The main purpose of this application is the thermodynamic analysis of possible heterodimers that can form between different primers used in a multiplex PCR reaction (many primers in the same PCR reaction).
It also analyzes the thermodynamic characteristics of the individual primers (melting temperature, dimers, hairpin-loop, base composition)


Syntax Data entry:
> name : primer_sequence [ optional ; primer_other_sequence] (written indifferently in lowercase or uppercase)    
- One primer sequence per line, or two primer sequences per line separated by ';'.
- Non-canonical bases (ACTG or actg) will be eliminated.
- If the name is not written, an alphabetical letter will be assigned as the name.    
- If two primers are written in one line, then 'F' and 'R' will be added to the name of the two primers
Example:
> Pr1 : ACCTATATGCTCCACGA ; ctttcggagagaatcg
> Pr2 : CCGTGGACCCACCAG
> ACCTGGGATCCACTC
etc

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

Annealing Temperature °C         
 
           

Attention, April 2024: different data for the PCR reaction could be obtained compared to the old version.
In fact, based on our experiences and to highlight the formation of any dangerous dimers or hairpin-loops, we have modified some default parameters, which can be modified in 'advance setting' relating to the formation of dimers and hairpin-loops of the primers.
In particular, the default data has been changed:
- Range Optimal Primer Melting Temperature --> For higher temperatures the Point with 50% penalty is ΔT changed from 10.0 to 5.0 ; Sloop is: changed from 0.08 to 0.1
- Dimers -> Extention factor at Annealing T. changed from 1.5 to 10.0
- Dimers --> penalty parameter: point with 50% penalty --> 3' extention -> Dimer fraction: changed from 0.15 to 0.12
- Hairpin --> Extention factor at Annealing T changed from 1.0 to 5.0

If you want to get the same data as the old version, change the values in 'advance setting'
 



Advance Setting (Under costruction)
To assign penalties, the application uses a sigmoid curve with 2 characteristic parameters: the point where the 50% penalty will occur and the slope of the curve at that point.
For each type of penalty these parameters will be assigned as specified in the table below.
Annealing temperature (Ann.Temp.):
the one entered in the submission form
Range Optimal Primer Melting Temperature:
Ann.Temp.+ °C     to    Ann.Temp.+ °C (Enable: )
If the Melting Temperature falls between these values, there is no penalty

Instead
For lower temperatures the Point with 50% penalty is: ΔT= Sloop is: )
For higher temperatures the Point with 50% penalty is ΔT= Sloop is:
  
Minimum fraction of
hybrid in the structures
f_thermo_min:
Dimeri
(*) Amplify_dimer_conc:
Extention factor at Low Temp.:
Extention factor at Annealing T.:
penalty_dimer_cutoff:
duplexDimer fraction cutoff
penalty parameter: point with 50% penalty
Dimer fraction:    sloop:
3' extention Dimer fraction cutoff
penalty parameter: point with 50% penalty
Dimer fraction:    sloop :
Hairpin
Extention factor at Low temp.:
Extention factor at Annealing T.:
penalty_hairpin_cutoff:
duplex Hairpin fraction cutoff
penalty parameter: point with 50% penalty
Hairpin fraction:    sloop :
3' extention Hairpin fraction cutoff
penalty parameter: point with 50% penalty
Hairpin fraction:    sloop :
 Vedi grafico sigmoide-penalità
Base content weight (Enable: )
penalty_cutoff
weight_imbalance
weight_stretch
weight_gc_at_stretch
weight_stabiliy_3p
Note:
(*) 'Amplify_dimer_conc' represents a forcing to highlight any formation of 'small' primer dimers
These seem to be, in theory, thermodynamically difficult to form, under reaction conditions. But it also seems, from some papers,
that any 'small' primer dimers can influence sequencing and PCR reactions. This parameter is simply a multiplication factor of the primer concentration.