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RFLP & RAPD

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Molecular Markers- Genetic mapping

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RFLP & RAPD

  1. 1. A SEMINAR ON MOLECULAR MARKERS RFLP-RAPD DR. ARUNIMA KARKUN(ASST. PROF). 1 1
  2. 2.  INRODUCTION  DEFINITION  HISTORY  ANALYSIS  TECHNIQUES  ADVANTAGES  DISADVANTAGES  APPLICATIONS  CONCLUSION  REFERENCE S Y N O P S I S MOLECULAR MARKER RFLP & RAPD 2
  3. 3.  RFLP - RESTRICTION FRAGMENT LENGTH POLYMORPHISM  RFLP & is very useful study in Genomic DNA Sequence.  RFLP has been developed for chromosomes mapping of humans, mice, maize, tomato,rice,etc.  RFLP is a non-PCR based method .  RFLP based Genetic Marker.  In this Method DNA is digested with restriction Enzymes.  RFLP is the co dominant marker.  RFLP is 1-10 loci detected. MOLECULAR MARKER RFLP & RAPD I N R O D U C T I O N 3
  4. 4.  “The Variations in the Restriction DNA Fragments length between individuals of a species is called RFLP.” MOLECULAR MARKER RFLP & RAPD D E F I N I T I O N 4
  5. 5.  BOTSTEIN.et. al.(1980)-The DNA sequence variation Detected by this method was termed RFLP.  GRODZICKER first Described this phenomenon for mutant strain of adenoviruses. H I S T O R Y MOLECULAR MARKER RFLP & RAPD 5
  6. 6. Genomic DNA isolation. Digestion with Restriction enzyme. Fractions on a Agarosegel. DNA in millions of restriction fragments fractionated in the gel by Molecular marker weight . DNA transferred out of the Gel on to a membrane filter southern Hybridization. Autoradiography RFLP pattern with positive Bands. MOLECULAR MARKER RFLP & RAPD A N A L Y S I S 6
  7. 7.  Results are based on reliable genotypic characteristics' rather than on phenotypes.  RFLP based Genetic Marker  RFLP is the co dominant marker  RFLP & is very useful study in Genomic DNA Sequence A D V A N T E G E S MOLECULAR MARKER RFLP & RAPD . 7
  8. 8. D I S A D V A N T E G E S  Time consuming.  Multistep procedure. MOLECULAR MARKER RFLP & RAPD 8
  9. 9.  SAMPLE– COLLECTIONS  Tissue are cells of individual are collected to extract their DNA.  The sample are collected separately.  ISOLATION OF DNA  DNA of the collected samples is isolated separately by standard procedure& purified to get genomic DNA.  RESTRICTION – DIGESTION  Genomic DNA of each samples is cut with a restriction enzyme separately To generate variable leanth of DNA fragments. MOLECULAR MARKER RFLP & RAPD R F L P - M E T H O D 9
  10. 10.  Restriction enzyme such as ECOR1, HINDIII ,Pst, etc ,are of much use for RFLP.  ELECTROPHORESIS  The Digested genomic DNA of the samples are loaded into separate cells in agarose or polyacrelamide gel &subjected to electrophoresis.  BLOTTING OF DNA  As in southern blotting the DNA is transferred to a nitrocellulose filter by using a blotting set up.  AUTOREDIOGRAPHY  Images of radioactive probes are captured on an x- Ray film using autoradiography. R F L P - M E T H O D MOLECULAR MARKER RFLP & RAPD 10
  11. 11.  Method involves:  a) digestion of extracted DNA by restriction enzymes,.  b) gel electrophoresis of fragments,.  c) southern blot by specific probes and detection of specific sequences. R F L P MOLECULAR MARKER RFLP & RAPD 11
  12. 12. R F L P MOLECULAR MARKER RFLP & RAPD 12
