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:
> [optional 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.
- The name can be omitted (in this case an alphabetical name will be assigned)br>
- 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         
 
           
 



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.