NSA-IPI
Non-Specific Amplification Implicated Primer Identification
NSA-IPI is an online tool designed to help researchers identify primer pairs responsible for non-specific amplifications in multiplex PCR. Click on the workflow icons below for detailed usage instructions.
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NSA-IPI
Mis-priming primer(s) prediction
After identifying the primer pair causing the non-specific amplification (NSA), predicting whether one or both primers are mis-priming is crucial for redesign. Below are illustrations of the three mis-priming scenarios, along with instructions for using the NSA-IPI mis-priming prediction feature:
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Specifically Annealed Forward Primer (FWR) and Mis-Annealed Reverse Primer (REV):
- Query: Assumed mis-annealing primer sequence, i.e., REV.
- Reference: A short sequence surrounding the assumed mis-priming site, located downstream (3') of the FWR binding site, starting at an offset equal to the NSA length.
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Specifically Annealed Reverse Primer (REV) and Mis-Annealed Forward Primer (FWR):
- Query: Assumed mis-annealing primer sequence, i.e., FWR.
- Reference: A short sequence surrounding the assumed mis-priming site, located upstream (5') of the REV primer binding site, starting at an offset equal to the NSA length.
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Both Primers Are Mis-Annealing:
- This can be inferred from the results of the scenarios above.