Restriction Enzymes 127
quantities of DNA can be seen on inspection of the gel under UV light.
Silver staining allows the detection of subnanogram quantities of DNA.
Alternatively, radioactively labeled DNA substrates may be used: After
digestion and electrophoresis, DNA bands are then visualized by auto-
radiography. The detection limit of this procedure is in the picogram
range with 32p-labeled DNA.
There have been efforts to devise spectrophotometric assays to
measure the activity of restriction enzymes. One assay makes use of
DNA immobilized on cellulose, which on incubation with restriction
enzymes leads to the release of DNA fragments into the solution and
a concomitant increase in UV absorbance (180). Although this assay
is less sensitive than the electrophoretic procedure, it allows a continu-
ous recording of the progress of the reaction. This is also possible with
a fluorimetric assay, which takes advantage of the fact that superheli-
cal DNA binds less ethidium bromide than nicked circular or linear
DNA (81,172). Therefore, cleavage of superhelical DNA in the pres-
ence of ethidium bromide leads to increased ethidium binding and
fluorescence. Both the spectrophotometric and the fluorimetric assays
suffer from the disadvantage that they do not discriminate between
specific and nonspecific endonucleolytic activities (see note added in
proof at end of chapter).
The following procedure may be used as a standard assay for most
restriction enzymes:- Incubate a solution of 250 ng ~. DNA in 10 ~L of the appropriate reac-
tion buffer at the desired reaction temperature; - Add 5 laL of a defined dilution of the restriction enzyme, freshly pre-
pared with reaction buffer; - Incubate 15 min at the desired temperature;
- Stop the reaction by the addition of 5 taL of a solution, containing 250 mM
EDTA, pH 8.0, 1.2% (w/v) SDS, 25% (w/v) sucrose, and 0.2% (w/v)
bromophenol blue; - Incubate at 70°C for 5 min;
- Chill on ice;
- Load 15 laL of the incubation mixture onto 1-2% agarose gels; and
- Carry out electrophoresis.
The agarose concentration for the electrophoresis gels depends mainly
on fragment length produced by each enzyme: e.g., EcoRI-~,-frag-
ments (ranging from 3530-21,230 bp) are resolved in 1% agarose