Presented by-V.L-AM. ScDepartment of biotechnologyCentral Mizoram university

RESTRICTION FRAGMENT LENGTH POLYMORPHISM

Restriction Fragment Length Polymorphism (RFLP) is a technique in which organisms may be differentiated by analysis of patterns derived from cleavage of their DNA.

If two organisms differ in the distance between sites of cleavage of a particular restriction endonuclease, the length of the fragments produced will differ when the DNA is digested with a restriction enzyme.

The similarity of the patterns generated can be used to differentiate species (and even strains) from one another.

A restriction fragment length polymorphism (RFLP) is a genetic variant that can be examined by cleaving the DNA into fragments (restriction fragments) with a restriction enzyme.

WHAT IS RFLP:

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.

Restriction Fragment LengthPolymorphism

Any mutation of a single nucleotide may destroy (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.

or A polymorphism is a clinically harmless DNA variation

that does not affect the phenotype.

RFLP analysis is the detection of the change in the length of the restriction fragments.

Cont..

RFLP detection relies on the possibility ofcomparing band profiles generated after Restriction enzymedigestion of target DNA. These differences in fragmentlengths can be seen after gel electrophoresis, hybridisationand visualization. The Laboratory steps involved are asfollows: Isolation of DNA Restriction digestion and gel electrophoresis DNA transfer by Southern blotting DNA hybridization

RFLP technology

Isolating DNA is the first step for many DNA-based technologies. DNA is found either in nuclear chromosomes or in organelles (mitochondria and chloroplasts).

To extract DNA from its location, several laboratory procedures are needed to break the cell wall and nuclear membrane, and so appropriately separate the DNA from other cell components.

When doing so, care must be taken to ensure the process does not damage the DNA molecule and that it is recovered in the form of a long thread.

Isolating DNA

Extracted DNA is digested with specific, carefully chosen, restriction enzymes.

Each restriction enzyme, under appropriate conditions, will recognize and cut DNA in a predictable way, resulting in a reproducible set of DNA fragments (‘restriction fragments’) of different lengths.

The millions of restriction fragments produced are commonly separated by electrophoresis on agarose gels. Because the fragments would be seen as a continuous ‘smear’ if stained with ethidium bromide, staining alone cannot detect the polymorphisms.

Hybridisation must therefore be used to detect specific fragments.

Restriction digestion and gel electrophoresis

DNA transfer is called ‘Southern blotting’, after E.M. Southern (1975), who invented the technique.

In this method, the gel is first denatured in a basic solution and placed in a tray. A porous nylon or nitrocellulose membrane is laid over the gel, and the whole weighted down.

All the DNA restriction fragments in the gel transfer as single strands by capillary action to the membrane. All fragments retain the same pattern on the membrane as on the gel.

DNA transfer by Southern blotting

The membrane with the target DNA is incubated with the DNA probe. Incubation conditions are such that if strands on the membrane are complementary to those of the probe, hybridisation will occur and labeled duplexes formed.

The DNA probe is a single-stranded molecule, conveniently labeled, using any standard method (e.g. a radioisotope or digoxygenin), and hybridized with the target DNA, which is stuck to the membrane.

Where conditions are highly stringent, hybridisation with distantly related or non-homologous DNA does not happen.

Thus, the DNA probe picks up sequences that are complementary and 'ideally‘ homologous to itself among the thousands or millions of undetected fragments that migrate through the gel.

Desired fragments may be detected after simultaneous exposure of the hybridized membrane and a photographic film.

DNA hybridisation: The procedure

RFLP technology in picturesAfter agarose has been poured into the gel mould, combs are immediately inserted to form wells and left until the gel hardens. The combs are then removed and the gel placed in an electrophoresis chamber.

Samples of digested DNA, with bromophenol blue dye added, are loaded into the wellswith a pipette.

After electrophoresis, the gel is treated with NaCl to break the DNA double helix bonds and make it single-stranded. This allows later hybridisation with a single-stranded DNA probe.

The blotting tray is first prepared by saturating sponges with NaOH. Safety glasses and gloves are required, and a laboratory coat recommended.

Absorbent paper is placed on top of the sponges to prevent direct contact with the gel.

Bubbles between the absorbent paper and sponges are removed by rolling a pipette or a glass rod across the paper. This ensures a complete transfer of the solution all throughthe gel.

The treated agarose gel is placed on top of the absorbent paper.

Membrane is cut into the appropriate size.The membrane is placed on top of the gel, then covered with a piece of absorbent paper.

The entire set-up is topped with a weight (here, a bottle of water standing on a piece ofglass) to promote good transfer. After some hours the transfer is complete, the blottingpaper is taken away, and the membrane stored until hybridisation with the probe.

The process of hybridisation begins. A DNA is boiled to denature it to single strands.

The labeled probe is added to the container with the hybridisation solution and membrane, and incubated overnight in an oven. The following day, the membrane is removed from the hybridisation set up, and washed with the appropriate stringency solution.

The membrane is then blotted dry and put into a cassette for holding X-ray film.

The cassette is wrapped, or sealed with tape, and stored in a freezer until the film issufficiently exposed, usually 1 to 4 days.

This is an RFLP autoradiogram.

RFLPs can be used in many different settings to accomplish different objectives. RFLPs can be used in paternity cases or

criminal cases to determine the source of a DNA sample. (i.e. it has forensic applications).

RFLPs can be used determine the disease status of an individual. (e.g. it can be used in the detection of mutations particularly known mutations).

In human population genetics, geographical isolates and comparison of genetical makeup of related species.

Applications of RFLP:

RFLPs can be used in criminal cases

Highly robust methodology with good transferability between laboratories.

No sequence information required.

highly recommended for phylogenetic analysis between related species.

Well suited for constructing genetic linkage maps.

Simplicity—given the availability of suitable probes, the technique can readily be applied to any plant.

Advantages of RFLPs

Large amounts of DNA required. Automation not possible. Low levels of polymorphism in some

species. Time consuming, especially with single-copy

probes Costly. Moderately demanding technically.

Disadvantages of RFLPs

References :- http://www.researchgate.net/publication/22

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learning_space/.../RFLPs.pdf www.kau.edu.sa/.../18591_Restriction%20Fr

agment%20Length%20Poly...

www.ncbi.nlm.nih.gov › ProbeDB › Technologie

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