ASEMINARON RAPD’sGUIDED BYDr.K.L.TIWARI (DIRECTOR)Dr. S.K. SEN (PRINCIPAL)Mrs.PRAGATISHUKLA(ASST.PROF)SUBMITTTED BYPREETHA SINGHAM.s.c 2nd semesterBIOTECHNOLOGYG.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 REFERENCESRAPD & ITS APPLICATION
INTRODUCTIONRAPD & ITS APPLICATIONMARKER- Any genetic trait that can be identified with confidence& relative case and can be followed in a mappingpopulation is called as genetic marker. Genetic marker is a specific location on a chromosome that isdefined by a naked eye polymorphism as differences inelectrophoretic mobility of specific proteins ,or as differences inspecific DNA sequence. There are three types of markers namely:- MORPHOLOGICAL MARKER BIOCHEMICAL MARKER MOLECULAR MARKER
DEFINITIONRAPD & ITS APPLICATION DEFINITION:- RAPD that is defined by differencesbetween individuals in terms of DNA regionseither being or not being amplified in a polymerasechain reaction primed by random oligonucleotidessequences. It is a type of PCR reaction, but the segments of DNA thatare amplified are random.RAPD creates several arbitrary, short primers (8–12 nucleotides), thenproceeds with the PCR using a large template of genomic DNA RAPD- The full form of RAPD is RANDOM AMPLIFIED POLYMORPHICDNAs are obtained by using a PCR equipment or a thermo cycler.RAPD - is a lab technique used to amplifyunknown(random) DNA segments
HISTORYRAPD & ITS APPLICATION1866- Mendel described the inheritancepattern of conceptual hereditaryunits now called genes.1980- Botstein et al suggested DNAmarker RFLP
TYPESOFMARKERRAPD & ITS APPLICATION MOLECULAR MARKERRFLP(Restriction Fragment LengthPolymorphism)RAPD(RANDOMLY AMPLIFIEDPOLYMORPHIC DNA SEGMENTS)AFLP(Amplified Fragment LengthPolymorphism)
PRICIPLeRAPD & ITS APPLICATIONRAPD analysis is a PCR based molecularmarker technique. Single shortoligonucleotide primer is arbitrarilyselected to amplify a set of DNA segmentsdistributed randomly throughout thegenome.
MOLECULARMARKERRAPD & ITS APPLICATION Molecular marker is a gene / DNA sequence witha known location on a chromosome that can beused to identify cells. A marker must be polymorphic that it must existin different forms so that chromosome carryingthe mutant gene can be distinguished from thechromosome with the normal gene by marker.
PROCEDURERAPD & ITS APPLICATIONRAPD involves following steps:-1.The DNA of a selected species is isolated.2. An excess of selected decaoligonucleotideadded.3. This mixture is kept in a PCR equipment andis subjected to repeated cycles of DNAdenaturation-renaturation-DNAreplication.4. During this process, the decaoligonucleotidewill pair with the homologous sequencepresent at different locations in the DNA.
PROCEDURERAPD & ITS APPLICATIONRAPD involves following steps:-5.DNA replication extend the decaoligonucleotide andcopy the sequence continuous with the sequence withwhich the selected oligonucleotide has paired.6.The repeated cycles of denaturation - renaturation-DNAreplication will amplify this sequence of DNA.7. Amplification will takes place only of those regions ofthe genome that has the sequence complementary tothe decaoligonucleotide at their both ends.8. After several cycles of amplification the DNA issubjected to gel electrophoresis.9. The amplified DNA will form a distinct band. it isdetected by ethidium bromide staining and visiblefluorescences under U.V. light
PROTOCOLRAPD & ITS APPLICATIONIsolation of DNAKeep the tubes in PCR thermocyclerDenature the DNA (94°C,1 minDNA strands separatedDecaoligonucleotide enzyme, primer, Taq DNApolymerase,Annealing of primer (36°C,2 minPrimer annealed to template DNA strandsDNA synthesis (72°C, 1.5 min
PROTOCOLRAPD & ITS APPLICATIONComplementary strand synthesis35 to 45 cyclesAmplified products separated by gelelectrophoresisBands detected by Ethidium bromide staining
RAPD & ITS APPLICATIONFig. no. 1 diagrammatic view of RAPD
APPLICATIONSRAPD & ITS APPLICATION1. Molecular genetic markers have been developed into powerfultools to analyze genetic relationships and genetic diversity.2. As an extension to the variety of existing techniques usingpolymorphic DNA markers, the Random Amplified PolymorphicDNA (RAPD) technique may be used in molecular ecology todetermine 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 ofDNA are available Efficiency and low expense.
ADVANTAGESfzRAPD & ITS APPLICATIONADVANTAGES1. Morphological character represents theactual phenotype importance while protein& DNA markers are important only asarbitrary loci used for linkage mapping &often do not corresponds directly tospecific phenotypes.2. It provides a quick and efficient screening for DNA sequencebased polymorphism at many loci.3. It involve no radioactive assays.
LIMITATIONRAPD & ITS APPLICATION1. Nearly all RAPD markers are dominant, i.e. it is not possible todistinguish whether a DNA segment is amplified from a locus that isheterozygous (1 copy) or homozygous (2 copies).2. Co-dominant RAPD markers, observed as different-sized DNAsegments amplified from the same locus, are detected only rarely.3. PCR is an enzymatic reaction, therefore the quality and concentrationof template DNA, concentrations of PCR components, and the PCRcycling conditions may greatly influence the outcome.4. Mismatches between the primer and the template may result in thetotal absence of PCR product as well as in a merely decreased amountof the product. Thus, the RAPD results can be difficult to interpret.LIMITATIONS
COMPARISIONRAPD & ITS APPLICATIONCHARACTERISTICS RAPD RFLP AFLPPRINCIPLE DNA amplification RestrictiondigestionDNA amplificationDETECTION DNA staining Southern blotting DNA stainingPRIMERREQUIREMENTYes (randomprimer)None Yes (selectiveprimer)PROBEREQUIREMENTNone set of specificprobesNoneDOMINANT/CODOMINANTDominant Co dominant Dominant (codominant )
conclusionRAPD & ITS APPLICATIONsummaryRAPD is a lab technique used to amplify unknown(random) DNAsegmentsIt is a technique firstly DNA is isolated, which is then treated withdecaoliganucleotide enzymes it act as a restriction enzymes whichis used to cleave a short ten nucleotide segments of DNA.Then mixture is taken to PCR equipment and the process of DNAdenaturation and the annealing of primer occcurs, then primerextension takes place for 35 to 45 cycles.DNA hybridizaion occurs at some segment of DNA,amplificationoccurs at a particular site.DNA is subjected to gel electrophoresis,the amplified DNA willform distinct band detected by ethidium bromide staining andvisible fluorescence’s under U.V.light
conclusionRAPD & ITS APPLICATIONconclusionRandom Amplified PolymorphicDNA (RAPD) markers are DNAfragments from PCR amplificationof random segments of genomicDNA with single primer of arbitrarynucleotide sequence.
REFERENCEsRAPD & ITS APPLICATIONREFERENCESBOOK NAME AUTHOR’S NAME PUBLICATIONNAMEYEARBOOK OFBIOTECHNOLOGYSATYANARAYANAU.2010BIOTECHNOLOGY,EXPANDINGHORIZONSSINGH B.D. KALYANIPUBLICATIONS2010INTRODUCTION TOPLANTBIOTECHNOLOGYCHAWLA H.S. OXFORD AND IBHPUBLISHING,THIRDEDITION2010