ResonSense®: simple linear fluorescent probes for quantitative homogeneous rapid polymerase chain reaction Original Research Article

Here we report a strand-specific fluorescent homogeneous assay format for rapid polymerase chain reaction (PCR). A number of similar assays are commonly used for research applications and are an ideal solution for a closed tube quantitative PCR. These assays use fluorescent resonant energy transfer (FRET) between donor and acceptor fluorescent moieties as the reporting mechanism. However, for different reasons these assays do not report amplification when very rapid cycling times are used. This is because current assays, such as TaqMan®, are limited, in terms of assay speed, by the 5′–3′ exonuclease activity of Taq DNA polymerase. Other assays based on hybridisation require either a complex de-conformational event to occur, or require more than one probe to report amplification. Reducing the complexity of the experiment reduces costs in terms of design, optimisation and manufacture. Here, we describe ResonSense® chemistries that use a simple linear fluorescent-labelled probe and a DNA minor-groove binding dye as either donor or acceptor moieties in a homogeneous assay format on the LightCycler®. This assay format will provide for rapid analysis of samples and so it is particularly well suited to point-of-use testing.