Eurofins Genomics' PCR Primer Design Tool uses Prime+ of the GCG Wisconsin Package originally written by Irv Edelman. The PCR Primer Design tool designs the pair of PCR primers to amplify the segment of interest in the input DNA sequence. Several constraints on the primer and amplified product are pre-set to default values during the primer design. These are suitable for most routine amplifications; however, you can change these parameters to further customize design parameters to your specific experiment.
Target Sequence
Enter the sequence of the target DNA here. DNA sequence as well as FASTA sequences (starting with an ">" followed by the name) with line breaks and blank spaces are allowed. While a target DNA with degenerate bases will be accepted, the tool will not design primers complementary to the degenerate/ambiguous sites on the template.
Design Parameters
Use the complete input sequence (default settings) as the amplification target or select a particular segment by providing start and end base positions. Select the base in the 3′-terminus (3′ clamp) and set the maximum acceptable difference in the Tm of the two primers.
Primer Criteria
Specify the minimum/maximum length (<100 nt), GC content, and Melting Temperature (Tm) for your primers. The primer melting temperature is calculated using the nearest neighbor model as described by Borer [J. Mol. Biol.; 86843-86853(1974)]. Thermodynamic parameters for DNA nearest-neighbor interactions and the salt dependence of oligonucleotides are determined using the protocol described by SantaLucia, Jr. [PNAS; 95; 1460-1465 (1986)]
Amplicon Criteria
You can specify a range of acceptable product sizes (max = 5,000 bases) and define the minimum and maximum GC content and melting temperature (
Tm) for the amplicons. The melting temperatures for the PCR products are calculated using the methods described by Baldino, et al. [Methods Enzymol. 168; 761-777 (1989)] and its slight refinement by Rychlik, et al. [Nucleic Acids Res. 18; 6409-6412 (1990)].
Reaction Conditions
Salt concentration (mM) is used in the determination of both primer and PCR product melting temperatures. While the default value is set at 50.0 (mM), it can vary from 0.1 mM to 50.0 mM. Primer concentration (nM) is used in the determination of melting temperature of the primer. The default value is 50.0 (nM) and the value may range from 0.1 nM to 50.0 nM.
To ensure efficient priming, the design tool evaluates extensively for and avoids sequences with a potential for self- and cross-dimer formations. After entering the target sequence and selecting the design parameters 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.