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

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: 77-83 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.

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

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, 30-50 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

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 30-50 bp long GC-rich sequence (GC-clamp) prevents the DNA from complete dissociation.

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

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

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

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

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)

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

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

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 127-141 (Nocker, A. et al, 2007) Genotypic microbial community profiling: A critical technical review, Microbial Ecology, Vol 54, Pages 276-289 (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 4405-4414 (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: 1-8599-6283-1, Published by Taylor and Francis Group