Restriction Enzymes 165
be detected (182). The radioactive products of these side reactions can
be quantitatively determined by homochromatography on DEAE- or
PEI-cellulose thin-layer plates or by HPLC on a reversed-phase column.
3.5.4. 3 '-Exonuclease Assay
~,-DNA restriction fragments with 5' protruding ends (see Section
3.5.3.) are labeled at their 3' ends by incorporation of ot [32p] - or c~[35S] -
dNTPs using Klenow polymerase (see Chapters 3 and 4). The 3'-labeled
DNA is incubated after separation of the unincorporated activity with
the enzyme preparation to be tested. Any Y-exonuclease activity attack-
ing the 3' terminally labeled fragments will liberate [32p]_ or [35S]-
labeled mononucleotides.
3.5.5. Ligation-Recut Assay
The functional integrity of the fragment termini produced after cleav-
age of any plasmid DNA with restriction enzymes is assayed in the
ligation-recut assay. Initially, the substrate DNA is completely digested
by incubating 10 Jxg of DNA with 50 U of the restriction enzyme in 50 ~L
of the appropriate reaction buffer over a period of 1-2 h. Restriction
fragments are extracted twice with phenol/chloroform (1:1) and preci-
pitated after addition of 1/10 vol 3M sodium acetate, pH 4.8, by 2 vol
ethanol. Sticky ends are ligated with 0.1 U, blunt ends with 1-10 U T 4-
DNA ligase/~g DNA in a total volume of 10 laL by incubation for 16 h at
12-16°C in 20 mM Tris-HC1, pH 7.5, 10 mM MgC12, 10 mM DTE or
DTT, and 0.6 mM ATP. Ligation is terminated by heating the reaction
mixture for 10 min at 70°C. An aliquot (approx 500 ng) is analyzed by
agarose gel electrophoresis to determine the extent to which the fragments
could be rejoined. The products of the ligation are recut with the same
restriction enzyme under the reaction conditions mentioned above. The
fragments produced are analyzed by agarose gel electrophoresis. A nor-
mal banding pattern indicates that 5' and 3' termini remained intact, dem-
onstrating the absence of contaminating endo- and exonuclease activities.
3.6. Nonspecific Inhibition of Restriction Enzymes
In general, restriction enzymes are inhibited by chelation of the essen-
tial cofactor Mg 2+. Some enzymes are very sensitive to variations in ionic
strength or can be specifically inhibited by thiol reagents. Impurities
introduced together with the substrate may cause a significant reduc-
tion of activity or, in extreme cases, completely inhibit restriction