Pick Primer ThermoHybrid (Release 2011_05)
pick primers from a DNA sequence (by Ivano Zara )

This application proposes the 'best' primers to achieve a PCR reaction
In addition to the template sequence, you can enter: the regions where you want to build the primers, the desired annealing temperature, the reagent concentrations, the optimal length, the type of polymerase used (hotStart, proofreading), etc.
The primers proposed by the 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.
The program also provides 'warnings' using the 'Amplicon Analysis' program (present in this same platform), for the possible hairpin-loop formations of the DNA template in the primer hybridization regions, at the annealing temperature, which could prevent the 'hybridization of the latter, and for the determination of possible hairpin-loops inside the amplicon, at the temperature of extension of the PCR, which could, instead, slow down or prevent the polymerization of the new filament.
Beware that this analysis, with long template sequences, could slow down the execution of the program too much. In this case, disable the analysis.

Another important aspect is the possibility of inserting sequences similar to the main one and asking to build primers that MUST or DO NOT need to amplify these sequences.


Try our examples
1) - Input Only Main Sequence
2) - Main Sequence and similar sequences that must all be amplified (Under construction)
3) - Only the main sequence must be amplified, not the similar ones (Under construction)

Sequence data
This section allows you to set the main sequence (on which the primers will be built) by selecting the central region.
It also offers the possibility of inserting secondary sequences that must or should not be amplified by the primers


MAIN SEQUENCE
no in FASTA format, see below the meaning of the symbols '>'
- Mark your sequence with '[' and ']': e.g. ...ATCT[CCCC]TCAT.. for that primers must flank the central CCCC.
- Mark your sequence with '<' and '>': e.g. ...ATCT<CCCC>TCAT.. for forbids primers in the central CCCC.
- Mark your sequence with '(' and ')' for locate primer 3' end
- Mark your sequence with '{' and '}' for locate primer 5' end
       e.g. ...ATCTCCC(CT)CAT means that 3' primer are only in C or T
Note: If you use round or curly brackets, it's need to use square brackets to indicate left and right part of template sequence
Note: You can input any character, but only simboli IUPAC character are accepted


sequence that must to be OR NOT to be amplifyHelp Similar Sequence

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



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

Min. pb Optimal pb Max pb
Penality for 100 bp of difference from optimal size
[ Primer ]   
 µM  
[ dNTPs ]   
 mM  
Salt concentrations
[Na+] or [K+] mM   [Mg++] mM


DNA Polymerase Proofreading DNA Polymerase Hot Start



Repeat Analyse              Use repeat library  

Analyse Hairpin Template Constrain   Disable
  At the end
  Always

This analysis is still under construction. Warning the 'Always' option slows down the execution of the program
We recommend that you use option 'Always' only if the regions where you build the primers are small
If you select 'At the end', a complete analysis of the possible template structures will be shown in detail.
If problems are found, 'disable' this analysis.

Primer
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

Display
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

Display
Number to Return        Max occurence same primer


ADVANCE SETTING
Under Costruction