Methods in Molecular Biology • 16 Enzymes of Molecular Biology

12 Weir

3.2. Assay of DNase Activity

Many methods have been used in the past to assay DNase activity:

Melgar and Goldthwaite (2a) used a somewhat complicated method

whereby T4 DNA was labeled with 32p and embedded in a polyacryla-

mide gel. After exposure of the gel to DNase, the gel was pelleted and

the amount of radioactive DNA fragments released into the superna-

tant was measured. More recent methods (e.g., ref. 5) have again used

radiolabeled substrates, but this time the rate of hydrolysis is monitored

by precipitating the DNA fragments produced out of aqueous solution

with trichloroacetic acid (TCA) in the presence of a carrier and then

measuring acid-soluble or acid-insoluble radioactivity. For most molecu-

lar biologists however, all that is required is an assay method to show

whether DNase is responsible for the loss of DNA from a preparation,

and if so, to show where the contamination is coming from. This is best

accomplished by using a covalently closed circular (ccc) plasmid as

substrate in a component analysis. In the following example, the compo-

nents of a restriction enzyme digestion ofa plasmid are to be analyzed.

1. Set up several reactions depending on the number of components to be
   tested, remember that the controls are all important.
   a. ccc plasmid in sterile distilled water (500 ng in 10 I.tL)
   b. ccc plasmid in buffer
   c. ccc plasmid in buffer + bovine serum albumin (BSA)
   d. ccc plasmid in buffer + enzyme
   e. ccc plasmid in buffer + BSA + enzyme


2. Incubate at 37°C for 30-60 min.


3. Run digests on an agarose gel.


4. Stain with ethidium bromide and view under UV illumination.

Endo-DNase activity is indicated by excessive conversion, as com-
pared to the controls, ofccc plasmid to open circles or to linears. Usually
endo-DNase contamination will be accompanied by exonuclease activity,
so there will be complete degradation of the plasmid to a smear of material
down the gel.

3.3. Removal of DNase Activity
DNase enzymes are heat labile and can be removed from solutions
by autoclaving and from glassware and the like by baking or autoclav-
ing. To make sure that solutions of RNase I are DNase free, boil the