Abstract

Improved qPCR performance using internally quenched probes

Mark Behlke, Vice President/Chief Scientific Officer, Integrated DNA Technologies Inc

Fluorescence-quenched probes are widely used in molecular biology applications and have particular utility in qPCR. The efficiency of quenching is a primary factor that determines probe performance. IDT developed a dark quencher that can be placed internally within an oligonucleotide probe (internal ZEN™ Quencher), which permits the quencher to be positioned close to the 5'-fluorophore and, thus, improves quenching and probe performance. Unlike most non-base modifiers that are inserted internally in a DNA sequence, an internal ZEN Quencher actually stabilizes duplex formation and increases the Tm of the probe. PrimeTime® qPCR Assays from IDT incorporate this technology and are available as either pre-designed assays for all genes in the human, mouse, and rat genomes or custom probes designed by the end user. The improved probes also can be combined with a blocked-cleavable primer strategy that increases the specificity of PCR, called rhPCR. The blocked primers contain a single ribonucleotide residue and must be cleaved by RNase H2 before polymerase extension can occur. The requirement of the primer to hybridize with the target sequence before cleavage eliminates the formation of primer-dimers and greatly reduces misamplification of closely related sequences. Mismatches near the scissile linkage decrease the efficiency of cleavage by RNase H2, further increasing the specificity of the assay. The rhPCR system can improve genotyping assays and can be applied to rare variant detection. This system can identify a mutant allele against a background of 10,000-fold excess of wild type sequences.