Designed primers with 3 bp mutations (codons) how to validate/visualize them?

Question

[morovatunc]

Dear all hi,

I was given a sequence of 1500 bps. I was asked to replace each amino acid with the rest 19 based on codon usage in human. To control what I have done, I tried to map them with bwa to my subject region and used blastn for the local alignment but for some regions i did not work.

my primer is consisted of 19 bp flanking regions as following.

19 bp + codon(19) + 19bp.

My aim is to visualise my primers correctly. Has anyone experience such a thing?

Thanks for the help,

Best regards,

Tunc.

[swbarnes2]

What command line options did you use? I would be surprised if it were impossible to relax the alignment stringency enough to allow something with 3 mismatches to align. But I wonder if bwa is making the alignment seed with your altered codon in the middle? Maybe making them again, with more than 19 bases in the front, will allow for them to align better.