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In general, PCR reactions were performed in a volume of 50 μl with 50 picomoles of each primer, dNTP's (Amersham Pharmacia Biotech, Inc., Piscataway, NJ) at a final concentration of 0.5 mM, 1 μl of template, 1X PCR buffer (10X PCR buffer contained 700 mM Tris pH 8.8, 20 mM MgCl 2 , 1% Triton X-100, and 0.1% Tween 20) and Taq polymerase (Perkin-Elmer Inc., Wellesley MA) diluted to the manufacturers recommendations.
[ 3H]-thymidine (20 Ci/mmol) was from Perkin-Elmer/NEN Life Sciences (Boston, MA).
PCRs were performed in a model 9600 Thermocycler (Perkin-Elmer Cetus) using the following cycling protocol: initial denaturation at 94°C for 10 min; 94.
Amplifications were done using either a Perkin-Elmer Amp PCR System 2400 or Model 480 DNA Thermal Cycler (Perkin-Elmer Cetus, Norwalk, Conn).
Samples underwent 30 cycles of amplification with Perkin-Elmer Taq polymerase in a PTC Thermal Cycler (MJ Research, MA) (94° for 40 sec, 57° for 1 min, 72° for 2 min).