PPI helps you design better PCR primers

PPI stands for polymerase preference index (patent pending). It is an algorithm we developed to help select the best performing PCR primers based on polymerase preference.

Most of the primer design softwares evaluate the melting temperature (TM) as the most important parameter. But TM, at its best, could only tell us if a primer would bind to the template at certain temperature; it tells us nothing about if the primers will be used or preferred by the DNA polymerase.

To evaluate polymerase preference, we analyzed high throughput sequencing data where random hexamers were used as primers to amplify genomic DNA. The results have shown that, of the 4096 possible hexamers, only small portion were “over utilized” and not all of them having higher CG content.

Publications have shown that, when synthesizing DNA, the polymerase covers about 10bp of templates – the 5’ end 6bp with primers, and the 3’ end 4bp (we call it “runway”) is the bases about to be synthesized. We hypothesized that the exact DNA sequences of this 10bp window may have significant influence on the polymerase “run on” rate. Using high throughput sequencing data (about 20 million reads), we can calculate the preference index of hexamers and tetramers by multiply their rate of them been used as primer (or runway) over the rate of the same sequence in the amplicons (genomic background).

Applying the PPI value to both the forward and reverse strands of DNA templateallows us to position PCR primers at those sequences with higher PPI values. Once primer position is identified, we will adjust the length of the primer to achieve balanced TM.

The online iC-Architect software helps registered users design mPCR (multiplex PCR) primers by incorporating TM, PPI, arm-PCR principles. And it is free.