Eprobes in multiplex detection.

<p>Regions of the human EGFR and KRAS genes were amplified from genomic DNA and amplification was monitored by specific Eprobes having different dyes. A: Amplification curves plotting random fluorescent units (RFU) obtained from LightCycler 480 against PCR cycle number (Red: genomic DNA, Green: negative control). B: Differential melting curve analysis by plotting –dF/dT against temperature (Red: genomic DNA, Green: negative control). Plots in yellow on the left show signals for KRAS using dye D514 (Eprobe KWT14m3 TO); plots in pink on the right show signals for EGFR using dye D570 (Eprobe 215-21 wt TP).</p

Agarose gel electrophoresis of PCR products.

<p>A: PCR products from real-time PCR experiments were analyzed on a 3% agarose gel. Lanes: 1: TaqMan Probe, 2:, Eprobe 203-10 wt TO 3: C3-blocked 203-10 wt oligonucleotide, 4: Eprobe 205-13 wt TO, 5: Eprobe205-13m TO, 6: C3-blocked 205-13 wt oligonucleotide, 7: Eprobe 215-21 wt TO, 8: Eprobe 215-21m TO, 9: C3-blocked 215-21 wt oligonucleotide, 10: Eprobe 215-21 wt TP, 11: SYBR Green I, M: marker. B: Partial sequence of human EGFR gene. Location of the primers (blue boxes) and the mutation L858R (red box) are indicated.</p

Eprobes used for this study.

<p>(T_M values for TO-labeled Eprobes were estimated using the following settings: 0.05 M Na⁺, 0.002 M Mg²⁺, 0.2 µM Eprobe, 60°C Temperature; *T_M values for Eprobe 215-21 wt TP are actual values; in Eprobe name: TO = D514, TP = D570; Z indicating position of modified T).</p

Eprobe derived background.

<p>A. Eprobe 215-21 wt TO labeled with D514 was digested as described in the Materials and Methods and fluorescence excitation spectra from 495 nm to 800 nm were recorded before (blue) and after Eprobe digestion (red). B. Eprobe 215-21 wt TP labeled with dye D570 was digested as described in the Materials and Methods and fluorescence excitation spectra from 550 nm to 800 nm were recorded before (blue) and after Eprobe digestion (red). C. Plotting random fluorescent units (RFU, mean values from triplicate data for each experiment) obtained from LightCycler 480 and Eprobe 205-13 wt TO at the beginning of the PCR reactions against plasmid DNA template concentration (from 150 to 150,000,000 copies per reaction). Eprobe concentrations are indicated by the different colors (dark blue: 100 nM, light blue: 200 nM, red: 300 nM, purple: 400 nM, light green: 500 nM). D. Plotting random fluorescent units (RFU) obtained from LightCycler 480 and Eprobe 205-13 wt TO at the beginning of the PCR reactions against Eprobe concentration (dark blue: 100 nM, light blue: 200 nM, red: 300 nM, purple: 400 nM, light green: 500 nM). For each data point, mean values plus error bars from triplicate values for each experiment and for all template concentrations are given in the graph.</p

Eprobe mediated mutation detection.

<p>Asymmetric PCR was performed to enrich the reverse strand for mutation detection using different templates and Eprobes. For further details refer to Materials and Methods. A: Differential melting curve analysis by plotting –dF/dT against temperature of asymmetric PCR experiments with 5×10⁴ copies of plasmid DNA using Eprobe 205-13 wt TO. Wild-type to mutation ratios are indicated by colors (red: 100% wild-type, Yellow: 100% mutation, blue: 50% wild-type and 50% mutation, green: negative control). B: Differential melting curve analysis by plotting –dF/dT against temperature of asymmetric PCR experiments with 1 ng genomic DNA using Eprobe 215-21 wt TP. Wild-type to mutation ratios are indicated by colors (red: 100% wild-type, Yellow: 100% mutation, blue: 50% wild-type and 50% mutation, green: negative control).</p

Web interface of Edesign.

<p>Design parameter fields are hidden and are only shown when a link to the corresponding category is checked. Changed parameter settings can be saved on a local disk and the saved setting can be reloaded for later use.</p

Structure and function of Eprimer and Eprobe.

<p>A. Chemical structure of modified deoxythymidine carrying two dye moieties. B. Chemical structure of D514 “Thiazole Orange”. C. Chemical structure of D570 “Thiazole Pink”. D. Signal generation by an arbitrary Eprimer (ECHO), where the two dye moieties are paired in the single-stranded oligonucleotide for signal suppression. Upon hybridization to complementary strand, dye moieties get separated and intercalate into the double-strand leading to emission of strong fluorescence. E. Structure of an arbitrary Eprobe having a blocked 3′ end to prevent primer extension during PCR.</p

Effect of mismatched base pair positions relative to the thiazole-orange labelled nucleotide on (A) fluorescence intensity (at 20, 40 and 60°C) and its <i>P</i> value (B) by comparison to full-match ECHO/DNA hybrids (Student’s T test, two-tailed), and (C) peak height in negative first derivative of melting curves and its <i>P</i> value (D) by comparison to full-match ECHO/DNA hybrids (Student’s T test, two-tailed).

<p>Each boxplot and point in (A-D) summarizes ECHO/DNA pairs with different ECHOs having the same distance from the labelled nucleotide to a mismatch, using mean values among replicates of each ECHO/DNA pair.</p

Evaluation of Eprobes and primers designed by Edesign.

<p>(A-D) Evaluation for mutation detection using pandemic 2009 H1N1 influenza virus genome segment 6 (neuraminidase) H275Y mutation; (E-H) Evaluation for low copy DNA detection using influenza B virus genome segment 7 (matrix protein) as a template. Upper panels (A, C, E, G) show results obtained by using Eprobes and primers designed by Primer3 with updated tables for <i>T</i>_M calculation of ECHOs. Lower panels (B, D, F, H) show results obtained by using Eprobes and primers designed by Edesign. Details on primers and Eprobes used are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146950#pone.0146950.s008" target="_blank">S7</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146950#pone.0146950.s009" target="_blank">S8</a> Figs.</p

Mechanism of Eprobe mediated PCR.

<p>During the denaturation step of a PCR reaction, an Eprobe is single-stranded and its signal is suppressed by an excitonic interaction between the two dye moieties (indicated in grey). During the annealing step, the Eprobe binds to its target sequence, and therefore emits a strong fluorescent signal (indicated in green). In qPCR experiments, an Eprobe-derived fluorescent signal is detected during the annealing step. Eprobes are designed in such a way that they commonly have a <i>T</i>_M below the elongation temperature. Therefore an Eprobe is normally single-stranded during the elongation step and hence does not interfere with the polymerase extension reaction. As indicated in yellow, the 3’ end of an Eprobe is blocked to avoid any primer extension reactions from the probe.</p