Restriction Enzymes 183
clea-
lr
.- ..... recog ............... hybridization ............
k II II II II II II^11 II II II It II II II II II II II II II II II II I1
....... nition-;>-- ~vage~ single stranded vector DNA
Fig. 2. Site-directed cleavage of single-stranded DNA using an adapter oligo-
deoxynucleotide containing a recognition site for a type IIs restriction enzyme (e.g.,
FokI) and a sequence complementary to the DNA sequence to be cleaved.
which has to be removed by phenol extraction, or with HKTM-phos -
phatase, which can be heat inactivated, before this end can be radio-
actively labeled using T4 polynucleotide kinase and 7132p] - or
7135S]-ATP (196). The yield of the phosphorylation reaction depends
on the accessibility of the 5'-OH group: It is higher for 5' overhanging
ends as compared to blunt or 3' overhanging ends. The DNA can be
dephosphorylated also by the kinase itself by enhancing the reverse
reaction using ADP in excess over the radioactive ATP (197).
Labeling at the 3'-OH group is carried out using calf thymus termi-
nal deoxynucleotidyl transferase and t~[32p]-cordycepine triphosphate,
which functions as a chain terminator. Also for this reaction, the yield
depends on the accessibility of the 3'-OH terminus; the yield can be
improved by thermal denaturation of the DNA ends.
Both these methods result in the labeling of both 5' or 3' ends of the
two strands forming the double-stranded restriction fragment. For
labeling only one end of a fragment, one can carry out a DNApolymer-
ization reaction (fill-in reaction) with a suitable mixture of dNTPs.
DNA polymerases can only work on a 5'-overhanging end. With two
restriction enzymes, two different ends are generated. This offers the
possibility of labeling only one of these ends by including only those
radioactive o~[32P] - or o~[35S]-dNTPs in the polymerization mixture
that are required by the fill-in reaction for one end.