Combined FAM-labeled TaqMan probe detection and SYBR green I melting curve analysis in multiprobe qPCR genotyping assays

摘要

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).