Selection of Hybridization Probes for Real-Time Quantification and Genetic Analysis

Abstract

The concept of LightCycler hybridization probes (HybProbes) is based on the detection of two adjacent oligonucleotide probes, whose fluorescent labels ‘communicate’ through fluorescence resonance energy transfer (FRET). The detection of a given nucleic acid is achieved by the near hybridization of two probes, or by an internal labeled PCR strand and a detection probe located on the opposite strand. The signal is dependent on the spatial approximation of the dyes, and is dependent on the amount of the target. Although hybridization is a very robust process and therefore most pairs of randomly chosen sequences will work satisfactorily, we believe that understanding how a probe finds its target will aid in the selection of probes with a better average performance. In the following text we will explain some rules for the selection of HybProbes. We will discuss their use for two possible applications, using HybProbes for the quantification of a target, or as probes for the detection of sequences variations. In both cases, effective competition of probe hybridization with rehybridization of the target and with elongation of the primers is important. In quantification, the probes can be placed anywhere within the amplicon. In contrast, the detection of sequence variants requires positioning the HybProbes over the variable site and difficult sequences cannot always be avoided. In the following text we will demonstrate how possible problems can be circumvented to increase the distinction of sequence variants. In any case, there may be a small number of molecular situations where no LightCycler-based assay can be constructed.