What is a restriction enzyme and how are they used?

This image is a cartoon rendition of the EcoRI restriction enzyme. Two catalytic magnesium ions are shown as magenta spheres and are adjacent to the cleaved sites in the DNA made by the enzyme (depicted as gaps in the DNA backbone).

restriction enzyme

Before the 1970′s the goal of isolating a single gene from a large chromosome was impossible. A gene is much different from a protein and it does not exist as a discrete entity within cells, but rather as a small region of a much larger DNA molecule.  DNA molecules could be randomly broken by mechanical force, but to isolate a single gene would be like finding a needle in a haystack. Since all DNA molecules consist of an approximately equal mixture of the same four nucleotides they cannot be readily separated. The solution to this problem began to be solved with the discovery of “restriction nucleases”. These enzymes could be purified from bacteria and can cut the DNA double helix and specific sites defined by the local nucleotide sequence. Restriction enzymes differ from species of bacteria such as EcoRI is from Escherichia coli or Hind III is from Hemophilius influenzae. 

These enzymes are capable of recognizing and cleaving specific DNA sequences of four to eight nucleotide bases. The sequences they recognize are palindromic. This means the base pairs are a palindrome, so EcoRI recognizes the base pair sequence of  5′GAATTC and 3′CTTAAG

 

From the 5′ end the sequence reads GAATTC so it base pairs with 3′ CTTAAG and thus the sequence is a palindrome. Once we know the complete DNA sequence for the gene of interest we can isolate it by finding restriction sites surrounding the gene. The technique has become an important part of the polymerase chain reaction (PCR) which is used to clone specific genes. Another interesting fact is restriction enzymes were used to discover DNA rearrangements of individual B and T lymphocytes.

 

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