1. A
SEMINAR
ON RAPD’s
GUIDED BY
Dr.K.L.TIWARI (DIRECTOR)
Dr. S.K. SEN (PRINCIPAL)
Mrs.PRAGATI
SHUKLA(ASST.PROF)
SUBMITTTED BY
PREETHA SINGHA
M.s.c 2nd semester
BIOTECHNOLOGY
G.D. RUNGTA GROUP OF SCIENCE AND TECHNOLOGY,
KOHKA, KURUD ROAD, BHILAI, (C.G.),490023

2. S
Y
N
O
P
S
I
S
 INTRODUCTION
 DEFINITION
 HISTORY
 PRINCIPLE
 TYPES OF MARKER
 PROCEDURE OF RAPD MARKER
 APPLICATIONS OF MARKER
 LIMITATIONS AND ADVANTAGES
 COMPARISION BETWEEN THREE MOLECULAR MARKERS
 CONCLUSION
 REFERENCES
RAPD & ITS APPLICATION

3. I
N
T
R
O
D
U
C
T
I
O
N
RAPD & ITS APPLICATION
MARKER- Any genetic trait that can be identified with confidence
& relative case and can be followed in a mapping
population is called as 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. D
E
F
I
N
I
T
I
O
N
RAPD & ITS APPLICATION
 DEFINITION:- RAPD that is defined by differences
between individuals in terms of DNA regions
either being or not being amplified in a polymerase
chain reaction primed by random oligonucleotides
sequences.
 It is a type of PCR reaction, but the segments of DNA that
are amplified are random.
RAPD creates several arbitrary, short primers (8–12 nucleotides), then
proceeds with the PCR using a large template of genomic DNA
 RAPD- The full form of RAPD is RANDOM AMPLIFIED POLYMORPHIC
DNAs are obtained by using a PCR equipment or a thermo cycler.
RAPD - is a lab technique used to amplify
unknown(random) DNA segments

5. H
I
S
T
O
R
Y
RAPD & ITS APPLICATION
1866- Mendel described the inheritance
pattern of conceptual hereditary
units now called genes.
1980- Botstein et al suggested DNA
marker RFLP

6. T
Y
P
E
S
O
F
M
A
R
K
E
R
RAPD & ITS APPLICATION
 MOLECULAR MARKER
RFLP(Restriction Fragment Length
Polymorphism)
RAPD(RANDOMLY AMPLIFIED
POLYMORPHIC DNA SEGMENTS)
AFLP(Amplified Fragment Length
Polymorphism)

7. P
R
I
C
I
P
L
e
RAPD & ITS APPLICATION
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.

8. M
O
L
E
C
U
L
A
R
M
A
R
K
E
R
RAPD & ITS APPLICATION
 Molecular marker is a gene / DNA sequence with
a known location on a chromosome that can be
used to identify cells.
 A marker must be polymorphic that it must exist
in different forms so that chromosome carrying
the mutant gene can be distinguished from the
chromosome with the normal gene by marker.

9. P
R
O
C
E
D
U
R
E
RAPD & ITS APPLICATION
RAPD involves following steps:-
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-DNAreplication.
4. During this process, the decaoligonucleotide
will pair with the homologous sequence
present at different locations in the DNA.

10. P
R
O
C
E
D
U
R
E
RAPD & ITS APPLICATION
RAPD involves following steps:-
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

11. P
R
O
T
O
C
O
L
RAPD & ITS APPLICATION
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

12. P
R
O
T
O
C
O
L
RAPD & ITS APPLICATION
Complementary strand synthesis
35 to 45 cycles
Amplified products separated by gel
electrophoresis
Bands detected by Ethidium bromide staining

13. RAPD & ITS APPLICATION
Fig. no. 1 diagrammatic view of RAPD

14. A
P
P
L
I
C
A
T
I
O
N
S
RAPD & ITS APPLICATION
1. Molecular genetic markers have been developed into powerful
tools to analyze genetic relationships and genetic diversity.
2. As an extension to the variety of existing techniques using
polymorphic DNA markers, the Random Amplified Polymorphic
DNA (RAPD) technique may be used in molecular ecology to
determine taxonomic identity, assess kinship relationships,
analyze mixed genome samples, and create specific probes.
3. Main advantages of the RAPD technology include:-
 Suitability for work on anonymous genomes
 Applicability to problems where only limited quantitie of
DNA are available
 Efficiency and low expense.

15. A
D
V
A
N
T
A
G
E
S
f
z
RAPD & ITS APPLICATION
ADVANTAGES
1. Morphological character represents the
actual phenotype importance while protein
& DNA markers are important only as
arbitrary loci used for linkage mapping &
often do not corresponds directly to
specific phenotypes.
2. It provides a quick and efficient screening for DNA sequence
based polymorphism at many loci.
3. It involve no radioactive assays.

16. L
I
M
I
T
A
T
I
O
N
RAPD & ITS APPLICATION
1. Nearly all RAPD markers are dominant, i.e. it is not possible to
distinguish whether a DNA segment is amplified from a locus that is
heterozygous (1 copy) or homozygous (2 copies).
2. Co-dominant RAPD markers, observed as different-sized DNA
segments amplified from the same locus, are detected only rarely.
3. 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.
4. 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. Thus, the RAPD results can be difficult to interpret.
LIMITATIONS

17. C
O
M
P
A
R
I
S
I
O
N
RAPD & ITS APPLICATION
CHARACTERISTICS RAPD RFLP AFLP
PRINCIPLE DNA amplification Restriction
digestion
DNA amplification
DETECTION DNA staining Southern blotting DNA staining
PRIMER
REQUIREMENT
Yes (random
primer)
None Yes (selective
primer)
PROBE
REQUIREMENT
None set of specific
probes
None
DOMINANT/
CODOMINANT
Dominant Co dominant Dominant (co
dominant )

18. c
o
n
c
l
u
s
i
o
n
RAPD & ITS APPLICATION
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

19. c
o
n
c
l
u
s
i
o
n
RAPD & ITS APPLICATION
conclusion
Random Amplified Polymorphic
DNA (RAPD) markers are DNA
fragments from PCR amplification
of random segments of genomic
DNA with single primer of arbitrary
nucleotide sequence.

20. R
E
F
E
R
E
N
C
E
s
RAPD & ITS APPLICATION
REFERENCES
BOOK NAME AUTHOR’S NAME PUBLICATION
NAME
YEAR
BOOK OF
BIOTECHNOLOGY
SATYANARAYANA
U.
2010
BIOTECHNOLOGY
,EXPANDING
HORIZONS
SINGH B.D. KALYANI
PUBLICATIONS
2010
INTRODUCTION TO
PLANT
BIOTECHNOLOGY
CHAWLA H.S. OXFORD AND IBH
PUBLISHING,THIRD
EDITION
2010