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Restriction Fragment Length Polymorphism

Restriction fragment length polymorphism (RFLP) is a method for estimating or identifying genetic variants in a population when there is no possibility of determining the exact DNA sequence in a region of the genome. This variation is a genetic polymorphism which is inherited from generation to generation and so provides information on relationships.

DNA can be fragmented in several ways; some methods break DNA at random positions, but there are certain bacterial enzymes, called restriction enzymes, which break DNA at very specific short sequences called restriction sites.

The following diagram shows how the EcoR I enzyme recognises and cuts a DNA sequence.

RFLP1.png

A restriction enzyme will cut a strand of DNA in many places, producing fragments of many different lengths. The fragments are separated by size (or weight) using electrophoresis. The fragments are transferred to a plastic sheet. We can determine which fragments are present. We use a short radioactive-labelled sequence of DNA (the probe) which will match and bind to most fragments. Then the probe is bound to the separated fragments. The sheet, with the bound probes, is placed on a radioactive-sensitive photographic film. The film is developed to reveal the positions of the bands. These bands correspond to the different fragments.

RFLP2.png

The RFLP method is useful to scientists because there are many different types of genetic variation which cause the loss or gain of restriction fragments. The following diagram shows some of these causes. The restriction sites might be gained or lost, or the region between the sites might get longer or shorter. Can you find the fragment which will no longer be recognised?

RFLP3.png

There are many different types of restriction enzyme, each recognizing a different sequence of DNA and so producing a different set of fragments for analysis.