NSA-IPI
Non-Specific Amplification Implicated Primer Identification
NSA-IPI is an online tool designed to assist end users (researchers) in identifying primer pairs that cause non-specific amplification in multiplex PCR settings.
NSA-IPI
Mis-priming primer(s) prediction
After identifying the Non-specific amplification causing primer-pair, predicting whether one primer or the whole pair is mis-priming (non-specifically annealing) is important in subsequent primer re-design. The figure below illustrates the three possibilities of primer mis-annealing in non-specific amplification, and based on these three possibilities, it is possible to predict the mis-priming primer(s) using this feature as follows:
- Assuming specifically annealed FWR & mis-annealed REV: Query sequence is the assumed mis-annealing REV & the reference sequence is tens to hundreds nucleotides AROUND the 3’ of the FWR primer binding site plus the NSA length.
- Assuming specifically annealed REV & mis-annealed FWR: Query sequence is the assumed mis-annealing FWR & the reference sequence is tens to hundreds nucleotides AROUND the 5’ of the REV primer binding site minus the NSA length.
- Both FWR & REV are mis-annealed either on the target DNA template or another DNA template in the sample: This is indicated by the results of the above.