Melting DNA-Hybrid Project
Amplicon Analysis (Release 2011_01)
Thermodynamics Analysis primer/template/amplicon structure)
by Ivano Zara


This application allows to determine the 'goodness' of a PCR product, analyzing the primers in relation to the template sequence..
It analyzes primers with 'Primer Structure Analysis' (see melting-program) and predicts the formation of any hairpin-loops of the template, which involve the region of hybridization of the primers, which can then create steric bulk and prevent the access of primers into the hybridization region.
The eventual hairpin-loops, inside the amplified sequence, that can obstruct the progression of the DNA polymerase are also analyzed.
Overview (only in the Italian language)


Tip: If you're working with the human sequence, maybe you might be interested in apps in Human variant Tab
An example is: Shows the Genomic Regions with polymorphisms

Amplicon Analysis(Beta TEST)
Please, try our example
      

Use our Pick Primer Thermo Hybrid app to select excellent primer pairs
Primer
It is possible to insert mismatched primers and see the effect in the PCR reaction
Primer FOR (5'-->3')

      Primer REV (5'-->3')
Note: the program automatically chooses the best primer alignment in template sequence
Template Sequence (strand '+')
OR Select Local File template

DNA in circular form
 
                 

Solution and PCR parameters
[Na+] mM       [Mg++] mM       [dNTPs] mM       [primer] mM    

Annealing Temperature °C            Extention Temperature °C         
Polymerase proofreading (exonucleasic 3' activity 3'-5' (proofreading)) (under construction)



Advance Setting
.
Cutoff_f_min_eff_start_PCR %
Cutoff_f_min_oligo_template_hairpin %
Cutoff_f_min_internal_hairpin %
Lateral Extention

Temperature factor to modify;:
template in duplex (0-10)
template in hairpin-loop in primer region (0-20)
template in hairpin-loop in internal amplicon (0-20)
Note: These values have been inserted to also consider the dynamism of the PCR reaction.
They modify the fractions of hybrid primer/template or the hybrid fractions of hairpin-loops
which are calculated with thermodynamic equilibrium methods.

For the prediction of the duplex fractions or for the prediction of the fractions of any secondary DNA structures (dimers or hairpin-loops), the program uses classic equations and thermodynamic parameters, which however do not consider the dynamics of the reactions (competitions and enzyme activity).
There are no particular studies that take into account these dynamisms of reactions.
The program, to improve the prediction, modifies the values of the thermodynamic equations. For this purpose particular parameters (Temperature factor) are used, which can be set in 'Advance Setting'.
By default the program uses values derived from our laboratory experience, these represent only a first attempt to obtain a 'significant' prediction.
These parameters and possibly other parameters will surely have to be applied and calibrated in the future.

Those who use our programs to analyze and/or design PCR reactions can contribute to the improvementof these predictions by sending the author (ivano.zara.bio@gmail.com) the results obtained in their real-time PCR experiments.