The Stoffel fragment of Taq @entity70099 lacks the 5' to 3' exonuclease activity that hydrolyzes potentially blocking DNA strands during primer extension. We there-fore 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: @entity526 , @entity3881 and Neisseria gonorrhoea. A single pair of primers was capable of amplifying a homologous 240-bp region from all three. Two non-extendable "blocking" oligonucleotides were synthesized with sequences complementary to the inter-primer regions of @entity526 and @entity3881 , respectively. Both blockers were shown specifically to prevent amplification of their complementary targets, but not of the reciprocal control targets or of the reciprocal control targets or of the @entity8664 . @entity2954 . Specificity was further confirmed by an internal positive control. Similar inhibition was seen with mixtures of targets in a single reaction. With intact Taq @entity70099 , 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.

Specific inhibition of PCR by non-extendable oligonucleotides using a 5' to 3' exonuclease-deficient XXXX .

@entity2954 :: ('MESH:D006069', 'Disease') :: ['gonorrhea'] @entity70099 :: ('13897297', 'Gene') :: ['DNA polymerase'] @entity8664 :: ('MESH:C536108', 'Disease') :: ['non-complementary N'] @entity3881 :: ('1423', 'Species') :: ['Bacillus subtilis', 'B. subtilis'] @entity526 :: ('562', 'Species') :: ['E. coli']
@entity70099 :: (13897297,Gene) :: ['DNA polymerase']
