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Example for Hyb-probe improvement using MeltCalc©

The ultimate thermodynamic modelling spreadsheet for Excel™

The following LightCycler™ melting curves have been achieved before and after thermodynamic Hyb-Probe assay optimization using MeltCalc© Professional which runs as Excel© "Add-In" in almost every Windows™ environment. Excel 7.0 or higher is required for execution. For this example
a 200 bp sequence surrounding the mutation of interest was given together with the mutation
of interest (For details please read the handbook). The program calculates 10 probe sets for each orientation (sense and antisense) and each sequence (mutation or wild-type) and will rank them according to the Tm-difference between mutation and wild-type. The detection probe and a 3' and 5' adjacent anchor oligonucleotide are calculated for each set. The program will by default ensure that the anchors will have a >15% higher Tm than the matching probe. You can choose other values on your own. Interested to get a fully featured license of MeltCalc©-professional?

In the example shown both probes (wild-type and the mutation compatible) hybridize with the antisense strand. The wild-type compatible probe (manual selection) shows no sufficient Tm difference between perfect match and mutation. However, the mutation compatible probe can reliably discriminate the Wild-type and was automatically chosen by MeltCalc©.

First probe set, manually selected Second probe set, selected with MeltCalc
Prediction of melting temperatures (Tm) with MeltCalc©
The ultimate thermodynamic modelling spreadsheet for Excel™
This correlation was calculated from 35 independent assays from us (n=25) and from data in the literature (n=10). All melting temperature were measured in LightCycler™ hybridization probe assays.
New features of this version are:
Calculation of Tm now including "dangling end"parameters (if applicable).
Maximum possible sequence length increased from 256bp to 1755bp for all features.
Maximum possible alignment for cross hybridization is shown graphically.
Mutations can be inserted by bp and base exchange.
A formula for calculation of Tm for long sequences is added .

Probe design:
Dangling end calculations are included into the algorithms.
(Highly important for optimization of hyb-probes; every probe has two dangling ends, since the DNA template is usually longer than the probes!).
Space between anchor and probes for LightCycler™ assays can be chosen (1 to 5).

Minor modifications:
Mismatches are highlighted automatically.
Calculation of sodium equivalents has been modified.
SYBR green-I™ influence on Tm is estimated, based on empirical data.

First page:
Cross-Hybridization Tms are highlighted using color coding in dependence of their hybridization temperature
Second page:
Cross-Hyb are shown graphically (partial display)
Mutations can be inserted by bp and base exchange