’5’
5’
5’3’
5’
3’5’
5’
5’
5’
5’3’
3’5’
3’5’
5’3’
5’3’
5’

18. RAPD
Melting
94 oC
Temperature
100
0
50
T i m e
5’3’
3’5’

19. RAPD
Melting
94 oC
Temperature
100
0
50
T i m e
3’5’
5’3’
Heat

20. RAPD
Melting
94 oC
Annealing
Primers
50 oC
Extension
72 oCTemperature
100
0
50
T i m e
3’5’
5’3’
5’
5’
Melting
94 oC

21. RAPD
Melting
94 oC
Melting
94 oC
Annealing
Primers
50 oC
Extension
72 oCTemperature
100
0
50
T i m e
30x
3’5’
5’3’
Heat
Heat
5’
5’
5’

22. RAPD
Melting
94 oC
Melting
94 oC
Annealing
Primers
50 oC
Extension
72 oCTemperature
100
0
50
T i m e
30x
3’5’
5’3’
5’
5’
5’
5’
5’
5’

23. RAPD
Melting
94 oC
Melting
94 oC
Annealing
Primers
50 oC
Extension
72 oCTemperature
100
0
50
T i m e
30x
3’5’
5’3’
5’
5’
5’
5’
5’
5’
Heat
Heat

24. Fragments of
defined length
RAPD
Melting
94 oC
Melting
94 oC
Annealing
Primers
50 oC
Extension
72 oCTemperature
100
0
50
T i m e
30x
5’
5’
5’
5’
5’
5’
5’
5’
3’5’
5’3’
5’
5’

25. Primers
• Primers are commercially available from various source
(ex.. Opéron Technologies Inc.,California; Biosciences,
Bangalore; Euro Finns, Bangalore; GCC Biotech,
Kolkata).

26. protocol Isolation of DNA
Keep the tubes in PCR thermocycler
Denature the DNA (94°C,1
min
DNA strands separated
Decaoligonucleotide enzyme, primer, Taq DNA
polymerase,
Annealing of primer (36°C,2 min
Primer annealed to template DNA strands
DNA synthesis (72°C, 1.5 min

27. Protocolcont. Complementary strand synthesis
35 to 45 cycles
Amplified products separated by gel
electrophoresis
Bands detected by Ethidium bromide staining

28. RAPD
Template
DNA
 Primer binds to many locations on the template DNA
 Only when primer binding sites are close and oriented in opposite
direction so the primers point toward each other will amplification
take place

29. RAPD
Template
DNA
Primers point away
from each other, so
amplification won’t
happen

30. RAPD
Template
DNA
Primers point in
the same
direction, so
amplification
won’t happen

31. RAPD
Template
DNA
Primers too far
apart, so
amplification
won’t happen > 2,000 bases

32. RAPD
Template
DNA
100 - 1,500 bases
Primers are just the
right distance apart, so
fragment is amplified

33. MM 2 3 4 5 6 7 8 9 10
Separated RAPD Fragments
4mM MgCl2
1.2 U Taq
5 pM OPA-16
4mM MgCl2
0.6 U Taq
10 pM OPA-16
2mM MgCl2
1.2 U Taq
10 pM OPA-16
Normal
concentrations are
shown in green text.
M = A size standard
Lowering
Magnesium ion
concentration
results in loss of
the largest
fragment visible
in lanes 2-7
RAPD reactions
were run in groups
of 3 using the same
template and primer,
but varying
Magnesium,
polymerase and
primer
concentrations

34. Fig. no. 1 diagrammatic view of RAPD

35. 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.

36. 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.

37. DEVELOPING LOCUS-SPECIFIC, CO-DOMINANT
MARKERS FROM RAPDs
• The polymorphic RAPD marker band is isolated from the
gel.
• It is amplified in the PCR reaction. The PCR product is
cloned and sequenced.
• New longer and specific primers are designed for the DNA
sequence, which is called the sequenced characterized
amplified region marker (SCAR).

38. 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.

39. Summary • RAPD is a lab technique used to amplify unknown(random)
DNA segments
• It is a technique firstly DNA is isolated, which is then treated
with decaoliganucleotide enzymes it act as a restriction
enzymes which is used to cleave a short ten nucleotide
segments of DNA.
• Then mixture is taken to PCR equipment and the process of
DNA denaturation and the annealing of primer occcurs, then
primer extension takes place for 35 to 45 cycles.
• DNA hybridizaion occurs at some segment of DNA
amplification occurs at a particular site.
• DNA is subjected to gel electrophoresis,the amplified DNA
will form distinct band detected by ethidium bromide
staining and visible fluorescence’s under U.V.light

40. Conclusion
• RAPD markers exhibit reasonable speed, cost and
efficiency compared with other methods and,
• RAPD can be done in a moderate laboratory. Therefore,
despite its reproducibility problem, it will probably be
important until better techniques are developed in terms
of cost, time and labour.

41. ReferenceS
BOOK AUTHOR PUBLICATION YEAR
BOOK OF BIOTECHNOLOGY
SATYANARAYANA U. UPALLA INTERLINK 2010
BIOTECHNOLOGY Susan R. Barnum Vikas Publishing House 2010
BASIC BIOTECHNOLOGY,
2nd Edition
Colin Rateledge and Bjorn
Kristiansen
Cambridge
Univ. Press
2008
MOLECULAR BIOLOGY Welsh J & McClelland M
Cambridge
Univ. Press 1990