INCREASING PROPORTIONS OF URACIL IN DNA SUBSTRATES INCREASES INHIBITION OF RESTRICTION ENZYME DIGESTS

Techniques that rely upon the incorporation of uracil into DNA are bei ng published with increasing frequency especially in PCR protocols. We report here the efficiency of 18 type II restriction enzymes to diges t PCR amplicons synthesized with varying proportions of TTP to dUTP in the PCR mixture. We find that most enzymes with A:T/U bp in their rec ognition site digest the amplicons less efficiently as the percentage of dUTP in the reaction mixture is increased. This effect is most dram atic when the proportion of dUTP in the nucleotide mixture exceeds 50% . All but one of the enzymes which fail to digest amplicons that are s ynthesized with 100% dUTP digest some amplicons which are synthesized with 90% dUTP.