degenerate PCR vs low stringency PCR - (Nov/24/2005 )
I'm trying to determine some nucleotide sequences of blow flies.
I use known nucleotide sequences of Drosophila, Tribolium, Anopheles and Schistocerca as reference sequences.
First I tried degenerate PCR but too many band were visible.
Now I'm considering low stringency PCR.
To determine unknown sequences using known highly conserved sequences, which one is better?
Is there anybody with much experience in low stringency PCR or degenerate PCR?
Ideally, you want to locate a pair of regions with highly conserved amino acid sequence using low degeneracy codons. Methionine is your friend if it is in such a conserved motif, because it has only a single codon. Memorize the low degeneracy amino acids. Take account of the codon usage table for your organism. Do a ClustalW alignment of as many nearby sequences as you can find. Locate the conserved motifs. If you have a protein with known active sites (see the conserved domain database that BLAST bring up) this can help in location.
The matching of the 3' end of the primer is most important, so concentrate on having the 3' end match very well with few degenerate bases. Make use of I as a base, even though the primer will be more expensive to make. Be prepared to fail and retry a few times.