1. A SEMINAR ON RAPD’s G.D. RUNGTA GROUP OF SCIENCE AND TECHNOLOGY,
SUBMITTTED BY
GUIDED BY
PREETHA SINGHA
Dr.K.L.TIWARI (DIRECTOR)
Dr. S.K. SEN (PRINCIPAL)
Mrs.PRAGATI
SHUKLA(ASST.PROF)
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 I RAPD & ITS APPLICATION INTRODUCTION DEFINITION HISTORY
PRINCIPLE
TYPES OF MARKER
PROCEDURE OF RAPD MARKER
APPLICATIONS OF MARKER
LIMITATIONS AND ADVANTAGES
COMPARISION BETWEEN THREE MOLECULAR MARKERS
CONCLUSION
REFERENCES

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

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

7. RAPD & ITS APPLICATION P R I Ce
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. RAPD & ITS APPLICATION a known location on a chromosome that can beU A R K
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. 1.The DNA of a selected species is isolated.
RAPD & ITS APPLICATION
PROCEDURE
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. RAPD & ITS APPLICATION PROCEDURE 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. RAPD & ITS APPLICATION 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

12. RAPD & ITS APPLICATION PROTOCOL 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. RAPD & ITS APPLICATION APPL ICAT IONS
Molecular genetic markers have been developed into powerful tools to analyze genetic relationships and genetic diversity.
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.
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. RAPD & ITS APPLICATION A D V N T G E Sfz 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. RAPD & ITS APPLICATION LIMITATIONS LIMITATION
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).
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.

17. RAPD & ITS APPLICATION COMPARISION CHARACTERISTICS RAPD RFLP AFLP
PRINCIPLE
DNA amplification
Restriction digestion
DETECTION
DNA staining
Southern blotting
PRIMER REQUIREMENT
Yes (random primer)
None
Yes (selective primer)
PROBE REQUIREMENT
set of specific probes
DOMINANT/
CODOMINANT
Dominant
Co dominant
Dominant (co dominant )

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

21. Thank you