174 Pingoud, Alves, and Geiger
3.8. "Star"Activity
"Star" activity, discussed in Section 2.4.2., is defined as a relaxed speci-
ficity of restriction enzymes under suboptimum reaction conditions
and is related to the naturally observed limited accuracy of these enzymes
when used in high concentration over a prolonged time. The relatively low
specific activities of most commercial enzyme preparations, normally
10 U/gL, do not lead to a"star" pattern under normal buffer conditions.
3.9. Terminating the Cleavage Reaction
Depending on the specific purpose for which a restriction digest has
to be carried out, the digestion reaction is terminated by inhibiting,
destroying, or extracting the restriction enzyme. This can be achieved
by chelation of the essential cofactor Mg 2÷, thermal denaturation of
the enzyme, chemical denaturation of the enzyme, or extraction of the
enzyme by phenol/chloroform. It should be kept in mind that chelation
only blocks catalytic activity, but does not prevent DNAbinding of the
restriction enzymes. DNA binding may interfere with the electro-
phoretic analysis of the digest. It is recommended, therefore, to dena-
ture the restriction enzymes prior to separation of the cleavage products
by gel electrophoresis by the addition of a loading buffer containing
1% SDS and 100 mM EDTA. Thermal denaturation after restriction
digestion and prior to further reactions, e.g., ligation, is achieved by
heating the reaction mixture to 65°C for 10 min, which is sufficient to
inactivate most enzymes or to 95°C for 10 min for some of them. This
is, however, not effective with thermostable enzymes (cf Table 5). In
this case, it is necessary to denature and remove the restriction enzyme
by a phenol/chloroform extraction normally followed by ethanol pre-
cipitation of the DNA fragments.
3.10. Analysis of the Cleavage Products
The analytical separation of DNA restriction fragments commonly
is carried out by electrophoretic techniques that allow a precise deter-
mination of the size of DNA molecules. For oligodeoxynucleotides,
homochromatography (183,184) or HPLC methods (for a review, cf
[185]) can be used as an alternative.
Electrophoretic separation of DNA cleavage products can be
achieved in agarose, polyacrylamide, or agarose-polyacrylamide com-
posite gels under native or denaturating conditions. Whereas condi-