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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
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 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
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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
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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
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RAPD & ITS APPLICATION
1866- Mendel described the inheritance
pattern of conceptual hereditary
units now called genes.
1980- Botstein et al suggested DNA
marker RFLP
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RAPD & ITS APPLICATION
 MOLECULAR MARKER
RFLP(Restriction Fragment Length
Polymorphism)
RAPD(RANDOMLY AMPLIFIED
POLYMORPHIC DNA SEGMENTS)
AFLP(Amplified Fragment Length
Polymorphism)
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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.
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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.
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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.
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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
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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
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RAPD & ITS APPLICATION
Complementary strand synthesis
35 to 45 cycles
Amplified products separated by gel
electrophoresis
Bands detected by Ethidium bromide staining
RAPD & ITS APPLICATION
Fig. no. 1 diagrammatic view of RAPD
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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.
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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.
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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
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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 )
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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
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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.
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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
Rapd ppt

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Rapd ppt

  • 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