A novel PCR method directly quantifies sequence features that block primer extension

Abstract

Many quantitative polymerase chain reaction (qPCR) assays indirectly quantify a target of interest as a drop in amplification signal relative to a larger total signal. The targeted sequence features include: DNA strand breaks introduced by enzymes that cleave at specific sequences or modifications, or caused by ionizing radiation or other DNA-damaging agents; DNA damage that does not break the DNA but blocks the DNA polymerase; and deletions of various sizes that prevent one or both primers from annealing. All of these loss of signal (a.k.a. negative) assays share the drawback of low sensitivity, as compared to positive assays, which generate an amplification signal that directly reflects the copy numbers of the sequence feature. Here we present a novel qPCR strategy that converts all of the above negative assays into positive ones. A mixture of three primers is added to genomic DNA, one primer pair that targets a sequence for strand-specific PCR, and a third, longer primer that prevents initiation of that PCR, by annealing at high temperature and extending across the target sequence, rendering it doublestranded and inaccessible for priming. Any sequence feature that blocks the third primers annealing or extension, while leaving the target sequence intact, allows the PCR to proceed and quantify the copy number of the sequence feature. This Primer Extension Blockade Enabled qPCR (PEBE-qPCR) method will facilitate many high-throughput, low-cost qPCR-based investigations in biology and medicine. As a first example, we present direct qPCR of the unmethylated allele at a MspI/HpaII site in the promoter of the human 45S rDNA gene.