I am using a pair of primers each oligo has a degeneracy of 4, so an overall degeneracy of 8 when they are used together.
The primers are for the amplification of 16S rDNA so are being used to identify the microbiota of particular tissues.
In my mind I feel there may be a a loss of sensitivity when each reaction is relying on one out of 8 primers to anneal to its target. This, in my mind, will have a greater impact when bacterial DNA present in my samples is particularly low.
Can someone please let me know one way or another, if I am just fussing over nothing, or better still if there is a study where LOD was investigated for primers sets with varying levels of degeneracy, that would be amazing!!
Thanks in advance
if you are saying that at 2 positions there is a 1:4 chance of any base being correct then I would expect the combined efficiency to be 1:16 of finding both polymorphisms existing not 1:8 and this might make a difference but it would depend on where, in the primer , the degeneracy is. Mismatches near the 3' end of a primer make a huge difference to whether it will anneal properly and the pcr will work and annealing temperatures will have to be lowered substancially. Mismatches more than 6 bases away from the 3' end should not make much difference and the annealing temperature lowered by only a couple of degrees.. If the primer has to anneal over a polymorphism could you use inositol base instead of 4 fold degeneracy as this should anneal well with any sequence. Given that you already have the primers I would increase the lrge excess of primer by a factor of 2, drop the annealing temperature and possibly use slightly more Magnesium in the reaction.
Hi Paul,
Thanks for your answer. I am no longer doing any work as I am finished with the lab and in the process of writing up my thesis. I have noticed part of my results are peculiar. When I was identifying the certain microbial sequences in tissue I used both universal primer sets and some genus and species specific primers too. I have noticed that I was able to amplify my target with the species specific primers but the universal primers (the ones with degeneracy) would not amplify certain species DNA. Therefore I am just thinking out loud and trying to understand what impact degeneracy has; whether it may in fact effect the sensitivity of the reaction or not. So if say certain bacterial DNA was close to low copy number range degenerate primers my struggle, whereas species specific would have little problem. My universal primers were well optimised and working great, but just wouldn't amplify certain species that were within the scope of the primers, especially considering these species could be amplified with species specific primers. Just makes me think its the degeneracy.
P.s. the degenerate bases were right in the middle of the oligo sequence, nowhere near the 3' or 5' ends
ok the degeneracy in the middle is good. specific primers must be better as they will produce only one product so amplification is efficient bbut if you have many products then some will amplify better depending on whether the product melts easily and the primer sticks well. Too many products will also lead to reagent depletion earlier in some cases.
You may want to consider that in the making of oligos the g base phosphoramidite ads slightly less efficiently than other bases so you will get slightly less of the g containing oligo in a redundant mixture. Tech help at the company that made them can advise how much difference this makes on long oligos but probably not great. you may need to check sequences of the non amplifying species for high cg content so poor melting of the product
paul
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