I'm working on deer fecal samples out of a tropical environment, so the DNA tends to degrade pretty quickly. I'm currently only doing one locus at a time in my PCRs which already yields low success for my samples, but it comes with the territory of using feces.
My problem is that I'm using expensive, high quality Taq since it seems as though it's the best way I can get things to work and my project is getting extremely costly. Does anyone have any experience in doing multiplexes using degraded DNA?
You may have better luck with qPCR as the primers can be designed to accommodate much shorter regions. You mention "PCR" - so does that imply you are running a gel afterwards and visualizing bands on a gel? qPCR becomes a better 'forensic' tool as it can be used on highly degraded samples with some success due to 70 to 150 bp target regions. Kappabiosystems has a highly aggressive Taq in its qPCR mixes - I don't use it, but I've seen it compared to other good mixes out there and it out-performed many of the other ones (gave Cq values 3 to 5 cycles earlier than others in bacterial soil samples).
If you are doing gel-based PCR and multiplexing -- you may want to design primers that are closer to one another -- and increase the % agarose in your gels so your products don't chase off into the abyss. (E.g. 8 to 10% agarose gels)... you have a challenging project.
Hi Vicki, did you run electrophoresis analysis to determine how bad the degradation was? As far as I know, ds DNA molecules are quite stable even exposed at 70℃ for half an hour. I believe most fecal DNA molecules are embedded in gut microbes which means your DNA should be well preserved for a while. Speaking of the low PCR success rate, did you measure the purity of your DNA templates? Also, which PCR kit do you use? Personally I will go with TOYOBO KOD series. We use that to amplify genes from marine sponges. It performs much better than Taq-based PCR kit, especially when there are plenty unknown contaminates in your DNA.
Hi Vicki,
DNA degradation is often not the major hurdle of PCRs with fecal samples. The low success with fecal samples were mainly due to the low host cell number in fecal samples, DNA in Fecal samples are mainly microbial DNA with very small quantity host DNA from epithelial cells of digestive tract walls. These host cells are present in the surface of the fresh feces. Further there is strong PCR inhibitor in the fecal extract. When setting up the PCR with more DNA extraction solution (due low concentration of host DNA), so increase the PCR inhibitors in the solution and kill the PCR.
In terms of multiplex PCRs, if the DNA works for single PCR, it should be fine for multiplex PCR. You can try the multiplex PCR kit from Qiagen or Lifescien (ABI).
Thank you for all of the help everyone! When I run my DNA on a 1% agarose gel, it rarely even shows up. However, I can still get the DNA to amplify. I'm using the Qiagen stool kit to reduce inhibitors.
Hi Vicki,
You may check the DNA concentration with Nanodrop, although the host DNA is low in the total DNA solution, there should be a certain amount of microbial DNA in the solution.
It depends on the aim of the your project, if you amplify microsatellite, low pcr yield will be fine to be visualized in Fragment analyser like ABI3700 or similar. For PCR-RFLP analysis, you may consider Agilent Bioanalizer that can detect DNA in 2-50ng with 1µl loading.
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