I'm currently trying to amplify 2 mitochondrial genes (COI, 16S) and 3 nuclear genes (H3, 28S, 18S) for a species of marine micromollusc. I'm able to get H3, 28S and 18S to work but I can't successfully amplify COI. I've designed primers, as well as trying universal primers, and using several different PCR regimes (mostly bottom-up approaches with annealing temps ranging from 40C to 50C), but nothing seems to work. Could this be a problem with low DNA yield given that it's a tiny micromollusc?
Thanks in advance for your help!
possibly more to do with you method of dna preparation not isolating or degrading small dna.what method are you using for dna preparation?
I agree with Paul.
If you think the problem was low DNA yield, just increase the number of individuals in the DNA purification.
For me, it sounds more like if your primers for COI were not good enough. Try a new design using the best possible reference sequence.
Nevertheless, annealing temps of 40-50ºC ar really low. Under my experience, it is better to adjust annealing temps to theoretically 60ºC (best sensitivity/specificity ratio), then try lower temps if this does not work
Thank you all for your help. I've been using the Qiagen DNeasy kits with 100uL elutions. It's worked well for other micromolluscs from the same family, but in this case yes I think it could be an issue with DNA degradation. I haven't tried another polymerase yet but I will add this to my list for troubleshooting. I've tried several different primers targeting different sized fragments of COI and annealing temperatures from 60-40 with both top-down and bottom-up regimes.
We experienced similar problems with some freshwater mussel samples and found the combination of tube extractions (no silica membrane - we like PureGene) and MyFi polymerase from Bioline to be a successful combination. Best of luck!
If you are talking about slugs. COI is can be very difficult for some species. There is some research into this issue. A new versatile primer set targeting a short fragment of the mitochondrial COI region for metabarcoding metazoan diversity: application for characterizing coral reef fish gut contents. best of luck
Thanks Nathan and Joshua. In this case these are small shelled gastropods that parasitize echinoderms. They are incredibly tiny so I use the whole animal for extraction. I will try to track down some more specimens and use PureGene in combination with MyFi- thanks for this suggestion!
As a side note I find that the Qiagen Kits don't work very well for larger PCR fragments -- so perhaps your fragment is too long and thus not amplifying (something to consider)? It seems odd to me that your high copy number nuclear genes would amplify, while your high copy number mitochondrial genes did not (high copy number because there is presumably an excess of mt genomes isolated from a single cell). I would assume that there is a problem with the specificity of your primers. I would certainly try a different polymerase and a temperature gradient in the thermocycler.
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