RFLP

Restriction fragment length polymorphism, Procedure and its application

1. Dr. T. Ramesh Assistant Professor PG & Research Department of Zoology Vivekananda College RESTRICTION FRAGMENT LENGTH POLYMORPHISM

2. Definition Post Graduate and Research Department of Zoology 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 1-10 loci detected

3. Post Graduate and Research Department of Zoology “The Variations in the Restriction DNA Fragments length between individuals of a species is called RFLP.” 1. BOTSTEIN.et. al.(1980)-The DNA sequence variation Detected by this method was termed RFLP. 2. GRODZICKER first Described this phenomenon for mutant strain of adenoviruses. Definition and History

4. Analysis Post Graduate and Research Department of Zoology STEPS: 1.Genomic DNA isolation. 2. Digestion with Restriction enzyme. 3. Fractions on a Agarosegel . 4. DNA in millions of restriction fragments fractionated in the gel by Molecular marker weight . 4. DNA transferred out of the Gel on to a membrane filter southern Hybridization. 5. Autoradiography 6. RFLP pattern with positive Bands.

5. Procedure 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 length of DNA fragments Restriction enzyme such as EcoR1, HINDIII, Pst , etc,are of much use for RFLP. Post Graduate and Research Department of Zoology

6. Procedure  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. Post Graduate and Research Department of Zoology

7. Procedure Post Graduate and Research Department of Zoology

8. Action of Restriction Enzymes Total DNA Size 6.5 kb EcoR1 3kb 3.5kb Two Fragments Hind III 2kb 4.5kb Two fragments EcoR1+HindIII 2kb 1kb 3.5kb Three Fragments A B C EcoR1- A+B (2+1 kb) EcoR1- C (3.5kb) Hind III- A (2kb) Hind III- B+C (1+3.5 kb) Post Graduate and Research Department of Zoology A B C

9. Advantages and Disadvantages 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 ADVANTAGES Time consuming. Multistep procedure. Post Graduate and Research Department of Zoology DISADVANTAGES

10. 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 Applications Post Graduate and Research Department of Zoology

11. THANK YOU .