Monday, November 28, 2005

Updated DGGE Guide

This is the first update of the DGGE Guide. I'm afraid some formating issues have been causing me problems and the figures in the guide are available only in the downloaded PDF and word formats. I have been happy to hear that people are interested in this guide, and I re-iterate my call for contributions to the website. Please send in a section if you feel like improving on those present. I will also be happy to post new articles relevant to the site.
Cheers, Stefan

Also, depending on your internet browser, links to PDF and word files may be at the very end of the page. Please check below for these links.

Highlighted Article This Month:

Neufeld, JD and WW Mohn. 2005. Fluorophore-labeled primers improve the
sensitivity, versatility, and normalization of denaturing gradient gel
electrophoresis. Appl. Environ. Microbiol. 71:4893-4896.


New items in this version of the DGGE manual:

GEL Transfer from buffer to UV Table
…..
ADD GLYCEROL TO INCREASE GEL FLEXIBILITY
Dr. Von Sigler of the Laboratory for Microbial Ecology (University of Toledo) has this to say about glycerol: “ I add glycerol to a final concentration of 2% (v/v) because it adds a great amount of flexibility to the gels. This added flexibility comes in handy when manipulating the gels (removal from the plates, staining, placement and positioning on transilluminator) and decreases the risk of tearing the gel. It does not impact DNA migration or the banding qualities.”
How much DNA to put in each PCR reaction?
………
Some situations can be far more complex. As Kim discusses in post #1267, one can have a situation where there is a variable amount of non-target genomic DNA. Thus, it may not be entirely fair to add the same total amount (ng) of genomic DNA to each PCR reaction, if the amount of target DNA varies from sample to sample. The example given is that of DNA extracts from plant samples. If one is looking at microbial communities associated with plant roots, one might recover variable amounts of root DNA depending on growth stage, etc. I don’t have a specific answer to the question of whether or not one should adjust the amount of genomic DNA added to PCR reactions based on the knowledge that variable amounts of non-target DNA are present. It seems likely that in many environments there will be variable amounts of non-target DNA (e.g. fungal DNA in bacterial population analyses, etc.) and that these cannot be readily quantified. Since standard PCR is not overly sensitive to starting concentration (thus the requirement for real-time PCR) this may not be something to worry about too much.


Primer dimerization
Some PCR primers form strong primer dimers than can be visualized by agarose electrophoresis [See figure below]. I have not found that these interfere with DGGE analyses as they appear to migrate out of the gel. However, these primer dimmers can be a problem if you run ligation/transformation/sequencing reactions directly from the PCR product. The high quantity of the primer dimer can decrease the efficiency of the cloning reaction because the dimer will compete for plasmid. This is because the primer dimers are double-stranded, and are A-tailed (if you are using Taq polymerase) just as the PCR product of the correct size. Since these primer dimers can be extremely abundant, and are double-stranded and a-tailed, they can compete with the primary PCR product in the ligation reaction. Thus, many of the clones will contain only the primer dimers, decreasing the efficiency of the ligation/transformation reaction. Thus, you may want to clean up your PCR product prior to cloning. Remember, if a band of smaller size (bp) is of the same intensity as a band of larger size, there are actually a greater number of copies of the smaller fragment.


GEL ANALYSIS

… I highly recommend a new manuscript in AEM by Josh Neufeld and William W. Mohn (“Fluorophore-labeled primers improve the sensitivity, versatility, and normalization of denaturing gradient gel electrophoresis”). This manuscript develops a new technique to increase our capacity to use DGGE gels for digital analysis by including standards within each lane. These standards have fluorescent tags that fluoresce at different wavelengths than the fluorescent molecules attached to the unknown PCR product. Brilliant! The caveat is as the authors note, besides expense, “that access to an expensive laser-scanning instrument is required, which may limit widespread use of this application at this time.” One other thing: since the sensitivity is increased by this method, the authors could reduce the number of PCR cycles required to generate enough DNA for DGGE. This is another advantage.


GC CLAMPS
….
I just happened across a website suggesting that having GC clamps on both PCR primers yielded better separation. I have had poor luck when I accidentally ordered GC clamps for both primers, but it appears this can vary according to the primer set. [See “Bipolar clamping versus monopolar clamping” on this website: http://www.charite.de/bioinf/tgge/ ]


Primer Mistmatches / PCR Bias

· Kousuke Ishii and Manabu Fukui. 2001. Optimization of Annealing Temperature To Reduce Bias Caused by a Primer Mismatch in Multitemplate PCR. Appl. Environ. Microbiol. 67(8): 3753–3755.
· Shinya Kurata, Takahiro Kanagawa, Yukio Magariyama, Kyoko Takatsu, Kazutaka Yamada, Toyokazu Yokomaku, and Yoichi Kamagata. 2004. Reevaluation and Reduction of a PCR Bias Caused by Reannealing of Templates. Appl. Environ. Microbiol. 70(12):7545-9.


DGGE OF HIGH GC PCR FRAGMENTS

· Per Guldberg1, Kirsten Grønbæk, Anni Aggerholm, Anton Platz, Per thor Straten, Vibeke Ahrenkiel, Peter Hokland and Jesper Zeuthen. 1998. Detection of mutations in GC-rich DNA by bisulphite denaturing gradient gel electrophoresis. Nucleic Acids Research. 26(6): 1548-1549.
· Ying Wu, Rein P. Stulp, Peter Elfferich, Jan Osinga, Charles H. C. M. Buys, Robert M. W. Hofstra. 1999. Improved mutation detection in GC-rich DNA fragments by combined DGGE and CDGE. . Nucleic Acids Research. 27(15): 9.


References:

Vanessa M. Hayes, Ying Wu, Jan Osinga, Inge M. Mulder, Pieter van der Vlies, Peter Elfferich, Charles H. C. M. Buys, Robert M. W. Hofstra. 1999. Improvements in gel composition and electrophoretic conditions for broad-range mutation analysis by denaturing gradient gel electrophoresis. Nucleic Acids Research. 27(20): 29.

William E. Holben, Kevin P. Feris, Anu Kettunen, and Juha H. A. Apajalahti. 2004. GC Fractionation Enhances Microbial Community Diversity Assessment and Detection of Minority Populations of Bacteria by Denaturing Gradient Gel Electrophoresis. Appl Environ Microbiol. 70(4): 2263–2270.

Kousuke Ishii and Manabu Fukui. 2001. Optimization of Annealing Temperature To Reduce Bias Caused by a Primer Mismatch in Multitemplate PCR. Appl. Environ. Microbiol. 67(8): 3753–3755.

Neufeld, JD and WW Mohn. 2005. Fluorophore-labeled primers improve the
sensitivity, versatility, and normalization of denaturing gradient gel
electrophoresis. Appl. Environ. Microbiol. 71:4893-4896.

Neufeld, JD, Yu, Z, Lam, W, and WW Mohn. 2004. Serial Analysis of
Ribosomal Sequence Tags (SARST): a high-throughput method for profiling
complex microbial communities. Environ. Microbiol. 6:131-144.

von Wintzingerode F, Gobel UB, Stackebrandt E. 1997. Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis. FEMS Microbiol Rev. 21(3):213-29

Janelle R. Thompson, Luisa A. Marcelino and Martin F. Polz. 2002. Heteroduplexes in mixed-template amplifications: formation, consequence and elimination by ‘reconditioning PCR’. Nucleic Acids Research. 30(9): 2083-2088.

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