Degnerate PCR and Sequencing Reactions - (Jan/07/2008 )
I am using degenerate primers to amplify and sequence the Cytochrome C. Oxidase 1 (C01) subunit for phylogenetic analysis.
I have ok to good PCR product (not great) on my gels with a single expected band around 800bp.
I run multiple reactions at 25ul volumes and combine them when purifying the PCR product so I can achieve enough DNA for sequencing.
Gel analysis of purified PCR products eluted in molecular water indicate an acceptable concentration of DNA (based off a sizing ladder) for sequencing.
My sequencing reactions come back very bad. Lots of noise with very little signal strength.
what is the degree of degeneracy of your primers? if they are too degenerate or if the degenerate nucleotides are too close then they may bind at multiple sites on the template.
Make sure the 3` base is highly conserved. Use primers that contain as much conservation at the 3` end as possible. This will promote annealing of the primer to your template and good extension products.
Good luck, Rob
Not sure how to calculate degree of degeneracy? Let me know and I'll get back to you. My primers have been and are used for insect C01 amplifications by other labs and show good results (per literature). My lab is just staring this molecular work (were new at it) and though trying these primers out would be a good way to get our feet wet. But we haven't designed the primers or anything.
On a separate note-
These primers tend to anneal at at a temperature around 47C with genomic DNA from different insects. The cycling conditions for my sequencing facility (UC Berkeley) are as follows: 96º for 1 minute then 25 cycles of: 96ºC for 10 second + 50ºC for 5 seconds + 60ºC for 4 minutes.
Looks like my annealing temperature is too low for these primers? This might make sense considering those few sequencing reactions where I have had success with theses primers had annealing temperatures at 52C?
degree of degeneracy is determined by the number of variable nucleotide sites and how many nucleotides can be fit into the site. for instance if you have one "n" site then you can have 4 different nucleotides in that spot. your degeneracy is 4-fold (4 distinct sequences). if you have 2 sites, one with 4 and one with 2 then you have 8-fold degeneracy. etc.
you can reduce the apparent degeneracy by inserting a dI into the sequence instead of a variable. it will complement any nucleotide so it will be nonspecific but you will have fewer species of the sequence around. this will allow for stronger response. insertion of dI is, however, expensive. you should only do it when you have too much degeneracy.
as for annealing conditions, are you running the reaction yourself? if so then reduce the annealing temperature to suit your primer. if not then you could request that your sequencing service reduce the annealing temperature.
also, are you using a modern or older thermocycler? if old then you may have to increase the denaturing and annealing times (the older cyclers are slower to temperature).