Melting DNA-Hybrid Project
Primer Multiplex PCR (Beta Release)
by Ivano Zara

This program analyzes the possible heterodimers that can form between the different primers used in multiplex PCR reactions (many primers in the same PCR reaction)

If the program is considered interesting, it can be implemented with new functions, for example, analyzing also possible DNA sequences and providing a simulation of the final electrophoretic run.

Data entry:
a pair of primers for each row. The line must start with '>' and the data must be separated by ';'
> ID_coppia; First primer sequence; Second primer sequence (;optional annealing temperature)
example:
> 1; ACCTATATGCGCGA; ACCTTGCGCGTAGGCTTG
> 2; ACCTATATGCGCGA; ACCTTGCGCGTAGGCTTG; 60

Note: in the sequences, all the characters that do not represent the canonical bases will be eliminated

Input file con le sequenza dei templati (in formato FASTA) (IN COSTRUZIONE)
Solution and PCR parameters
[Na+] mM    [Mg++] mM    [dNTPs] mM    [primer] mM    

Annealing Temperature °C         
 
           
Bassa sensibilità     Media sensibilità     Alta sensibilità
 
 
 



Advance Setting (Under costruction)
Annealing temperature: 58.0Range Optimal Melting Temperature:
T.Ann.+ °C (Point with 50% penalty: DT= Sloop =)
- to -
T.Ann.+ °C (Point with 50% penalty: DT= Sloop =) (Enable: )
There is no penalty if the T.melting of the primer is lower than the allowed Ta + DTm.
On the other hand, there is a 50% penalty if Tm - (Ta + DTm) is equal to 'DT'. At this point the penalty varies with slope given by 'Sloop'
  
Minima frazione di
ibrido nelle strutture 		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.