Methods in Molecular Biology • 16 Enzymes of Molecular Biology

100 Grosse and Manns

3. Experimental Procedures
   In the following, we will give some examples for the practical use of
   terminal transferase. We readdress the issue of homopolymer synthe-
   sis (cf ref. 4) by providing data for the use of phosphorothioate deriva-
   tives of the four dNTP substrates (dNTPaS). Homopolymers containing
   phosphorothioate diester linkages are interesting mainly because they
   are expected to be more stable against nuclease degradation than
   phosphatediester homopolymers (22,23). Their thermodynamic behavior
   in an annealed state is not yet known. Furthermore, (dCS)28 has been
   shown to display anti-HIV-1 activity, both in vivo and in vitro (24).
   We further describe reaction conditions for the addition of dNTPo~S
   to 3'-hydroxyl ends of linear double-stranded DNA. The insert DNA
   will be tailed uniformly with (dG)a 0. The less valuable linearized vec-
   tor will be tailed with oligo(dC). After annealing, the dG/dC-tailed
   recombinant molecule will be used to transform competent E. coli cells.
   Within the bacteria, remaining single-stranded DNA will be filled up
   by DNA polymerase I and ligated to covalently closed circular DNA.
   As a sidereaction, intracellular nucleases degrade the recombinant
   molecule, preferably at its vulnerable single-stranded regions. This
   reduces transformation efficiencies. Replacing the dNTPs by their
   o~-phosphorothioate analogs yields single-stranded DNA intermedi-
   ates that are less susceptible to degradation.

3.1. Materials

1. Terminal deoxynucleotidyl transferase (Stratagene, Heidelberg, Germany).


2. Cacodylic acid (Sigma, Deisenhofen, Germany).


3. Solid KOH.


4. Chelex 100; 200--400 mesh, sodium form (BioRad, Richmond, CA).


5. p(dC)10 (Pharmacia, Freiburg, Germany).


6. 0.1MCoC12.


7. 0.1MMnCI a.


8. 0.1 M Dithiothreitol (Boehringer, Mannheim, Germany).


9. Deoxynucleoside triphosphates (Boehringer).


10. [3H]Deoxynucleoside triphosphates, 600 Ci/mmol (Amersham,
    Braunschweig, Germany).


11. Deoxynucleoside-5'-O-(1-thio)-triphosphates, dNTPaS (Amersham).


12. [35S]Deoxynucleoside-5'-O-(1-thio)-triphosphates, 1300 Ci/mmol
    (NEN, Bad Homburg, Germany, Amersham).


13. DE81 filter disks (Whatman, Maidstone, UK).