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Restriction Enzymes

The isolation, characterization, and commercial production of restriction enzymes represents a cornerstone of molecular biology. Three classes of these endonucleases are used:

• Class I enzymes cleave 100 to 1000 base pairs away from the recognition site and currently are of limited use
• Class II enzymes cleave within or very near a specific recognition sequence and are used to cleave double stranded DNA at specific sites; these enzymes consist of one protein which cleaves the DNA and a second one that methylates the DNA (nearly all the commercially available enzymes are class II)
• Class III enzymes cleave 25 to 27 base pairs from their recognition site and are gaining popularity in some cloning strategies; the SAME protein carries out both the modification (methylation) and the ATP-dependent restriction

The restriction enzymes cleave the phosphodiester bonds in each strand of double-stranded DNA:

• cleavage at adjacent sites produces a "blunt end"
• cleavage that is offset by 1 to 4 bases, leaving either a 3' overhang or a 5' overhang of a single strand, yields a "sticky end"

Examples of class II restriction enzymes

HaeIII from Haemophilus aegytius		Blunt end
HindIII from Haemophilus influenzae Rd		5' overhang sticky end
PvuI from Proteus vulgaris		3' overhang sticky end

Restriction enzymes are obtained from many prokaryotes and about 1500 enzymes with known sequence recognition sites have been isolated. Naming these endonucleases follows a system proposed by Nathans and Smith. Each name contains at least one capital letter and two small letters followed by a Roman numeral. The letters are initials of the genus and species of origin and the number represents the number of enzymes discovered in the organism. (Historically the numeral identified the protein peak in which the enzyme eluted during chromatography.) Additional information may be added as a letter. For EcoRI, the R indicates the particular strain of E. coli.

Restriction enzymes from different organisms may recognize the same DNA sequence. If the enzymes recognize the same site and cleave at the same position, they are labeled isoschizomers. Ones that recognize the same site but cleave in different positions are heteroschizomers or neoschizomers.

Iso- or heteroisoschizomers add flexibility to experimental design. Cost, methylation sensitivities, and types of "ends" are considerations as well as buffer conditions for optimal activity.

A few buffer conditions suit nearly all the restriction enzymes but no single buffer allows activity of every enzyme. Suppliers of enzymes always provide a reaction buffer (10x concentrate) that is optimum for the enzyme. Components of the 1x buffer usually are 10-100 mM Tris at pH 7.3 to 8.5, various levels of salts like KCl and NaCl (10 to 150 mM), 10 mM Mg(2+), 2 mM beta-mercaptoethanol. Sometimes 0.01% Triton- X100 (a detergent) and bovine serum albumin are included as a stabilizers. (Alternatively, swine skin gelatin can be used and offers the advantages that it is stable to autoclaving and costs about 1/15 as much as BSA.)

Since restriction enzymes can require different buffer conditions, some strategy must be used to do double digests. The preferred method is to simultaneously digest with both enzymes in a compatible buffer. This method can be used even if one enzyme is not fully active (e.g., 75% active). More of one enzyme can be added (e.g., 1 U of enzyme A + 1.33 U enzyme B) for equal cutting efficiency. There are limits to the excess enzyme due to increased glycerol in the reaction that can reduce specificity of some enzymes.

An alternative method is to digest with the "low salt" enzyme then add more buffer and the "high salt" enzyme to complete the digest. This obviously doubles the time required for digestion. In extreme cases the DNA can be precipitated after one digest and dissolved in the second digest buffer. Digests are carried out at 37 degrees C unless otherwise noted for the enzyme.

Non-specific or relaxed specificity cleavage or "star" activity can occur if non-optimal conditions are used. Conditions that encourage star activity include:

1. high pH (>8.0)
2. low ionic strength buffer (<25 mM)
3. high enzyme concentration (ratios >100 U/µg DNA)
4. high levels of glycerol (>5%) or other organic solvents that affect the DNA helix
5. long incubation times
6. low temperature (increased temperature can decrease star activity)
7. substitution of Mg(2+) with other divalent cations [Mn(2+), Co(2+), Cu(2+), Zn(2+)]

The state of the DNA also influences the ability of restriction enzymes to cut efficiently. "Dirty" DNA contaminated with protein, salts, and RNA is not cut by some enzymes. DNA from sources that methylate adenine or cytosine residues may not be digested (methylated sensitive, Table 6, pp. 126-134 Lab Fax or a catalogue appendix). Some strains of E. colimethylate the N(6) position of adenine in the sequence, GATC, and are denoted as dam . Others methylate the C(5) position of the internal cytosine in sequences of CCAGG, CCTGG and are designated as dcm. DNA from other sources (e.g., mammals, plants) can be methylated at other sites. Some restriction enzymes will not cut near these methylations if their recognition site overlaps with the methylation site.

Miscellaneous information on restriction enzymes

• By definition, a unit of restriction enzyme will completely cleave 1µg of Lambda DNA (or other substrate DNA) in one hour in the recommended buffer and temperature.
• Reaction volumes should be 25-50 µl and the amount of enzyme added should not exceed 10% of the volume due to the glycerol content. The amount of DNA in a digest should not exceed 250 µg/ml because the increase in ionic compounds in the DNA preparations will decrease the efficiency of cleavage.
• Restriction enzymes are most stable when stored in a non-cycling -20 degrees C freezer. (Frost-free freezers are not recommended.) Most enzymes are stored in 50% glycerol and therefore are not frozen solid, allowing removal of the enzyme without thawing. Always keep the enzyme solution in a freezer block (Stratacooler) and return it to the freezer as quickly as possible.
• Catalogues from suppliers represent an accessible (and free) source of valuable information on restriction enzymes. A wealth of information is usually found in table form in appendices in the back of the catalogue or near the pages listing the restriction enzymes. These tables give buffer concentrations, isoschizomers, compatible ends, methylation sensitivities, E. coli strains and genotype tables, explanations and more. Even extinction coefficients for DNA and RNA are listed. Promega, New England Biolabs, Stratagene, USB, and Pierce are exceptionally efficient at providing information. Both Promega and Pierce supply useful technique manuals free of charge.
• New England BioLabs provides several useful web-based resources:
  • Enzyme Finder
  • NEBcutter
  • Double Digest Finder
  • NEBuffer Chart
  • DNA Sequences and Maps

Copyright, Acknowledgements, and Intended Use
Created by B. Beason (bbeason@rice.edu), Rice University, 6 February 2008
Updated 10 February 2008