DGGE

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Denaturing gradient gel electrophoresis (DGGE) is a technique used for separating DNA fragments according to their mobilities under increasingly denaturing conditions (usually increasing formamide/ urea concentrations).

Contents

Principle

Small samples of DNA (or RNA) are added to an electrophoresis gel that contains a denaturing agent. The denaturing gel induces melting of the DNA at various stages. As a result of this melting, the DNA spreads through the gel and can be analyzed for single components.

DGGE (Muyzer et al. 1993) analyses are employed for the separation of double-stranded DNA fragments that are identical in length, but differ in sequence.

In practice, the DNA fragments are usually produced via PCR amplification. The DGGE technique exploits (among other factors) the difference in the stability of G-C pairing (3 hydrogen bonds per pairing) as opposed to A-T pairing (2 hydrogen bonds). A mixture of DNA fragments of different sequence is separated by electrophoresis on an acrylamide gel containing a linearly increasing gradient of DNA denaturants (usually urea and formamide). In general, DNA fragments richer in GC will be more stable and remain double-stranded until reaching higher denaturant concentrations. Double-stranded DNA fragments migrate better in the acrylamide gel, while denatured DNA molecules slow down or stop in the gel. In this manner, DNA fragments of differing sequence can be separated in an acrylamide gel. DGGE is commonly performed for partial 16S rRNA gene, but also functional genes may be used. A GC (guanine plus cytosine) rich sequence can be incorporated into one of the primers used in the PCR to modify the melting behaviour of the fragment of interest and to improve the separation of the fragments. The DGGE gels can be stained with DNA binding fluorescent dyes, such as SYBR Green and visualized under UV light. Known standards may be used for comparing the samples on different gels. Ideally one band on the gel corresponds to one species, and therefore the number of bands gives an idea of the diversity of the sample. The gene fragments can be excised from the gel, eluted e.g. into sterile water and amplified for sequencing. The relative abundance of various microorganisms can be estimated by measuring the intensity of their bands relative to the intensity of all bands in the corresponding sample.

Advantages

  • Very sensitive to variations in DNA sequence;
  • Allows simultaneous analysis of multiple samples;
  • Useful method for monitoring shifts in community structure over time;
  • Community profiles can be analyzed with cluster analysis;
  • The use of universal primers allows the analyses of microbial communities without any prior knowledge of the species;
  • The fragments separated by DGGE can be excised, cloned and sequenced for identification;
  • It is possible to identify constituents that represent only 1 % of the total community;
  • DGGE can be applied to phylogenetic and functional genes.

Disadvantages

  • DGGE analysis is rather time consuming;
  • DGGE analysis suffers from the same drawbacks as all PCR-based community analysis techniques, including biases from DNA extraction and amplification;
  • The variation in 16S rRNA gene copy number in different microbes makes this technique only “semi-quantitative”;
  • Microheterogeneity in rRNA encoding genes present in some species may result in multiple bands for a single species and subsequently to an overestimation of community diversity;
  • Heteroduplexes can cause biases to the observed diversity;
  • No method for automated analyses currently available;
  • Works well only with short fragments (<600 bp), thus limiting phylogenetic characterization;
  • Gels of complex communities may look smeared due to the large number of bands;
  • Band position does not provide reproducible taxonomic information;
  • Results difficult to reproduce between gels and laboratories.

DNA-based DGGE as a monitoring tool

It is especially suitable for samples with few species and gives complementary information on species diversity when combined with other methods. It is also well suited for analysing community changes over time.

  • Very suitable technique for the identification of novel or unknown organisms. The detection sensitivity is limited due to PCR and primer artefacts, usually the most abundant species are detected.
  • Easy to perform
  • The most abundant species can be readily detected.
  • Limited information about the abundance of detected species and no information about the activity of the detected species.
  • Long analysis time (several days) that makes it difficult to use it as an “on-line” tool.

Current use in bioleaching studies

DGGE has been used to elucidate and characterize microbial populations in many bioleaching environments, including bioreactors (Kinnunen and Puhakka 2004), acidic mining-impacted environments (González-Toril et al. 2003), and bioleaching heaps (Hawkes et al. 2004).

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