PrimerDigital

If necessary for an individual control for the PCR primers or probes design for each sequence individually, you can find “Quick PCR Commands Help” at menu bar – Help:

General PCR primer options you can find – “Ctrl-K”

• Ends Nucleotide Composition: you can choose with 5’ or 3’ ends is most preferable for all primers or probe. Minimal size is 2 letter (maximum – primer length), “5’-nn-3’” is any, “5’-sww-3’” – all variants for 5’-(C/G)(A/T)(A/T)-3’”. You can type one or more variants with space between them and with the same length. 
• Primer’s Tm Optimisation: allow automatically design best primer for current Tm (in practise - FastPCR select primer length from 18 to 28 bases). 

• C >> T bisulphite conversion: bisulphite modified genome sequence, design of specific PCR primers for in silico bisulphite conversion for both strands - only cytosines not followed by guanidine (CpG methylation) will be replaced by thymines:
  

5’aaaCGaagtCCg     5’aaaCGaagtTTg
  ||||||||||||  ->   ||||||| |  | 
3’tttGCttCaggC     3’tttGCttTaggC

• Primer quality (amplification efficiency): this abstract value of the ‘primer quality’ describes thus the level of primer/PCR successfulness; this value varies from 100% for the “perfect or ideal” to 0% for the "worst" primer. A “perfect” primer has a wider range of executable temperatures. A program is select the best primer with optimal range of executable temperature, which allowed to design qualified primers (probe) for any target sequences with any CG and repeat contents. Primer Quality Control: help eliminate “weak” primers. 
• Linguistic Complexity Control the complexity values were converted to a percentage value, in which 100% means maximal ‘vocabulary richness’ of a sequence. The ‘primer quality’ values of 80 and higher allow for the rapid choice of the best PCR primer pair combination. No adverse effects, due to the modification of the reaction buffer, sourced thermostable polymerases or variations in annealing temperature, have been observed on the reproducibility of PCR amplification using FastPCR-designed primers.