Sometimes, you may not get a good amplicon because one primer shows better binding at an off target site. Doing the BLASTn in the way you suggested may not pick up on this problem early.

The reason a BLASTn check is reccomened is to be sure that your primers do not show a high similarity to off target sequences. Since the primers are commonly used in pairs, it is better to validate each primer in the set for its unique homology individually.

I'd do it pairwise. If one primer is a good match to several areas, you'll still see it.

Thank you for your answers Katie A Burnette Praveesh Valissery . I have a scenario where if I run the primer individually, I get multiple hits with 100% query cover and % identity (one of which is the sequence I want to be amplified), but when I run it in a pair (ie. both primers with 20 N's separating them) I only get one hit (the sequence I want amplified).

If I am understanding this correctly, the reason for these different results is that the individual primers may be binding at different off-target sites, but together they should produce my desired amplicon. If this is true, then it should be fine to use these primers for PCR. Is this correct?

Andrew Chong . Checking primers one at a time ensures that the primer set you have chosen is uniquely specific to your target sequence. Even if one primer binds at an off target site with better specificity than your target then you may not get efficient amplification of your desired sequence.

The idea is to make sure the primer sequence you have chosen outputs your desired gene with the best score and low expect value in nBLAST against the genome/template library. If you get an unwanted hit with a better score and lower expect value then the primer sequence needs to be moved or increased to ensure specificity to your target.

I wouldnt say that this screening process guarantees sucessful amplification of your target sequence but it is an effective step in that direction.