Primer design and degeneracy - Are synthesized sets identical? (Sep/14/2008 )
Dear all,
Consider the following primer
GA{A,G}TTCGTG = GARTTCGTG
Now, to send it for synthesis, how'd it be synthesized? They can't add an R but either a Guanine or Adenine. Do they make 50% of primer sets containing G and another containing A?
For higher degeneracy, things would get really diverse wouldn't they?
I'm looking at the possibility of me fishing for genes in a genome, thus primer design. Thank you people!
Yes, nominally 50% of each is made. With many degenerate bases, the variety explodes exponentially.
To design degenerate primers, start by doing an alignment of the protein coding regions from many species, which will show you highly conserved regions of the protein. Then, you can back-translate those into degenerate nucleotide sequences. Look for places with Met and Trp residues, since these have only one codon. Avoid Arg, Ser, Leu, with six codons. You may know something about the codon usage of your organism class that will be useful. Try to make the 3' end of the primers as non-degenerate as possible, and limit total degeneracy to the 4000 or so range. Use more than the usual primer concentrations for the PCR, since the primer you really want is there in low concentration.
To design degenerate primers, start by doing an alignment of the protein coding regions from many species, which will show you highly conserved regions of the protein. Then, you can back-translate those into degenerate nucleotide sequences. Look for places with Met and Trp residues, since these have only one codon. Avoid Arg, Ser, Leu, with six codons. You may know something about the codon usage of your organism class that will be useful. Try to make the 3' end of the primers as non-degenerate as possible, and limit total degeneracy to the 4000 or so range. Use more than the usual primer concentrations for the PCR, since the primer you really want is there in low concentration.
Thank you phage434.