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Rflp 2513
- 2. WHAT IS RFLP • The term Restriction Fragment Length Polymorphism, or RFLP refers to a difference between two or more samples of homologous DNA molecules arising from differing locations of restriction sites, and to a related laboratory technique by which these segments can be distinguished.
- 3. Cont…. • Commonly pronounced “rif-lip”. • Its analysis was the first DNA profiling technique cheap enough to see widespread application. • It is an important tool in genome mapping. • Localization of genes for genetic disorders. • Determination of risk for disease, and paternity testing.
- 4. WHAT IS DNA • DNA is a genetic material. • It is a nucleic acid. The structure of a DNA is double helix, two long strands makes the shape of double helix. • Chemically, DNA consist of two long polymers of simple units, called nucleotides, with backbones made up of base, sugars & phosphate groups.
- 5. Restriction Fragment Length Polymorphism • A restriction enzyme cuts the DNA molecules at every occurrence of a particular sequence, called restriction site. • For example, HindII enzyme cuts at GTGCAC or GTTAAC. • If we apply a restriction enzyme on DNA, it is cut at every occurrence of the restriction site into a million restriction fragments each a few thousands nucleotides long.
- 6. Cont… • Any mutation of a single nucleotide may destroy or create the site(CTGCAC or CTTAAC for HindII) and alter the length of the corresponding fragment. • The term polymorphism refers to the slight differences between individuals, in base pair sequences of common genes. • RFLP analysis is the detection of the change in the length of the restriction fragments.
- 7. ANALYSIS TECHNIQUE • The basic technique for detecting RFLPs involves fragmenting a sample of DNA by a restriction enzyme, which can recognize and cut DNA wherever a specific short sequence occurs, in a process known as a restriction digestion. • The resulting DNA fragments are then separated by length through a process known as agarose gel electrophoresis. • Then observed the DNA fragments in UV illuminator
- 10. DNA EXTRACTION PROCEDURE Bogenvillia leaf Crush the leaves with the motor and pestle Add CTAB buffer solution in it Transfer it into clean eppendrof and incubate at 55 C for 20 min in dry bath Centrifuge it at 8000 rpm for 10 min Collect supernatant &add chloroform, phenol & isoamylalcohol (25:24:1) Mix it by inversion Centrifuge at 10000 rpm for 5 min Collect upper layer & add chilled ethanol in equal volume Incubate at -20 C for 24 hours & than centrifuge for 1min at 10000 rpm Collect pallet &resuspended in TE buffer & than use for RFLP analysis
- 11. PREPARATION OF RFLP MIXTURE Assay buffer 10x = 20 micro liter Template DNA or phase DNA = 100 micro liter EcoR I = 3 micro liter Hind III = 3 micro liter BSA(Bovine Serum Albumin) = 5 micro liter Mix it and incubate at 37 C for 1 hour and now prepare the agarose gel and TAE buffer. Preparation of TAE buffer ,Take 2ml of TAE & add 98ml distilled water
- 12. Gel-Electrophoresis • DNA is cut into fragments using an enzyme • The cut DNA is put on a Gel material • An electric current is applied on the Gel • DNA fragments will start moving towards the positively charged side • It shows that DNA is negatively charge • Smaller fragments move faster • After some time, we have a separation of the different fragment lengths
- 13. DNA Sample • Some cells are obtained by DNA extraction technique Restriction Enzyme • A restriction enzyme is used to cut the DNA into fragments • Hind III restriction site is AAGCTT
- 14. RESTRICTION ENZYME • EcoR I restriction site is GAATTC EcoR I • BSA is used in restriction digest to stabilize some enzymes during digestion of DNA and to prevent adhesion of the enzyme to reaction tube. BSA
- 15. Apply Enzyme • RFLP mix are put together in a tube. • The tube is shaken by rotation for DNA, Hind III ,BSA,EcoRI & assay buffer to mix.
- 16. Water Bath • The tube is put on a plate floating on water at 37 C. • It is left for 60 minutes. • This is needed for the apply enzymes reaction to take place
- 17. After incubation collect the eppendrof and add gel loading dye Preparation of DNA dye(gel loading dye) 3ml = 30% glycerol 0.025gm = bromophenol blue 7ml distilled water Now prepare the agarose gel for loading the sample
- 18. Preparing the Agarose Gel • In the meantime, we prepare the Gel. • Agarose powder is the basic substance for making the Gel. • For 2% agarose gel take 2gm agarose powder and dissolved in 100ml boil distilled water.
- 19. • The powder is mixed with water in a container. • The container is heated until the powder completely dissolves in the water. • The solution becomes clear.
- 20. • Now add Etbr.The DNA is visualized in the gel by addition of ethidium bromide. This binds strongly to DNA by intercalating between the bases and is fluorescent meaning that it absorbs invisible UV light and transmits the energy as visible orange light.
- 21. • The liquid Gel is poured into the inner box. • A comb like piece is put at the edge of the inner box. • The liquid Gel is left to cool and solidify. • When the Gel solidifies, the comb will create wells for the DNA samples to be put.
- 22. Gel casting • Fill the H shaped container with TAE buffer solution • Remove comb
- 23. Putting DNA on the Gel • DNA samples mixed with colored solution and UV reactive solution i.e.is DNA dye which we added. • DNA samples inserted into wells • A sample DNA containing only specific fragments (called ladder) can be used for comparison original uncut DNA DNA cut by enzymes DNA with rflp mix ladder
- 24. Run the Gel • Apply electric current • the DNA Fragments will migrate towards the positive charge which means that the DNA is negatively charged • Small fragments move faster
- 25. DNA Fragments Move • The colored solution provides an indication to how much the DNA has traveled on the Gel.
- 26. Observation • Gel can be viewed under UV light in UV illuminator.
- 27. Observation • Original uncut DNA sample makes a sharp band at the beginning (one big fragment) • DNA sample cut with enzymes makes s smear (lots of fragments of all sizes) • Ladders are used for comparison (they contain specific fragments) • We could run it for a longer time to achieve better separation Hind III band EcoR I band Original uncut DNA
- 28. Original uncut DNA Hind III bandEcoR I band RFLP mix
- 29. Restriction fragment length polymorphism (RFLP) is most suited to studies at the intraspecific level or among closely related taxa. Presence and absence of fragments resulting from changes in recognition sites are used identifying species or populations.