Specific Inhibition of PCR by Non-Extendable Oligonucleotides Using a 5′ to 3′ Exonuclease-Deficient DNA Polymerase

Abstract

The Stoffel fragment of Taq DNA polymerase lacks the 5′to 3′exonuclease activity that hydrolyzes potentially blocking DNA strands during primer extension. We therefore asked whether by using this fragment in the PCR, non-extendable, base-paired oligonucleotides could inhibit amplification in a sequence-dependent manner. Model targets were chosen from the partially conserved ribosomal 16S rDNA of three bacterial species: E. coli, Bacillus subtilis and Neisseria gonorrhoea. A single pair of primers was capable of amplifying a homologous 240-bp region from all three. Two nonextendable “blocking” oligonucleotides were synthesized with sequences complementary to the inter-primer regions of E. coli and B. subtilis, respectively. Both blockers were shown specifically to prevent amplification of their complementary targets, but not of the reciprocal control targets or of the non-complementary N. gonorrhea. Specificity was further confirmed by an internal positive control. Similar inhibition was seen with mixtures of targets in a single reaction. With intact Taq DNA polymerase, no blocking was observed. Primers and blockers targeting specific regions of N. gonorrhoea rDNA were used to confirm the requirement that blockers be directed to the inter-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.