NSA-IPI
Non-Specific Amplification Implicated Primer Identification
Unpredicted non-specific amplification (NSA) can occur when constructing multiplex PCRs, even when utilising primer optimisation tools. This makes it challenging to determine which specific oligonucleotide primers within the mixture are causing the non-specific annealing.
NSA-IPI is an online tool designed to help researchers efficiently identify the primer pairs responsible for NSAs in a multiplex PCR. Click on the workflow icons below for a quick usage guide, or click here for the detailed user manual.
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.
