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.
Moreover, The user can add to the known polymorphisms, even of their own stored in a VCF format file.
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 -------]
--> --> <-- <-- NO primer here
--> <-- correct primer
prime for primer rev
|- - - - - - - - amplicon - - - - -- - - - - |

Other note: (EN) (IT)      PickPrimerThermoHybridPCR Help


Main default data: considering all variants, Temp.annealing is 56 C,
optimal amplicon length is 500 pb, standard PCR solution.

Genome data
This section allows to set the genomic data and select a central region (core region).
The primers will be built laterally to this region..

Core region:
    Chromosome   Start:   End:
   around Free
(region around the core excluded from the primers)

Mark the genomic variants
This section allows to mark the genome with the variants (according to their frequency)
in order to prevent the construction of the primes where these variants exist.

Make variations if their frequency is greater than:
All 1e-6 1e-5 1e-4 1e-3 1e-2 1e-1 no mark

RepeatMasker **(new)**
mask region with RepeatMasker DataBase (from UCSC)

in addition, you can insert your variants in a file vcf format

Genomic polymorphism were derived from:
- dbSNPS 151 (GRCh37 and GRCh38) from NCBI (file 00_All.vcf, only SNPs validate)
- Frequencies from GnomAD:
gnomAD v2.1.1 for GRCh37/hg19 (exomes and genomes)
gnomAD v3.0 for GRCh38/hg38 (genomes)

PCR reaction
this section allows to select the parameters of the PCR reaction

Annealing Temperature °C (Ta) Delta melting temperature (Tm-Ta) °C (ΔTm)
Difference from primer melting temperature and
experimental annealing temperature

[ Primer ]   
[ dNTPs ]   
Salt concentrations
[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

click on the suggested links to get explanations about the choices

this section allows to characterize the primers and/or choose their own

Primer length min max

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

If you use your primers you have to say on which strand you built them
   '+'    OR    '-'
Primers built on the strand '-' will be converted into the strand '+'. The program uses the strand '+'

click on the suggested links to get explanations about the choices

this section allows to select the desired type of display (on screen or on file),
the quantity of copies of primers to be listed and
how many times the same primer must be used in combinations

number of primer pairs returned       Max occurence same primer
      Output on file
click on the suggested links to get explanations about the choices