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ProbeMelt - Outputs

FISH
  • Calculations are based on a single reaction model (SRM) that predicts the Gibbs free energy (ΔG0) change of DNA/RNA duplex formation at 46°C and 1M sodium concentration. The free energy change is then used to estimate the fraction of targets bound by probe (hybridization efficiency) at varying levels of the denaturant formamide ([FA]; %, v/v).
  • Summary display: The summary includes the probe/target sequences, ΔG0 for 0% [FA] and 46°C, and the hybridization efficiency (HE) at 0%, 15%, 30%, and 45% [FA]. Each probe/target row is color-coded by its chance of hybridization under stringent conditions, with perfect match targets shown in alternating white and blue, those with a high chance of cross-hybridization in red, moderate chance in yellow, and low chance in green.
  • Complete results download: A comprehensive list for each probe/target pair with ΔG0 and HE at varying concentrations of formamide is available via the download link. After unzipping the tab-delimited text file it can be opened with a spreadsheet program such as MS Excel to analyze the results (e.g., to plot the melting curve).
    Descriptions of the columns are as follows:
    1. Number: The probe/target pair number in the same order as entered in the input.
    2. Probe: The input probe sequence(s) oriented in 5' to 3' positive (+) sense and aligned to their respective target sequence.
    3. Target: The target site sequence(s) oriented in 5' to 3' positive (-) sense and aligned to their respective probe sequence.
    4. The following outputs are given:
      • Risk: Gives the approximate risk of cross-hybridizing for mismatched non-targets under stringent conditions. For perfect match targets the "Risk" column is not applicable ("NA").
      • dG1: The free energy change of duplex formation at 0% formamide ([FA]).
      • FAm: The formamide melt point (FA concentration where hybridization efficiency is predicted to be 50% at 46°C).
      • dFAm: The difference in formamide melt points between the perfect match and mismatched target molecules.
      • HybEff_[FA]: The predicted hybridization efficiency of the probe/target pair at 0% to 70% (v/v) formamide and 46°C.
  • The model (SRM) is optimized for predicting formamide curves of perfect matches. For mismatches, the curve should be used with care. We specifically recommend using the difference between formamide melting points of a probe with mismatched non-targets and the perfect match target (Δ[FA]m). The relative stability of mismatched non-targets is indicated by the coloring in the results summary, and the Risk column in the detailed outputs.
Microarray
  • The Linear Free Engergy Model (LFEM) first calculates the standard Gibbs free energy (ΔG0) change of binding at 42°C as a linear function of the concentration of the denaturant formamide ([FA]; %, v/v). Then hybridization efficiency (HE) of target capture, defined as the ratio of probe bound targets to total target molecules, is obtained using ΔG0 based on equilibrium thermodynamics.
  • Summary display: The summary includes the probe/target sequences, ΔG0 for 0% [FA] and 42°C, and the hybridization efficiency (HE) at 0%, 10%, 20%, and 30% [FA].
  • Complete results download: A comprehensive list for each probe/target pair with ΔG0 and HE at varying concentrations of formamide is available via the download link. After unzipping the tab-delimited text file it can be opened with a spreadsheet program such as MS Excel to analyze the results (e.g., to plot the melting curve).
    Descriptions of the columns are as follows:
    1. Number: The probe/target pair number in the same order as entered in the input.
    2. Probe: The input probe sequence(s) oriented in 5' to 3' positive (+) sense and aligned to their respective target sequence.
    3. Target: The target site sequence(s) oriented in 5' to 3' positive (-) sense and aligned to their respective probe sequence.
    4. The following outputs are given:
      • HybEff_[FA]: The predicted hybridization efficiency of the probe/target pair at 0% to 50% (v/v) formamide and 42°C.
      • dG_[FA]: The predicted free energy change of duplex formation at 0% to 50% (v/v) formamide and 42°C.
  • Thermodynamic parameters of ProbeMelt were estimated using high density 4-plex NimbleGen microarrays hybridized at 42°C. Model predictions are not recommended to be used for hybridizations at temperatures significantly different than 42°C. Application to platforms other than NimbleGen 4-plex arrays should also be handled with care.