Hello People
I am extracting host DNA from fecal samples. However, feces contains lot of microbes and their DNA too gets extracted during DNA extraction procedure.
I want to get rid/minimise the microbial DNA. It is causing a lot of issues in my PCRs (lots of misamplification). Is there an inexpensive method to do so?
What are you looking to do with your data? What regions are you amplifying? Species specific primers would eliminate amplification of other contaminant DNA, but need more information about your intent.
Yes this question can really only be addressed in a helpful manner with more information about your needs and unfortunately your budget. But basically eDNA techniques are emerging that allow simultaneous amplification/sequencing of all of the OTUs that successfully amplify, so by definition this approach uses universal primers for markers usually such as 16S rRNA and ITS, internally transcribed spacer region, or COI cytochrome c oxidase I, then the resulting sequences are blasted against NCBI database to determine biodiversity in the sample. But if you are using a more traditional PCR approach where you want to specifically amplify only certain target lineages, one possibility is use species of specific primers. Depending on the species of interest, this can be tricky if such PCR primers are not available, but you can design your own primers that specifically amplify only the species of interest, provided there are DNA sequences available for these taxa.
I agree with Brenden S Holland. You must have read this paper, i am giving this link in case Article A Method for Selectively Enriching Microbial DNA from Contam...
Thanks everyone for your inputs. What I am trying to do here is amplify a few microsatellite loci from a particular primate species and I do have specific primers for all of them. Some of the primers work beautifully but for others, I get a lot of misamplification. I did try troubleshooting with my PCRs, but nothing seems to work. So, I thought there might be a possibility that the microbial DNA is getting amplified (given the size of the markers is
I never tried it myself for fecal samples, but generally in environmental microbiology, we used to separate microbial cells (mostly bacteria) by applying several cycles of sample (soil, sediment, water...) dispersion and differential centrifugation. Can be used also a series of PES filters of different pore size. It is NOT easy job and always we have gotten fractions enriched with small bacteria and bigger eukaryotic/host cells, NOT complete 100% separation! Note, it is also expected considerable amount of free extracellular DNA that could be loosely bound to non-cellular solids of fecal mass. Thus, physical separation of smaller prokaryotic from bigger host cells is possible, but it is a hard work which is often not much rewarding. I agree with all people above, the crucial thing is what is the ultimate goal of the project.
- Badges
- Science topic
- Similar topics
- Biological Science
- Evolutionary Biology