Restriction enzymes are DNA-cutting enzymes found in bacteria (and harvested from them for use). Because they cut within the molecule, they are often called restriction endonucleases.
A restriction enzyme recognizes and cuts DNA only at a particular sequence of nucleotides. For example, the bacterium Hemophilus aegypticus produces an enzyme named HaeIII that cuts DNA wherever it encounters the sequence 5'GGCC3'
3'CCGG5'
The cut is made between the adjacent G and C. This particular sequence occurs at 11 places in the circular DNA molecule of the virus phiX174. Thus treatment of this DNA with the enzyme produces 11 fragments, each with a precise length and nucleotide sequence. These fragments can be separated from one another and the sequence of each determined.
| Link to page describing DNA sequencing. |
HaeIII and AluI cut straight across the double helix producing "blunt" ends. However, many restriction enzymes cut in an offset fashion. The ends of the cut have an overhanging piece of single-stranded DNA. These are called "sticky ends" because they are able to form base pairs with any DNA molecule that contains the complementary sticky end. Any other source of DNA treated with the same enzyme will produce such molecules.
Mixed together, these molecules can join with each other by the base pairing between their sticky ends. The union can be made permanent by another enzyme, DNA ligase, that forms covalent bonds along the backbone of each strand. The result is a molecule of recombinant DNA (rDNA).
The ability to produce recombinant DNA molecules has not only revolutionized the study of genetics, but has laid the foundation for much of the biotechnology industry. The availability of human insulin (for diabetics), human factor VIII (for males with hemophilia A), and other proteins used in human therapy all were made possible by recombinant DNA.
| Link to discussion of recombinant DNA. |
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