I have designed primers using the program Oligo 7 and I want to evaluate their specificity using primer-BLAST. However, I'm not sure how to interpret the output from BLAST and determine whether I should go through with using my primers. One of the first predicted targets is a human gene with a product length of 20 nts. Both my primers are 20 nts in length. It looks like this:
>XM_005245982.1 PREDICTED: Homo sapiens complement component 1, q subcomponent, B chain (C1QB), transcript variant X1, mRNAproduct length = 20
Forward primer 1 AGTGGTTCAGAGAGGTTAAG 20
Template 24 ..A..C.............. 43
Reverse primer 1 CTTAACCTCTCTGAACCACT 20
Template 43 ..............G..T.. 24
I don't get why the 3rd nt of my primer matches with correctly with the A, but it is considered as a mismatch since the letter is shown.
In general, what are the steps you would use to interpret this data and determine whether there is a good chance the primers will amplify an off-target gene?