Check for Binding Specificity

Once primer pair design using any of the PRIMEGENS algorithm (SSPD,FSPD and PSPD) is accomplished, the model checks for potential amplicons amplified by each primer pair across whole genome. The chance that a primer pair will amplify another region in addition to the target region is relatively low because only a certain order and orientation of primer binding can result in an amplicon. In addition, even if the primer pair binds in correct order and orientation, the amplicon size should fall within the designed size criterion. For example, a primer pair which shows cross-hybridization with amplification size of more than 10,000 bases other than target region can be safely assumed to be specific to the target region. Figure below shows an example of primer oligo blast hit, which results in successful or failed amplification in PCR. As shown, out of 4 different cases of primer pair hits, only one case results in successful amplification. Further, it occurs only if the amplicon size is less than maximum possible amplicon size for RT-PCR. In current model, we use 10,000 bases as cutoff to avoid false negatives during computations.

After applying all above mentioned filters, exact sequence similarity at 3'-end of each potential primer pair hybridization is checked. It considers only those hybridization hits that have last 2 and first 2 bases in left and right primers respectively are exact comlementary to each respective primer and there is no adjacent mismatch in sequence hybridization of next 3 bases in 3'-end is present.

Next, energy/thermodynamics of each of thus obtained potential primer pair hybridizations is calculated for their stable binding. For energy calculation, single internal mismatch parameters(NN-model) are used and "Minimum 3'end Stability" value provided by the user (also used by Primer3 for primer design) is used as energy threshold. Hybridizations that pass these specificity filters are reported as cross-hybridizations for each primer pair designed.

Figure-1

Fundamental restriction for PCR amplification. Of the 4 scenarios, (A) alone will produce amplicon, and only if the inter-primer distance is less than maximum amplicon size as determined by the user.