Restriction enzymes--permit the cutting of DNA molecules at precise sites

1. Restriction enzymes could be considered a form of antibodies for bacteria. "Self" DNA is distinguished from foreign DNA by methylation.

• Types I and III have methylation activity in the same molecule as the nuclease.
• Type I enzymes cut at random sites flanking recognition sites.
• Type II enzymes are most useful for cloning. The activities that methylate Type II recognition sites are carried on different enzymes, which are frequently available commercially.

2. More than 600 type II enzymes have been isolated; a large fraction of these is commercially available. How to keep track:

• Manufacturers' catalogs (contain much additional information)
• Richard Roberts' annual supplement to Nucleic Acids Research
• Roberts' restriction enzyme database, or REBASE, available by clicking here
• Updates from manufacturers of sequence analysis software

3. Recognition sites are usually symmetric. Cut sites may occur at the center of symmetry (generating blunt ends) or off-center (generating cohesive ends with 5' or 3' overhangs).

4. There are many special manipulations that are frequently required in working with restriction enzymes. These are too numerous to cover here. Some examples are presented in this list. Details of the methods used to carry out these special manipulations can be found in techniques manuals.

• Converting cohesive ends to blunt ends
• Converting blunt ends to cohesive ends
• Cohesive joining of normally incompatible cohesive ends

5. Isoschizomers are enzymes from different sources that have the same recognition site. Frequently an isoschizomer of the enzyme that you need may accomplish the same purpose for a lower price. Most commercial suppliers list available isoschizomers.

6. In some cases, compatible cohesive ends may be generated by enzymes with different recognition sites or recognition sites of different complexities.

7. It is good to be aware of the effects of methylation. Some type II enzymes will not cut when their recognition site is methylated, but others will. It is important to note that many bacterial hosts, from which cloned DNA preparations are made, may methylate some restriction sites. If your restriction enzyme doesn't seem active, consider the possibility that the substrate DNA may be methylated!

• Nucleotide sequences prone to methylation:
  • E. coli: GATC; CCAGG or CCTGG
  • Mammalian DNA : CG
• Dealing with methylation:
  • Manufacturers' information
  • Cloning manuals
  • Methylation-deficient E. coli  strains
• Utilizing methylation:
  • Restriction enzyme recognition site specificities can be altered by appropriate methylation.
  • Internal sites in libraries can be protected by appropriate methylation during cloning.

This page was updated August 30, 1998.

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