1. Denaturing Gradient Gel Electrophoresis
Tool to describe the unknown and to look for the known
Andrea Bagi , 2008

2. History Medical fileds of science:
(BORRESEN AL, HOVIG E, BROGGER A, 1988) Detection of base mutations in genomic DNA using denaturing gradient gel electrophoresis (DGGE) followed by transfer and hybridization with gene-specific probes MUTATION RESEARCH   Vol: 202 Iss: 1 Pages:
Field of environmental microbiology :
Employed as a genetic fingerptinting tool for analysis of microbial communities
- barcode like ID for communities 1 2 3 4 1. 2.
Fingerprinting 4. 3.

3. Environmental sample Phylogenetic trees
In situ hybridization
Extraction
Community nucleic acids (DNA or RNA)
PCR, RT-PCR
Dot-blot
Nucleic acid probes
Genetic fingerprints,
separated DNA bands
DGGE
Community rDNA
Cloning
rDNA clones
Sequencing
Probe design
Sequencing
rDNA sequences, database
Phylogenetic trees

4. Prior to DGGE Sample Extraction
Environmental samples (soil, seawater, freshwater)
Samples from microcosm studies
Extraction
mRNA – gene expression
rRNA – metabolically active memebers
rDNA – predominant members of the community
PCR or RT-PCR (depending on DNA or RNA)
Primers with GC-clamp (GC-rich sequence, bp)
Primers anneal to highly conserved regions of 16S rDNA and flanking highly variable region (careful selection – database analysis)
Primers for functional genes
Group-specific primers
PCR products with same length but different sequence

5. DGGE In theory - principals
PCR amplified DNA fragments of the same length (~ 500 bp) but with different sequences are separated in a polyacrylamid gel containing a linear gradient of DNA denaturants (formamid and urea).
Separation is based on the decreased electrophoretic mobility of a partially melted double-stranded DNA.
Melting proceeds in melting-domains – sequence variation within such domains causes melting temperatures to differ (one nucleotid difference can be enough).
Migration of the molecule will stop at a particular position in the gel according to its melting temperature.
Attachment of a bp long GC-rich sequence (GC-clamp) prevents the DNA from complete dissociation.

6. What happens? Electrophoresis Denaturant
1. PCR products with GC clamp are applied on the top of the gel. 1.
2. Double stranded DNA fragments are moving in the gel due to the electrophoretical power . 2.
Denaturant
Electrophoresis 3.
3. DNA molecules meet with increasing denaturing concentrations – partially melting, decreasing mobility 4.
4. DNA fragments are melted, practically GC clamp holds the strands together – molecules stop moving
Polyacrylamide gel with a linear denaturant gradient – increasing from top to bottom

7. In practice – how does it look like?
Electrophoresis chamber
Power supply for heating
Polyacrylamide gel
Heating elements

8. Optimization – melting behaviour of DNA fragments
Denaturing concentration / gradient
Denaturant
A steep transition in mobility occurs at the denaturant concentration corresponding to the melting temperature of the lowest melting domains.
0 %
100 %
Electrophoresis
Different DNA sequences with same length
Denaturant
30 %
70 %
Temperature and duration of electrophoresis is optimized on paralell gels
Electrophoresis

9. Processing the gel Hybridization with specific oligonucleotide probes
1. Staining the gel (Ethidium bromide, Syber gold)
Hybridization with specific oligonucleotide probes 2.
Sequencing approach
1. Excising bands under UV light
2. Reamplification of the fragments
3. Sequencing the DNA fragments
4. Phylogenetical analysis, probe design
DGGE gel

10. Computer enhanced graphic representation of the banding patterns
Statistical analysis of DGGE
banding pattern
Ecological indicies like Shannon-Weaver index of diversity, Simpson index of dominance
Principal component analysis
DGGE gel photo
Computer enhanced graphic representation of the banding patterns
Dice’s coefficient of similarity
(Sei, K. et al., 2004)

11. Main fields of application
Studying community complexity – pattern of bands, number of bands correspond to the number of predominant species
Studying community changes – monitoring community behaviour after environmental changes over a long time
Monitoring the enrichment and isolation of bacteria
Detection of microheterogenity in rRNA encoding genes
Comparison of different DNA extraction protocols
Screening of clone libraries
Determining PCR and cloning biases

12. Limitations Sample handling, extraction, PCR
Electrophoretic techniques only display the rDNA fragments obtained from the predominant species
Separation of relatively small fragments (~500 bp) limits the amount of sequence information
It’s not always possible to separate DNA fragments with a certain amount of sequence variation
Presence of multiple rrN operons with sequence microheterogenity – overestimation of species richness
Besides the DGGE conditions, resolution of separation depends on the region of 16S rRNA selected for analysis
Co-migration of DNA fragments, gel-to-gel variations

13. References
(Muyzer, G. and Smalla, K. 1998) Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology, Antonie van Leeuwenhoek, Vol 73 Pages
(Nocker, A. et al, 2007) Genotypic microbial community profiling: A critical technical review, Microbial Ecology, Vol 54, Pages
(Sei, K. et al., 2004) Monitoring behaviour of catabolic genes and change of microbial community structures in seawater microcosms during aromatic compound degradation, Water Research, Vol 38, Pages
(Torsvik, V. et al., 1998) Novel techniques for analysing microbial diversity in natural and perturbed environments, Journal of Biotechnology, Vol 64, Pages 53-62
(A. Mark Osborn and Cindy J. Smith, 2005) Molecular Microbial Ecology, ISBN: , Published by Taylor and Francis Group