title: Evaluation of DNA extraction methods for freshwater eukaryotic microalgae.
abstract: 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.
authors.affiliation: Newcastle University, School of Civil Engineering of Geosciences, Newcastle upon Tyne NE1 7RU, United Kingdom. l.e.eland@ncl.ac.uk
authors.forename: Lucy E
authors.initials: LE
authors.lastname: Eland
authors.forename: Russell
authors.initials: R
authors.lastname: Davenport
authors.forename: Cesar R
authors.initials: CR
authors.lastname: Mota
mesh_terms: D002623:Chemistry Techniques, Analytical / Q000379:methods*; D004247:DNA / Q000302:isolation & purification*; D058645:Denaturing Gradient Gel Electrophoresis; D005618:Fresh Water / Q000382:microbiology*; D058086:Microalgae / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D011933:Reagent Kits, Diagnostic; D013045:Species Specificity
chemical_list: D012337:RNA, Ribosomal, 18S; D011933:Reagent Kits, Diagnostic; D004247:DNA