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Ideas - mtDNA amplifies well - microsats won't amplify at all

mtDNA microsatellites fecal extractions FTA cards wont amplify

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#1 punkybugsy



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Posted 24 January 2012 - 06:04 PM

I work with non-invasive fecal samples from primates. Former lab members had the exact opposite problem - where they had no issues getting microsatellite data, but the mtDNA sequences were difficult for them to amplify - so I'm a bit stumped. I use Qiagen stool kits for my extractions and have followed the same procedure as has always been used in the lab. All the reagents have are fresh and have been changed out. I get very strong amplification for mtDNA of a 1200-1500 bp section of DLOOP as well as about a 1000 bp region of 12S/16S. However, the nuclear microsats won't amplify at all. They work for my positive controls that I've extracted from blood - so I know the primers are fine.

When I posed my problem to some PIs their initial thought was that the samples from this past field season had gone bad. But I've since done FTA card blood extractions as well as fecal extractions from older samples that had been genotyped using the same microsats and cycling conditions as the previous lab member - none of them will work for these markers and all of them work well for both mtDNA regions. I've been playing around with my own optimization using temperature gradients and concentrating the DNA - nothing has worked so far. I used Qiagen's DNEasy protocol for dried blood spots for my FTA extractions - I haven't attempted using the actual punch outs from the FTA samples yet.

Is there anything extremely obvious that I'm missing here? This is the first time I'm working with microsats and I realize mtDNA is typically easier to amplify - but the fact that they worked better in the past for former lab members than the mtDNA is what concerns me. If anyone can weigh in I'd greatly appreciate it.

Also tagged with one or more of these keywords: mtDNA, microsatellites, fecal extractions, FTA cards, wont amplify

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