Abstract
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.
Highlights
Many PCR applications directly monitor the amplification reaction in real-time such as research applications, commercial tests, and medical diagnostics
In non-hybridized TaqMan probes the signal of a fluorescent dye is suppressed by a quencher, which leads to a low background signal as long as the fluorescent dye and quencher are in close contact
The signal of Molecular Beacons depends on a conformation change, where the fluorescent label and quencher at the ends of the probe are separated upon hybridization of the probe to the PCR template
Summary
Many PCR applications directly monitor the amplification reaction in real-time such as research applications, commercial tests, and medical diagnostics. More specific real-time detection methods use labeled primers for incorporation into the amplicon, e.g. Sunrise primers [2] or Scorpion primers [3], or add additional probes to the reactions that hybridize to the amplicon in a sequence-dependent manner [4,5] Such DNA probes commonly carry a fluorescent label and a quencher for background control, e.g. TaqMan probes [6], and Molecular Beacons [7,8], or two separate oligonucleotides have been used like in the case of HybProbes [9] where one oligonucleotide carries a donor dye and the other oligonucleotide carries an acceptor dye. Many other approaches to real-time PCR and fluorescent probes have been described in the literature [4,5,10,11] following similar principles as outlined above for the most commonly used tools
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