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

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

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

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

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

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

RAPD & ITS APPLICATION P R I C e 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.

RAPD & ITS APPLICATION a known location on a chromosome that can be U 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.

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.

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

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

RAPD & ITS APPLICATION PROTOCOL 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

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.

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.

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). 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.

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 )

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

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.

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

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