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Evaluation of DNA extraction methods for freshwater eukaryotic microalgae

Eland, Lucy E. ; Davenport, Russell ; Mota, Cesar R.

Water research (Oxford), 2012-10, Vol.46 (16), p.5355-5364 [Periódico revisado por pares]

Kidlington: Elsevier Ltd

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Título:
Evaluation of DNA extraction methods for freshwater eukaryotic microalgae
Autor: Eland, Lucy E. ; Davenport, Russell ; Mota, Cesar R.
Assuntos: Applied sciences ; blood ; cell walls ; Chemistry Techniques, Analytical - methods ; Communities ; Community analysis ; Culture ; Denaturing Gradient Gel Electrophoresis ; Deoxyribonucleic acid ; DGGE ; DNA ; DNA - isolation & purification ; DNA extraction ; Electrophoresis ; ethanol ; Ethyl alcohol ; Exact sciences and technology ; Extraction ; Fresh Water - microbiology ; freshwater ; Freshwaters ; genes ; Microalgae ; Microalgae - genetics ; microscopy ; Molecular techniques ; Pollution ; Polymerase Chain Reaction ; Reagent Kits, Diagnostic ; ribosomal RNA ; RNA, Ribosomal, 18S - genetics ; Species Specificity ; waste lagoons ; Waste stabilization ponds ; Water treatment and pollution
É parte de: Water research (Oxford), 2012-10, Vol.46 (16), p.5355-5364
Notas: http://dx.doi.org/10.1016/j.watres.2012.07.023
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Descrição: The use of molecular methods to investigate microalgal communities of natural and engineered freshwater resources is in its infancy, with the majority of previous studies carried out by microscopy. Inefficient or differential DNA extraction of microalgal community members can lead to bias in downstream community analysis. Three commercially available DNA extraction kits have been tested on a range of pure culture freshwater algal species with diverse cell walls and mixed algal cultures taken from eutrophic waste stabilization ponds (WSP). DNA yield and quality were evaluated, along with DNA suitability for amplification of 18S rRNA gene fragments by polymerase chain reaction (PCR). QiagenDNeasy® Blood and Tissue kit (QBT), was found to give the highest DNA yields and quality. Denaturant Gradient Gel Electrophoresis (DGGE) was used to assess the diversity of communities from which DNA was extracted. No significant differences were found among kits when assessing diversity. QBT is recommended for use with WSP samples, a conclusion confirmed by further testing on communities from two tropical WSP systems. The fixation of microalgal samples with ethanol prior to DNA extraction was found to reduce yields as well as diversity and is not recommended. [Display omitted] ► Kits tested for DNA extraction from eukaryotic microalgae in stabilization pond and pure cultures. ► Quality, quantity and use in downstream analysis, PCR and DGGE were tested. ► Seeking to minimise bias caused by variability of algal communities in mixed samples. ► Qiagen's Blood and Tissue kit outperformed other kits tested. ► Ethanol fixing has negative effect on DNA yield, fast −20 °C storage is suggested for cell storage.
Editor: Kidlington: Elsevier Ltd
Idioma: Inglês

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Evaluation of DNA extraction methods for freshwater eukaryotic microalgae

Eland, Lucy E. ; Davenport, Russell ; Mota, Cesar R.

Kidlington: Elsevier Ltd

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