Eurofins Genomics' PCR Primer Design Tool is using Prime+ of the GCG Wisconsin Package originally written by Irv Edelman.
The PCR primer desgin tool analyses the entered DNA sequence and chooses the optimum PCR primer pairs. In selecting appropriate primers, a variety of constraints on the primer and amplified product sequences are already considered and taken as default values
. You either can use the default constraint values or modify those values to customise the analysis. Target Sequence
Copy & paste the target sequence from an external source. DNA sequence information as well as FASTA sequences ( starting with an ">" followed by the name) are possible. Line breaks and blank spaces are allowed. The template sequence may contain ambiguous bases, but the design tool will not select primers complementary to any ambiguous sites on the template sequence.Design Parameters
You can design PCR primers from the whole template (= target sequence) or limit the choices to a particular region. For PCR primer pairs, you can specify any required bases at the 3' end of the primer (3' clamp), and a maximum difference in primer melting temperatures.Primer Criterias
For the PCR primer pairs you can specify minimum and maximum primer length, primer GC content and primer melting temperature (Tm). The maximum primer length you can search for are 100 bases. Primer melting temperatures is calculated by using the nearest-neighbor model of Borer, and thermodynamic parameters for DNA nearest-neighbor interactions and the salt dependence of oligonucleotides determined by SantaLucia [Proc. Natl. Acad. Sei. USA. 95; 1460-1465 (1986)]Amplicon Criterias
For PCR products, you can specify a range of acceptable product sizes and define the minum and maximum GC content and melting temperature (Tm). The maximum product length to enter is 5,000 bases. PCR product melting temperatures are calculated by using the formula of Baldino, et al. [in Methods Enzymol. 168; 761-777 (1989)] as modified slightly by Rychlik, et al. [Nucleic Acids Res. 18; 6409-6412 (1990)]. Reaction ConditionsSalt concentration
specifies the mM salt concentration in the reaction. This value is used in the calculation of both primer and PCR product melting temperatures. The default value is 50.0 and the value may range from 0.1 to 50.0.Primer concentration
specifies the nM concentration of primer DNA in the reaction. This value is used in the calculation of primer melting temperature. The default value is 50.0 and the value may range from 0.1 to 50.0.
For efficient priming, the design tool avoids primers with extensive self-dimer and cross dimer formations in order to minimize primer secondary structure and primer dimer formation. Click on the "Design Primers"
button to get a list of appropriate PCR primer pairs. The output includes a proposed annealing temperature for each listed primer pair.