[Letter to the editor] Combined FAM-labeled TaqMan probe detection and SYBR green I melting curve analysis in multiprobe qPCR genotyping assays.

Since its first use, real-time quantitative PCR (qPCR) has evolved into a flexible, application-made method for the quantification and identification of nucleic acids (1-2). Depending on the application, most researchers choose between fluorescent nucleic acid binding dyes or labels that interact by fluorescence resonance energy transfer (FRET) as nucleic acid detection methods (2-3). Binding dyes are relatively cheap, easy to use, and generate high signals. The fact that they display sequence independent binding properties can be considered an advantage or disadvantage, depending on the application. Currently, SYBR green I is the most frequently used fluorescent nucleic acid binding dye (3-5). Among all available FRET-based nucleic acid detection methods (e.g. hybridization probes, molecular beacons, hydrolysis probes, dye-primer systems), TaqMan probes are currently the most popular (3,6-7). They are more expensive, more difficult to design, but sequence specific. Although both detection systems can generate melting curves in closed tube reactions without the need for post-qPCR gel electrophoresis, only those produced by binding dyes provide a total PCR amplicon readout, including nonspecific amplicons and oligo dimers, whereas those produced by probes only reflect the amount of PCR product detected by the probe (8-9). So far, only two reports applied both types of detection in a single assay (10-11). Lind et al. described the combination of the nonspe-cific dsDNA binding dye BOXTO with sequence-specific FAM-labeled TaqMan and LNA probes (10), and Cheah et al. described a two-tube combined TaqMan/SYBR green assay with a ROX-and a Cy5-labeled probe (11). For those applications, they needed to combine specific sequence detection by fluorescently labeled probes with the generation of the total PCR amplicon readout by binding dyes. However, both papers mention the incompatibility of combining the most popular inter-calating dye SYBR green I with a TaqMan probe labeled with the most common fluoro-phore, FAM, due to their overlapping spectra (10-11; Figure 1). Here, we performed qPCR assay 1 of the dual fluorescent multiprobe assay for prion protein genotyping in sheep, as described by Van Poucke et al. to show that FAM-labeled probe detection can be combined with SYBR green I melting curve analysis if the detection of the probe is performed at a temperature higher than the melting temperature of the amplicon. In addition, we formulated some compatibility guidelines for TaqMan probe detection and SYBR green I melting curve analysis in qPCR genotyping assays in general, which provide extra qPCR design flexibility for future assays. As a …