Multiplexed real-time PCR using universal reporters.

Abstract

Real-time quantitative PCR is a sensitive and accurate method for gene expression studies (1). The detection chemistries of all real-time PCR procedures are based on one of two principles for monitoring amplification products: binding to double-stranded DNA or hybridization to single-stranded DNA. Small molecules bind to double-stranded DNA either as intercalators or as minor groove binders, e.g., ethidium bromide (2), Hoechst 33258 (3), or SYBR® Green I (4). Several approaches using target-specific hybridization to single-stranded DNA have been introduced, including Molecular Beacons (5), Scorpions (6)(7), the TaqMan or hydrolysis/5′-nuclease assay (8)(9), the AEGIS probe system (10), labeled primers (11)(12), and light-up probes (13). In contrast to binding of dyes to double-stranded DNA, these methods are suitable for multiplexing approaches because they use differentially labeled fluorescent dyes. However, as they require a unique probe or modified primer for each target, currently used hybridization-based methods for real-time quantitative PCR have high reagent costs and require large developmental efforts. Here we present a real-time PCR assay that uses universal hybridization-based probe sets suitable for any target. Because the assay uses tailed locus-specific nonmodified amplification primers, PCR products can be monitored via common reporters (cr) hybridizing to the common tails. The general principle of combining tailed PCR primers with universal probes has been introduced for other genetic applications, such as single-nucleotide polymorphism genotyping (14)(15) and in situ amplification (16), but these methods have not been applied to quantitative gene expression studies. Our system, which is similar to the method developed by Whitcombe et al. (15), leads to a more flexible and low-cost setup than conventional hybridization-based approaches. Using differentially labeled universal reporting reagents, we have developed a multiplex setup for simultaneous analysis of target gene and internal control (housekeeping gene), which is an accepted method for …