I have been following the protocol from Hu, et al 2007 (nature protocols). Using their 40R and 5F and 42F 39R primers I have managed to amplify complete mitochondrial genomes from two strongylid lung worms from porpoises and seals. I am currently attempting to amplify mitochondrial genomes from all of my nematode samples, including representative families Anisakidae (order Ascaridida), Tetrameridae (order spirurina), Onchocercidae (spirurina), and metastrongyloides. The problem I am having is knowing how long of an extension time I need, as the gene order in mt genomes is variable between species, so I don't actually know the expected amplicon size. The paper suggests designing primers to the large rRNA subunit and ND1... I'm currently running PCR with variable anneal/extend time to try and amplify the genome, but am wondering if there is any better way to design primers and execute PCR not knowing the amplicon size. I am currently using TaKaRa LA Taq (I have tried BD advantage and LA Taq with no success). Thank you for any guidance!
For the extension time, I would calculate with the worst case, 15-17 kb. Of course, the side effect could be that you may amplify DNA unspecifically. But here you could still clean it up over an agarose gel, by cutting away at least the weaker bands.
Anyway, I think the bigger problem is that you cannot be 100% sure that your primers target your DNA. The paper just talks about genes "known to be relatively conserved among selected nematodes". Just a few base pair substitutions may be enough to degrade the ability of the primer to anneal to the template DNA. Just have a point mutation in your GC clamp, and amplification should be crippled.
One solution to that problem is to try different sets of primers, maybe one sets targets perfectly conserved regions while the other set does not. In fact, Hu et al. used different sets of primers to amplify mt-DNA from different parasites.
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