Restriction Enzyme Lab Report
Function of Restriction Enzymes:
Restriction endonucleases cleave the phosphodiester bond between an adjacent phosphate and deoxyribose group in the phosphate backbone of the DNA. The active site of the endonuclease perform this cleavage by binding to the side chain of certain amino acids to the phosphate group through a chemical bond. This dissolves the preexisting bond between the deoxyribose sugar and the phosphate resulting in a breakage with in the DNA chain at a specific location. (3, 7)
One characteristic feature of restriction endonucleases is that they cut at a very particular site having a specific DNA sequence. This specific sequence that allows the enzyme to attach is known as the recognition site. Consider the example of the first restriction enzyme discovered, EcoRI.
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Pulse field electrophoresis is used for the separation of larger fragments of DNA. These fragments result from digesting a bacterial genome with a rare-cutting restriction enzyme. The pattern of DNA produced on the gel is used to differentiate different strains of bacteria. For the identification of individuals like humans or other organisms, Restriction fragment length polymorphism (RELP) analysis has turn out to be very practical. After isolation of DNA from the source it is digested enzymatically with the help of restriction endonucleases. Enzyme treated DNA is then separated by size in an agarose gel and shifted to a membrane. A radioactive or fluorescently labeled probe is bonded with the DNA on the membrane. They target specific sequences that are marked by the restriction enzyme sites. The size of these fragments varies hence generate a biological bar code of restriction enzyme- digested DNA fragments. This pattern is unique to each individual. Restriction enzymes are fore sighted to be an integral part of the modern genetics. (3,
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