degenerate primer design - (Feb/13/2006 )
I'm designing degenerate primers. I have only two suitable conserved domains in the alignment of amino acid sequences (BTW, what's the requirement for the length of conserved regions and the product?). There are a lot of 6 fold sites in these two domains. It's my first time to design it and I'm not sure the correct way to decrease the degeneracy. Also, I'm wondering if there is a popular software to do this task. I searched some, but they did not work well. Please let me know your suggestions. Thanks a lot!
Two conserved domains in the alignment of amino acid sequences does not ensure you a conserved domain in DNA sequences. Perhaps you should look at the DNA sequence of the coresponding amino acid domain.
Length of the conserved domain should be at least 20bp (my opinion), which is a suitable length for you primer. Length of your PCR product doesn't matter.
What do you mean by "6 fold site in these two domians" ?
Beaware that if you design a conserve degenerate primer, the specificity of your primers will decrease. In the end you might picking up something unspecifically.
Performe a BLAST to make sure your primer specificity.
Thanks for your reply.
"6 fold site in these two domians"-I mean Arginine, Leucine and serine exist in these domains, which bring high degeneracy. You mentioned " Performe a BLAST to make sure your primer specificity". Could you explain it in more details? Thanks.
there's an interesting page on the web
I think to reduce the degeneracy in the primers you can reduce the length by few bases.
kindly go through this doc.