PrimerDigital

The major aspects of primer features include unique nucleotide structure and the melting temperature. Usually PCR primers 18-35 bases in length (PCR primer or probe length can vary from 12 to 500 bases) should be designed such that they have complete sequence similarity to the desired target fragment to be amplified.
The parameters controllable by the user are primer length, melting temperature with different nearest neighbour thermodynamic parameters or simple formulas, sequence linguistic complexity and pyrimidine-purine linguistic complexity, primer CG content, CG 3’end terminal enforcement and preferable 3’ or 5’ ends nucleotide sequence composition in degenerated formula, polypyrimidine (T, C) or polypurine (A, G) stretches, repeats of identical bases in primers.
Also other main parameters used in FastPCR for primer selection are: the general nucleotide structure of the primer such as linguistic complexity (nucleotide arrangement and composition); specificity; the melting temperature of whole primer and the melting temperature at the 3’ and 5’ termini; a self-complementarity test; secondary (non-specific) binding choosing primer. Software dynamically optimizing the best primer length for entered parameters.

All PCR primer (probe) design parameters are flexible and changeable according specific of analysed sequence and the task.
Primer pairs analysed for cross primer hybridization, specificity of both primers and optionally for similar melting temperature. Designing primers with balanced melting temperatures (within 1-4°C of each other) is desirable but not mandatory.
The default primer design selection criteria showed at table 1.

Table 1. Default primer design selection criteria.

Criteria	Default	Ideal
length (nt)	20 – 25	22 – 27
Tm range (°C)a	55 – 65	60 – 68
Tma at 3’-end, 12 bases	32 – 48	41 – 45
GC (%)	45 – 65	50
3’-end composition (5’-nnn-3’)	sws, wss, ssw, swa	wss, ssa, sws
Sequence linguistic complexity b	>80%	>95%
Sequence “quality”	80	>85

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 (Shit-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.