PCR primers design generalities

Primer design is one of the key steps for successful PCR. For PCR applications, primers are usually 18-35 bases in length and should be designed such that they have complete sequence identity to the desired target fragment to be amplified. The parameters, controllable either by the user or automatically, are primer length (12-500 nt), melting temperature for short primers calculated by nearest neighbour thermodynamic parameters, the theoretical primer PCR efficiency (quality at %) value, primer CG content, 3'end terminal enforcement, preferable 3'terminal nucleotide sequence composition in degenerated formulae, and added sequence tags at 5' termini. The other main parameters used for primer selection are: the general nucleotide structure of the primer such as linguistic complexity (nucleotide arrangement and composition); specificity; the melting temperature of the whole primer and the melting temperature at the 3' and 5' termini; self-complementarity; secondary (non-specific) binding.

The software can dynamically optimize the best primer length for entered parameters. All PCR primer (probe) design parameters are flexible and changeable according to the specifics of the analysed sequence and task. Primer pairs are analysed for cross-hybridization, specificity of both primers and, optionally, selected with similar melting temperatures. Primers with balanced melting temperatures (within 1-3°C of each other) are desirable but not mandatory. The default primer design selection criteria are shown in Table. It is possible to use pre-designed primers or probes or, alternatively, pre-designed primers can act as references for the design of new primers. The programme accepts a list of pre-designed oligonucleotide sequences and checks the compatibility of each primer with a newly designed primer or probe.

Table. Default primer design selection criteria.
Criteria Default Ideal 
length (nt)       19 – 23 >21
Tm range (°C)a53 – 55 55 – 57
Tma at 3’-end, 12 bases 30 – 45 30 – 42
3’-end composition (5’-nnn-3’)swh ssw wsh sww wwwswh ssw wsh sww www
Sequence linguistic complexity b>75>80
Sequence “quality”         >75>80
a Nearest neighbour thermodynamic parameters (Allawi and SantaLucia, 1997).
b Sequence linguistic complexity measurement was performed using the alphabet-capacity l-gram method.

In many cases it is necessary to use predesigned single or a list of primers (probe). The program accept the list of predesigned oligonucleotide sequences for checking it compatibility with a newly designed primers or probes. Other case is predesigned oligonucleotides will includes as part of final PCR primer design result. The software automatically checked primer sequences location (with local alignment) on a target sequence and add correct primers to a list of selected primers. Predesigned primers or probes imported in all formats accepted at generally for FastPCR from a clipboard with keyboard (Shift-Insert or Ctrl-V) or right-click mouse displays a contextual menu or from file:


The program is able to generate either long oligomers or PCR primers for the amplification of gene-specific DNA fragments of user-defined length. Up to now, several primer/oligo design programs have been developed. All of them are specialized for either the design of PCR primers or oligomers.

Our FastPCR software provides a more flexible approach of designing primers for many applications with a quality and speed. If either primers or probe have secondary binding sites that may give raise to an additional PCR product somewhere else. The evaluation of potential secondary binding sites of each primer performing with local repeats dataset sequences.

The selection of the optimal target region for the design of long oligomers is performed in the same way as for PCR primers. The basic parameters in primer design are also used as a measure of the oligomer quality; however, the thermodynamic stability of the 3’ and 5’ terminal bases and central part of oligomer is evaluated.

The user can vary the product size or design primer pair for whole sequence without specifying parameters or using default or pre-designed parameters.

The pre-designed parameters are specified for different situation, for example: sequence with low CG content, or long distance PCR, or degenerated sequence, or manual options.