SPECIFIC-INHIBITION OF PCR BY NON-EXTENDIBLE OLIGONUCLEOTIDES USING A5'-EXONUCLEASE-DEFICIENT TO 3'-EXONUCLEASE-DEFICIENT DNA-POLYMERASE

The Stoffel fragment of Taq DNA polymerase lacks the 5' to 3' exonucle ase activity that hydrolyzes potentially blocking DNA strands during p rimer extension. We therefore asked whether by using this fragment in the PCR, non-extendable, base-paired oligonucleotides could inhibit am plification in a sequence-dependent manner. Model targets were chosen from the partially conserved ribosomal 16S rDNA of three bacterial spe cies: E. coli, Bacillus subtilis and Neisseria gonorrhoea. A single pa ir of primers was capable of amplifying a homologous 240-bp region fro m all three. Two non-extendable ''blocking'' oligonucleotides were syn thesized with sequences complementary to the inter-primer regions of E . coli and B. subtilis, respectively. Both blockers were shown specifi cally to prevent amplification of their complementary targets, but not of the reciprocal control targets or of the non-complementary N. gono rrhea. Specificity was further confirmed by an internal positive contr ol. Similar inhibition was seen with mixtures of targets in a single r eaction. With intact Taq DNA polymerase, no blocking was observed. Pri mers and blockers targeting specific regions of N. gonorrhoea rDNA wer e used to confirm the requirement that blockers be directed to the int er-primer region. Sequence-dependent amplification inhibition, such as that demonstrated here, would be applicable to PCR-related strategies using primers capable of using multiple targets, where such selective inhibition could be useful.