I am looking for a free program that will search a known DNA sequence with known PCR primers, and find degenerate matches. If I put my DNA sequence and primers into NCBI Primer Blast, for example, it says my PCR primers don't anneal to that sequence. What I'm looking for is a program that will show where my PCR primers will anneal to my sequence if only 18 of the 20 bases of the primer anneal, or if only 16 of the 20 bases anneal. I have looked on the web for such a program, but can't find one.
(My problem is that I'm getting a PCR band when I shouldn't be, and I'd like to know if my primer is binding non-specifically.)
Hello Juli, I think BLAST and NCBI Primer BLAST are the best options in your case. Maybe Did you try with all the data bases available in Primer BLAST?
I told you this, because sometimes when I have been assessing primers I got alignments just with one or two of the data bases of Primer BLAST.
Another advice is to use the option "Somewhat similar sequences" when you are doing the BLAST.
I hope you can solve this problem soon.
For this purpose, I use ampliFX software for its virtual pcr feature.
Hello Juli! I agree with Santiago. PrimerBLAST is good to pick degenerate primers considering that it will make use of all the sequences in GenBank, you just have to change the options and select "Somewhat similar sequences" before running it. The you can see if something you don't want may amplify with your primers. I hope it helps. Alternatively, try to redesign your primers and try it experimentally (PCR is always like that haha)
Cheers!
- Badges
- Science topic
- Similar topics
- Computer Science and Engineering
- Bioinformatics
- Bioinformatic Tools