Eprobe mediated real-time PCR monitoring and melting curve analysis

Real-time monitoring of PCR is one of the most important methods for DNA and RNA detection widely used in research and medical diagnostics. Here we describe a new approach for combined real-time PCR monitoring and melting curve analysis using a 3' end-blocked Exciton-Controlled Hybridization-sensitive fluorescent Oligonucleotide (ECHO) called Eprobe. Eprobes contain two dye moieties attached to the same nucleotide and their fluorescent signal is strongly suppressed as single-stranded oligonucleotides by an excitonic interaction between the dyes. Upon hybridization to a complementary DNA strand, the dyes are separated and intercalate into the double-strand leading to strong fluorescence signals. Intercalation of dyes can further stabilize the DNA/DNA hybrid and increase the melting temperature compared to standard DNA oligonucleotides. Eprobes allow for specific real-time monitoring of amplification reactions by hybridizing to the amplicon in a sequence-dependent manner. Similarly, Eprobes allow for analysis of reaction products by melting curve analysis. The function of different Eprobes was studied using the L858R mutation in the human epidermal growth factor receptor (EGFR) gene, and multiplex detection was demonstrated for the human EGFR and KRAS genes using Eprobes with two different dyes. Combining amplification and melting curve analysis in a single-tube reaction provides powerful means for new mutation detection assays. Functioning as "sequence-specific dyes", Eprobes hold great promises for future applications not only in PCR but also as hybridization probes in other applications.