Mismatch discrimination using blocked-cleavable primers with an RNA:DNA base-pair mismatch versus standard unmodified allele-specific PCR primers. Four synthetic 103 base oligonucleotide targets were employed where a single base was varied (A, C, G, or T) within the primer binding site. For each DNA target, a single common For primer was paired with four different "rDDDDx" blocked-cleavable Rev primers, varying the RNA base (A), or with four unmodified allele-specific primers terminating in a different 3'-DNA base (B). Amplification reactions were performed in real-time mode using SYBR® Green detection; all reactions were run in triplicate. ΔCq values are shown in the tables and represent the difference between the Cq value for the allele-specific primer for each template and the Cq value for the perfect match template. The concentration of RNase H2 was 1.3 mU per 10 μL.
Dobosy et al. BMC Biotechnology 2011 11:80 doi:10.1186/1472-6750-11-80