Restriction endonucleases [local] are defense proteins produced by bacteria to protect against viral infections. They are named for the bacterial species from which they are isolated (e.g. EcoR I from Escherichia coli and Kpn I from Klebsiella pneumoniae) They are dimeric proteins [local] that hydrolyze DNA in sequence-specific locations. The dimeric endonucleases bind to locations that thave the same specific sequence on both strands. These sites where the same sequence is present on both strands are palindromes.

Examples:

5'-----G^AATTC-----3'

3'-----CTTAA^G-----5'

This is recognized and cleaved by EcoR I

5'-----GGTAC^C-----3'

3'-----C^CATGG-----5'

This is recognized and cleaved by Kpn I

Thus, restriction endonucleases produce pieces of DNA with matching overlaps at the ends. These are termed 'sticky ends' and two pieces of DNA with complementary sticky ends can bind to one another.

DNA Mapping

DNA Mapping [local] is something that can be done with restriction endonucleases. It is often called restriction mapping. The technique also relies on the ability to fractionate DNA fragments by electrophoresis. You have seen electrophoresis used to fractionate and analyze proteins. The methods to fractionate proteins and DNA are somewhat different. Proteins are usually fractionated in gels of polyacrylamide held between two glass plates oriented in a vertical position. The pattern of fractionated proteins is usually revealed by staining the gel. Polynucleotides are usually fractionated in gels composed of agarose held in a horizontal orientation within a buffer environment. Fractionated DNA and RNA is commonly visualized by fluorescence produced by an intercalating agent like ethidium bromide.

To make a restriction map, the piece of DNA must be treated with a restriction endonuclease and the digestion allowed to proceed to completion. The fragments are then fractionated by electrophoresis on an agarose along with marker fragments of known size. This produces information about the number of restriction enzyme sites on the DNA of interest, but not their location. By analyzing the patterns produced by multiple restriction enzymes, both separately and together, and by isolating single fragments and digesting them again with different enzymes, it is possible to produce a map that locates all of the restriction sites on that DNA.

Limitations: Very small fragments tend not to be resolved by electrophoresis. Very large pieces of DNA (> 20 kb) tend not to be resolved. Two fragments of about the same size are also not resolved from one another, but it is frequently possible to discern that since such a 'band' will have twice as much fluorescence as expected.