  13. 13.  RFLP technique can be used in Forensic Medicines.  To characterize Germplasm resources .  Use of genotype specific RFLP patterns can be done to distinguish verities in crop like maize, rice, soya bean, potato etc.  To assess genetic diversity.  To identify Breeding lines & varieties. MOLECULAR MARKER RFLP & RAPD A P P L I C A T I O N S 13
  14. 14. - I N R O D U C T I O N  RAPD –Random Amplified Polymorphic DNA.  RAPD are of much use to construct genetic maps.  The RAPD is a PCR based Method.  RAPD is a very general method for obtaining a molecular fingerprinting or a strain species .  RAPD analysis is a PCR based molecular marker technique.  It is a Dominant marker.  It s a 1-3 loci detected. MOLECULAR MARKER RFLP & RAPD 14
  15. 15.  DEFINITION  “The set of DNA generated by the Random PCR is called RAPD.”  “If primers with arbitrary sequences are used for amplification, DNA segments to be amplified will be selected at random and this provides truly random sample of DNA markers and so is described as random amplified polymorphic DNA (RAPD)”.  HISTORY  The method was Developed by J.G.K.Williams et.al in (1991). MOLECULAR MARKER RFLP & RAPD R A P D 15
  16. 16. Genomic DNA Isolation / Cells or tissues . Keep the tubes in PCR thermo cycler. Denature DNA 940C 1 minutes DNA Strands separated. Annealing of primers. Complementary DNA Synthesis (35 -45 cycles). Amplified product separated by Gel Electrophoresis R A P D - A N A L Y S I S MOLECULAR MARKER RFLP & RAPD 16
  17. 17.  Method involves: a) extraction of DNA, b) amplification by PCR using random primers. c) gel electrophoresis of amplified DNA and visualization of markers. R A P D MOLECULAR MARKER RFLP & RAPD 17
  18. 18. MOLECULAR MARKER RFLP & RAPD R A P D 18
  19. 19.  RAPD is a Quick Method.  RAPD Analysis can be performed in crude DNA Sample also.  It detect Dominant variation in the Genome.  It involves non-radioactive assays.  RAPD required only a small amount of DNA samples. A D V A N T A G E S MOLECULAR MARKER RFLP & RAPD 19
  20. 20. U S E S MOLECULAR MARKER RFLP & RAPD  RAPD is used as Genetic markers for constructing genetic maps of higher organisms.  Example – pines, rice etc.  RAPD analysis help as to identify genes of high economic values through comparison of RAPD fragments .  Example- rice, wheat, maize , pea etc.  RAPD Markers help to determine specific genes in chromosomes.  RAPD can be used for identification of somatic Hybrid among the developing regenerates .  RAPD can be used for evaluation &character of genomic resources . 20
  21. 21. D I F F R E N C E R F L P & R A P D MOLECULAR MARKER RFLP & RAPD RFLP RAPD 1-3 loci detected.  1-10 loci detected Can detect allelic variants. Cannot detect allelic variants Technique comparatively more reliable. Technique comparatively less reliable. Large quantity of purified DNA required i.e. 2-10µg. Quantity of DNA required for analysis is small 10-50µg. Different species specific probes are required. Same primers with arbitrary sequence can be used for different species. 21
  22. 22.  RFLP - RESTRICTION FRAGMENT LENGTH POLYMORPHIS C O N C L U T I O N MOLECULAR MARKER RFLP & RAPD RAPD –RANDOM AMPLIFIED POLYMORPHIC DNA. RFLP is a non-PCR based method . RAPD is a Quick Method. RAPD required only a small amount of DNA samples. RFLP based Genetic Marker. 22
  23. 23. MOLECULAR MARKER RFLP & RAPD R E F R E N C E S AUTHER NAME EDITION BOOK NAME H.S.CHAWLA 1998 BIOTECNOLOGY S.N.JOGDAND 2009 BIOTECNOLOGY B.D.SINGH 2010 BIOTECNOLOGY V.KUMARESAN 2011 BIOTECNOLOGY 23
  24. 24. THANK YOU 24

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