Capillary Electrophoresis single-strand conformation polymorphism

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Capillary Electrophoresis Single-Strand Conformation Polymorphism.

Contents

Principle

CE-SSCP is a structural analysis technique in which single-stranded DNA fragments of the same length are separated based on their sequence. The principle is based on the fact that the electrophoretic mobility of nucleic acids in a non-denaturing polymer is sensitive to both size and shape. Single-stranded DNA will thus adopt a conformation determined by intramolecular interactions and base stacking that is uniquely dependent on sequence composition. This conformation can be affected by a single base change. Conformational changes are detected as changes in the electrophoretic mobility. About 200 bp of the V3 region of the 16S rRNA genes are amplified using a fluorescently labelled primer. The labelled fragments thus obtained are denatured to yield singlestranded labelled DNA, which are then separated by capillary electrophoresis under nondenaturing conditions, and detected by laser-induced fluorescence detection. Migration position of each single-stranded DNA is defined by comparison with an internal standard labelled with a different fluorochrome.

Advantages

  • High sensitivity of multicolour (up to five different fluorochromes) laser-induced detection;
  • High reproducibility between runs; internal standard eliminates run-to-run variability;
  • Automation allows easy and rapid process of 96 samples at the time;
  • Identification of unknown microorganisms from environmental samples by comparison of the mobility of the unknown DNA to that of known sequences;
  • Relative abundance of detected sequences (microorganisms) is determined by measuring the fluorescence of each peak relative to the sum of the fluorescence;
  • Suitable for unknown microorganisms;
  • Suitable for population monitoring and identification of microorganisms.

Disadvantages

  • CE-SSCP analysis suffers from the same drawbacks as all PCR-based community analysis techniques;
  • Limited to DNA fragments of 200 to 400 bp in size, thus limiting phylogenetic characterization;
  • Not quantitative, only semi-quantitative;
  • Two different sequences may have same electrophoretic mobility.

Current use in bioleaching studies

CE-SSCP analyses have been previously used to study the evolution of a bacterial consortium during batch bioleaching of the Kasese cobaltiferous pyrite (Battaglia-Brunet et al., 2002; Foucher et al., 2003).

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