Characterization of Aberrant Melting Peaks in Unlabeled Probe Assays

An unlabeled probe assay relies on a double-stranded DNA-binding dye to detect and verify target based on amplicon and probe melting. During the development and application of unlabeled probe assays, aberrant melting peaks are sometimes observed that may interfere with assay interpretation. In this report, we investigated the origin of aberrant melting profiles observed in an unlabeled probe assay for exon 10 of the RET gene. It was determined that incomplete 3′ blocking of the unlabeled probe allowed polymerase-mediated probe extension resulting in extension products that generated the aberrant melting profiles. This report further examined the blocking ability of the 3′ modifications C3 spacer, amino-modified C6, phosphate, inverted dT, and single 3′ nucleotide mismatches in unlabeled probe experiments. Although no 3′ blocking modifications in these experiments were 100% effective, the amino-modified C6, inverted dT, and C3 spacer provided the best blocking efficiencies (1% or less unblocked), phosphate was not as effective of a block (up to 2% unblocked), and single nucleotide mismatches should be avoided as a 3′ blocking modification.