I am using ITS1f-ITS4 primer set to amplify the ITS1-5.8S-ITS2 region from genomic DNA extracted from soil/sludge sample expected to contain at least some fungi. To the best of my understanding, the ITS1f primer is fairly specific to fungi.
PCR reaction consisted of: Platinum Hot Start PCR 2X mastermix (diluted to 1X), BSA (0.4 mg/ml), primers (0.4 uM ea.), template (1ng/uL).
Thermocycler Program:
Cyc1(1X) 94C for 5 minutes
Cyc2(35X) 94C for 30s
55C for 30s
72C for 30s
Cyc3(1X) 72C for 5 minutes
Cyc4 4C forever
I've attached a picture of the gel. I ran 5 uL of the PCR products on a 0.8% agarose gel at 80V for 60 min, along with a 1kb ladder (G Biosciences). Samples in lanes 1&5 and 2&6 are the same environmental samples extracted by different methods. Subscripts on the additional sample 5 labels indicate the (increased) concentration of template in the PCR reaction for those lanes (e.g. 5 ng/uL and 3 ng/uL). Lane 0 was a negative control (template from DNA extraction with no sample).
From my understanding, I should expect ITS amplicons of around 600 bp for fungi; however, most samples show bands at much lower sizes (200-400 bp). These appear to be too large to be primer dimers, so I expect amplification occurred (also based on negative control, where primer dimers are not visible on the gel).
Are there any methodological issues that could lead to smaller than expected fungal ITS amplicons?
Thanks for taking the time to consider!
https://tools.thermofisher.com/content/sfs/manuals/Platinum_Hot_Start_PCR_Master_Mix_UG.pdf
I am sending one reference , hope this will be useful.
What is the sequence of your primers ?
Hi
I use the PCR conditions proposed by Hedh et al. (2008), using the primers ITS1 and ITS4 (White et al., 1990), and I have very good results.
Hedh, J., Samson, P., Erland, S. and A. Tunlid. 2008. Multiple gene genealogies and species recognition in the ectomycorrhizal fungus Paxillus involutus. Mycological Research 112: 965-975.
White, T.J., Bruns, T., Lee, S. and J. Taylor. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis, M.A., Gelfand, D.H., Sninsky, J.J. and T.J. White. (eds.). PCR Protocols: a guide to methods and applications. Academic Press, San Diego. pp: 315-322
The size of the ITS of some fungi species could vary a lot off (e.g.: genus Leccinum), try to find some information about it. And finally what tissue are you using for the extraction (mycelium, fruit bodies, mycorrhizas,...) is also have influence in the results.
Good luck
Try different primers to check if your DNA extract is any good. (like ITS1)
or one of these: http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6941.2012.01437.x/pdf
Hi Andreas Altenburger, thanks for taking the time to comment. This an an old thread related to some of my early graduate research in molecular biology. In the image above, the direction of migration through the gel is from the bottom of the image to the top, so the products are indeed smaller than 500 bp.
I never directly resolved this issue. Based on the suggestions of others above, I acquired new reagents, including primers targeting just ITS2 region (ITS3-ITS4) and a new DNA ladder with better resolution, and everything looked reasonable from that point forward.
- Badges
- Science topic
- Similar topics
- Biological Science
- Bioscience