Note: Pick Primer VariantGenomic program should 'be able' to build primers for the PCR reaction. The primers will be built externally to a genomic region, selectable by the user through chromosome, position and width (see the chart below).
They will also be built in areas without known polymorphisms, derived from dbSNPs, GnomAD, 1000 genomes, etc. the user can select them based on frequency threshold.
 
The primers proposed by the program, analyzed with thermodynamic parameters with the Nearest-Neighbor method by our PinkPrimerThermoHybridPCR program, will have a length appropriate to the set temperature and will be analyzed for the content in bases, stability at 3 ', presence of any stretch of similar bases, possibility to form hairpin-loops and dimers (self and hetero).
The program assigns a penalty for each aspect analyzed and the primers with the minor penalty will be proposed.


                                around free              around free      
                                  |.......|              |......|               
               p=polymorphism          Pos_start      Pos_end                     p=polymorphism
  --SEQ: --------p-p--------p-----[-------(--------------)------]---------------p----------p-pp------------
                                  [-------- free  region -------]       
                -x->                                                           <-x- NO primer in polymorphism
                       -->                                             <--   correct primer for and rev
                      |- - - - - - - - amplicon - - - - -- - - - - - - - |
Other note: (EN) (IT)      PickPrimerThermoHybridPCR Help

Genome Data Section
- - - - - - - - - - - - Genome Core region - - - - - - - - - - - - - - GenomeRelease:
Chromosome
Start:
  End:
  
   around Free

(region around the core excluded from the primers)

Mark the genomic variants Section

Make variations if their frequency is greater than:
All 1e-6 1e-5 1e-4 1e-3 1e-2 1e-1 no mark
mask region with
RepeatMasker DataBase
(from UCSC)

PCR Reaction Section
click on the suggested links to get explanations about the choices
Annealing Temperature °C (Ta) Delta melting temperature (Tm-Ta) °C (ΔTm)
[ Primer ]: µM   [ dNTPs ]: mM   [Na+] or [K+] mM   [Mg++] mM
Min. pb Optimal pb Max pb Penality for 100 bp
of difference from optimal size
DNA Polymerase Proofreading DNA Polymerase Hot Start

Primer Section
Primer length min max
Display     number of primer pairs returned       Max occurence same primer       Output on file
Pick primer or use your primer
Left primer (5'->3')
 Pick left primer
Use this primer
Rigth primer (5'->3')
 Pick rigth primer
Use this primer