Wednesday, December 20, 2006

Some more publications of interest and silver staining

- Immediate link to DGGE Guide -

* Please notify me if there are links that are no longer valid. - Thanks, Stefan

Some newer publications of potential interest to the DGGE community:

A double-gradient (acrylamide and denaturant):
Analysis of Bacterial Communities in Seagrass Bed Sediments by Double-Gradient Denaturing Gradient Gel Electrophoresis of PCR-Amplified 16S rRNA Genes
. Microbial Ecology: Volume 52, Number 4 / November, 2006. J. B. James, T. D. Sherman and R. Devereux.

A new approach combining both sequencing and DGGE in a clever manner:
Denaturing Gradient Gel Electrophoresis Can Rapidly Display the Bacterial Diversity Contained in 16S rDNA Clone Libraries
. Microbial Ecology: Volume 51, Number 4 / May, 2006. M. D. Burr, S. J. Clark, C. R. Spear and A. K. Camper.

In addition, I have had a very nice correspondance with Ing. Oscar de Vos from the Biological Farming Systems group at the Wageningen University. Oscar wrote me to inform me that silver staining is still being used. He stated:

"I would like to mention that I still use silver staining (oxidizer, silver nitrate and developer from Bio-Rad) to retrieve nice pictures of our soil/manure samples with about 100 to 150 bands per sample. I once verified our Phoretix software; does the software detect bands that I can detect by eye? Using a vertical illuminator with my gel attached to it I found sometimimes 3 distinct bands per mm. Nice thing about using silver in combination with Gelbond (Amersham Biosciences) is that you can cover it after preservation with cellophane sheet (also from Amersham) and dry it at room temperature for 3 days. Result is a well-preserved gel with an example in the attachment."

As you see, the diversity detected in these samples is tremendously high, and this raises questions about our capacity to observe shifts in microbial communities and to compare levels of diversity. I inquired how Oscar dealt with these issues, and he responded:

"I added a very recent correspondence with the technical support from Nonlinear Dynamics (Phoretix, TotalLab). I was having a bad time comparing lanes across gels, even comparing lanes within a gel is hard with such a high diversity. We are looking for alternative ways, but until then we just use species diversity/richness, which is a valuable parmeter for out research (in the added mails, you may find a brief outline of our research). Zooming in with DGGE for interesting regions is an option, but we are very much interested in the whole eubacterial community for example of manures derived from cows with different feeding regimes and relate this to the survival of the human pathogen E. coli O157:H7 in this manure. It is nice that you have seen the difficulties in this research.

Arjen Speksnijder was in the lab lately and liked the sharp bands and diversity in the gels I sent you very much, it is quite a difference with the sometimes fuzzy bands he retrieves with Ethidium Bromide, but with GelCompar and his low diversity gels he has the advantage of having a good comparison across gels..."

I think the article by Burr et al. 2006 (Microbial Ecology), may help resolve some of these issues by making sequencing/DGGE more of an integrated approach. I highly recommend looking at it as a way to help overcome some of these difficult issues in characterizing communities with high levels of diversity.

Happy holidays to all.