“RESTRICTION FRAGMENT LENGTH POLYMORPHISM”

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.

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.

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.

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.

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.

A restriction fragment length polymorphism (RFLP) The DNA molecule on the left has a polymorphic restriction site (marked with the asterisk) that is not present in the molecule on the right. The RFLP is revealed after treatment with the restriction enzyme because one of the molecules is cut into four fragments whereas the other is cut into three fragments.

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

ANALYSIS TECHNIQUE cont.. Then transferred to a membrane via the Southern blot procedure Hybridization of the membrane to a labeled DNA probe then determines the length of the fragments which are complementary to the probe. Each fragment length is considered an allele, and can be used in genetic analysis.

DNA EXTRACTION PROCEDURE Transfer it into clean eppendrof and incubate at 55 C for 20 min in dry bath Add CTAB buffer solution in it Crush the leaves with the motor and pestle Bogenvillia leaf Mix it by inversion Collect supernatant &add chloroform, phenol & isoamylalcohol (25:24:1) Centrifuge it at 8000 rpm for 10 min Collect pallet &resuspended in TE buffer & than use for RFLP analysis Incubate at -20 C for 24 hours & than centrifuge for 1min at 10000 rpm Collect upper layer & add chilled ethanol in equal volume Centrifuge at 10000 rpm for 5 min

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

DNA Sample Restriction Enzyme 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

RESTRICTION ENZYME EcoR I restriction site is GAATTC BSA is used in restriction digest to stabilize some enzymes during digestion of DNA and to prevent adhesion of the enzyme to reaction tube. EcoR I BSA

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.

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

Preparation of DNA dye(gel loading dye) 3ml = 30% glycerol 0.025gm = bromophenol blue 7ml distilled water After incubation collect the eppendrof and add gel loading dye Now prepare the agarose gel for loading the sample

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.

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.

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.

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.

Gel casting Fill the H shaped container with TAE buffer solution Remove comb

Putting DNA on the Gel DNA cut by enzymes 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 with rflp mix ladder

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

DNA Fragments Move The colored solution provides an indication to how much the DNA has traveled on the Gel.

Observation Gel can be viewed under UV light in UV illuminator.

Observation Original uncut DNA Hind III band Original uncut DNA sample makes a sharp band at the beginning (one big fragment) DNA sample cut with enzymes makes 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 EcoR I band

EcoR I band Hind III band Original uncut DNA RFLP mix

Applications: RFLPs can be applied in diversity and phylogenetic studies ranging from individuals within populations or species, to closely related species. RFLPs have been widely used in gene mapping studies because of their high genomic abundance due to the ample availability of different restriction enzymes and random distribution throughout the genome. RFLP markers were used for the first time in the construction of genetic maps by Botstein et al.1980.

CONCLUSION 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. 

THANK YOU -PHARMA STREET