Degenerate Primers - Help with Degenerate Primers (Jul/31/2007 )
I'm very new to molecular biology and I'm helping start up a lab. My supervisor is also new to molecular biology and we are learning as we go so please excuse me if I sound ignorant.
I have a few questions about degenerate primers.
1.) Is there an advantage of using the AA sequence over Nucleotide sequences when aligning sequences for degenerate primer design?
2.) When aligning sequences do we pick the most highly conserved area and design the primers around that area?
3.) Is Vector NTI a good program for this? I have also heard of using ClustralW??
4.) Degenerated Primers are generally the same price as regular primers? Where is a good place to purchase the primers if I'm on the East Coast of Canada.
Any tips or advice would be greatly appreciated as I am very new to this area of research.
first of all if you have the nucleic acid sequence of whatever you wish to amplify it's better to use that instead of a.a. because of the degeneracy of the genetic code (the same amino acid can be coded for by multiple = 1-6 different codons). I'm guessing you have the cDNA sequence of a protein in one species and you want to amplify a homologue in a different or several different species. You can align multiple sequences using clustalw, which is pretty fast and easy (here is a link to a japanese site http://align.genome.jp/) or you can download a software package called BioEdit and do all that there. What you want to do is find stretches of conserved nucleotides between different species (or whatever else you want to amplify) and wherever there is a position where the nucleotides are different you insert a degenerate site. For example if two genes have this sequence AAAAAATAAAAA and AAAAAAGAAAAA you would design degenerate primers that would be AAAAAAKAAAAA (where K is an equimolar ratio of T and G). Of course most of the time it's not that simple, because there are usually positions where there is a preferred but not the only nucleotide for example the third A is present in 75% of all known genes, but in 1/4 you find T. In this case you can either say OK I'm going to take A anyway, or put in a degenerate site. That depends on the overall degeneracy of your primers. When this is too high, you'll have trouble setting up the PCR annealing temperature for all the different kinds of primers in your mix. I normally work with an overall degeneracy of 256-fold, but higher than that I don't know. I've seen people use much more than that, but I don't know what you get. Because with all those different kinds of primers, you can get non-specific amplicons just by chance.
I hope this helps,
oh and for primer synthesis, you can check out Invitrogen, but it's not cheap.