I am new in DGGE analysis. I am evaluating fungi profile from root samples and doing 2 PCRs with ITS primers, including the nested PCR, before run the DGGE. I wonder if two or three bands in an agarose gel are problematic in the DGGE analysis, or if it can be arbitrary, just related to the sample diversity. Is it possible to find it, or I'd better avoid those samples?
Thank you for attention!
In essence, yes it is a potential problem. If you have different sized fragments, as suggested by having separate bands in an agarose gel, then the separation in DGGE can be by size rather than sequence. This can be a potential problem with fungi because the lengths of (1) 18S rRNA gene (from NS1 to NS6) and (2) ITS regions can vary in size. Here the primers are OK and the variation is real, not PCR induced. It is normal to check the size of fragments before performing DGGE so that only similar sized fragments are compared. If you are separating environmental DNA, I guess I have to ask whether the method of separation (sequence or length) is that important? Here you just want to separate fragments from a heterogeneous mixture so that they can be sequenced. A more difficult problem with DGGE I have noticed for some groups of fungi is no band resolves - just a smear is obtained. This is true for 28S rRNA gene regions for some members of the Dothideomycetes. I hope that this is informative.
Hi Erica,
Could you include a picture of your gel to give us an idea how close together the bands are? Following on from Julian and Ali's questions, you need to work out if the multiple bands are
- unspecific or cross amplification - this will be bad for your analyses as these samples do not contain the sequences you wish to compare. Adjusting your PCR conditions or changing primers may help.
- ITS sequences of different length - if this is the case, there is already some selection going on between genera that fall into either of those sequence lengths. In this case I'd recommend loading your entire PCR product on a gel, getting that resolution between the two (or three) ITS lengths then cutting out those bands. Those can then be purified using a gel purification kit (or simply leaving the band in eppendorfs with molecular water in the fridge over night) and then load each band on a different lane in the DGGE.
DGGE is a massive pain to work with. If there is any way you can change that part of your experimental design, I would highly recommend doing so.
Also, one common problem with community analysis is that your answer is very dependent on the primer set you use. It's worth checking that the set you have selected is adequate at amplifying your target fungi so you don't unintentionally loose some information. Depending on what taxon resolution you're after, ITS may be too short a sequence to give you an ID down to species level when you get to the sequencing stage.
All the best!
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