Combining Sequence-Specific Probes and DNA Binding Dyes in Real-Time PCR for Specific Nucleic Acid Quantification and Melting Curve Analysis

Abstract

Currently, in real-time PCR, one often has to choose between using a sequence-specific probe and a nonspecific double-stranded DNA (dsDNA) binding dye for the detection of amplified DNA products. The sequence-specific probe has the advantage that it only detects the targeted product, while the nonspecific dye has the advantage that melting curve analysis can be performed after completed amplification, which reveals what kind of products have been formed. Here we present a new strategy based on combining a sequence-specific probe and a nonspecific dye, BOXTO, in the same reaction, to take the advantage of both chemistries. We show that BOXTO can be used together with both TaqMan probes and locked nucleic acid (LNA) probes without interfering with the PCR. The probe signal reflect formation of target product, while melting curve analysis of the BOXTO signal reveals primer-dimer formation and the presence of any other anomalous products.

The Digital MIQE Guidelines: Minimum Information for Publication of Quantitative Digital PCR Experiments

“…Alternatively, internal probes can be used. When using hydrolysis probes, a compatible dye can be added for additional melting curve analysis in a separate spectral channel of the instrument (47 ).…”

Simultaneous Detection and Differentiation of Human Rhino- and Enteroviruses in Clinical Specimens by Real-Time PCR with Locked Nucleic Acid Probes

“…2). Addition of the dsDNA-binding dye to the PCR mixture reveals the formation of primer dimers or other nonspecific products with deviating T m values (37). Furthermore, specific amplicons with mismatches preventing probe binding or hydrolysis are likely to be detected.…”

Molecular-Genetic Approaches to Identification and Typing of Pathogenic Candida Yeasts

“…Traditionally, one has to choose between the use of a sequence-specific probe and a non-specific dsDNA binding dye, because of spectra overlap in fluorescent dyes used for labelling of probes and for dsDNA staining. Recently, however, the use of BOXTO dsDNA binding dye has been reported as compatible with the use of probes labelled by FAM 23 .…”