PCR primers and probes design command lines

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:

  • Quality limit (PCR primer 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.
  • Linguistic complexity control: the complexity values were converted to a percentage value, in which 100% means maximal ‘vocabulary richness’ of a sequence.
  • 3'-End composition (5'-3'): 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.
  • С >> 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’aaCGaagtCC-3' 5’aaCGaagtTT-3'
      |||||||||| ->   |||||| |

    3’ttGCttCagg-5' 3’ttGCttTagg-5'

  • Alternative amplification (non-specific binding ) control: the program will analyse primers at current sequence and at "Reference dataset (non-specific primer binding test)" (using quick hash-table alignment) and look for the presence of possible additional complement sites for each primer (probe), which would result in non-specific PCR product. Usually, the site-specificity of the primer can be checked by performing a sequence homology search (e.g. blastn) through all known template sequences in the public genome database such as National Center for Biotechnology Information (NCBI), but it is no necessity, our experience, the almost all problems can be simply solving using any the BAC sequence(s) (about 100,000 or more bases) from analysing genome, because only high copy number repeats will compromise the performance of unique PCRs so, unless the experiments are planned specifically to exploit them, they are best avoided.