A SEMINAR

ON RAPD’s

GUIDED BYDr.K.L.TIWARI (DIRECTOR)

Dr. S.K. SEN (PRINCIPAL)Mrs.PRAGATI SHUKLA(ASST.PROF)

SUBMITTTED BYPREETHA SINGHA

M.s.c 2nd semesterBIOTECHNOLOGY

G.D. RUNGTA GROUP OF SCIENCE AND TECHNOLOGY,

KOHKA, KURUD ROAD, BHILAI, (C.G.),490023

SYNOPSIS

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

INTRODUCTION

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

DEFINITION

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

HISTORY

RAPD & ITS APPLICATION

1866- Mendel described the inheritance pattern of conceptual hereditary units now called genes.

1980- Botstein et al suggested DNA marker RFLP

TYPES

OF

MARKER

RAPD & ITS APPLICATION

MOLECULAR MARKER RFLP(Restriction Fragment Length

Polymorphism)

RAPD(RANDOMLY AMPLIFIED POLYMORPHIC DNA SEGMENTS)

AFLP(Amplified Fragment Length Polymorphism)

PRICIPLe

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.

MOLECULAR

MARKER

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.

PROCEDURE

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.

PROCEDURE

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

PROTOCOL

RAPD & ITS APPLICATION

Isolation of DNA

Keep the tubes in PCR thermocycler

Denature the DNA (94°C,1 min

DNA strands separatedDecaoligonucleotide 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

PROTOCOL

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

APPLICATIONS

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.

ADVANTAGESfz

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.

LIMITATION

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

COMPARISION

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 )

conclusion

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

conclusion

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.

REFERENCEs

RAPD & ITS APPLICATION

REFERENCESBOOK NAME AUTHOR’S NAME PUBLICATION

NAMEYEAR

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

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