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  • 1.
    Godhe, Anna
    et al.
    Marine Botany, Department of Marine Ecology, Göteborg University.
    Anderson, Donald M.
    Biology Department, Woods Hole Oceanographic Institution, Woods Hole.
    Rehnstam-Holm, Ann-Sofi
    Biology Department, Woods Hole Oceanographic Institution, Woods Hole.
    PCR amplification of microalgal DNA for sequencing and species identification: studies on fixatives and algal growth stages2002In: Harmful Algae, ISSN 1568-9883, E-ISSN 1878-1470, Vol. 1, no 4, p. 375-382Article in journal (Refereed)
    Abstract [en]

    Cultured strains and individually isolated dinoflagellate cells from field samples were preserved in different fixatives to find a method of cell preservation that could provide DNA template in PCR reactions and preserve cell morphology for microscopic studies. Lugol’s solution and various ethanol concentrations all showed shortcomings, whereas an initial formalin preservation step followed by storage in 100% methanol fulfilled both demands. Cells could be stored up to 1 year and still provide functional DNA template for positive PCR reactions. The amplified fragment was approximately 700 bp of the D1/D2 region of the LSU rDNA, which is to our knowledge significantly longer than the low-molecular-weight DNA typically reported from formalin preserved samples. By cloning and sequencing the PCR product and subsequently aligning the sequences with previously sequenced fragments of the same or similar species, we confirmed that no base pair alteration had taken place during the time that the cells were fixed and frozen. In another experiment it was demonstrated that the growth phase of cultured Alexandrium minutum did not have any influence on the result of PCR reactions. This was true for extracted DNA from cultures and for direct PCR with a small number of disrupted cells. Phenol/chlorophorm/isoamylalcohol extraction proved to be an unpredictable method for DNA extraction, whereas direct PCR on isolated cells was more reliable. Extracted DNA purified with a commercial DNA cleaning kit always rendered a positive PCR. The environmental condition for cells to be used as DNA template in PCR is discussed.

  • 2.
    Godhe, Anna
    et al.
    Department of Fishery Microbiology, College of Fisheries, University of Agricultural Sciences, Mangalore.
    Rehnstam-Holm, Ann-Sofi
    Kristianstad University, School of Education and Environment, Avdelningen för Naturvetenskap. Kristianstad University, Forskningsmiljön Man and Biosphere Health (MABH).
    Karunasagar, Indrani
    Department of Fishery Microbiology, College of Fisheries, University of Agricultural Sciences, Mangalore.
    Karunasagar, Iddya
    Department of Fishery Microbiology, College of Fisheries, University of Agricultural Sciences, Mangalore.
    PCR detection of dinoflagellate cysts in field sediment samples from tropic and temperate environments2002In: Harmful Algae, ISSN 1568-9883, E-ISSN 1878-1470, Vol. 1, no 4, p. 361-373Article in journal (Refereed)
    Abstract [en]

    Species-specific primers were constructed for Scrippsiella trochoidea, Protoceratium reticulatum and Lingulodinium polyedrum, which all are common cosmopolitan cyst forming dinoflagellates. The designed primers amplified a product of expected size from cultured planktonic cells of the three species, and did not yield any product with a wide range of other algal species used as negative controls. The PCR method for detection and identification of dinoflagellate cysts from the three species was applied on field samples. Undisturbed surface sediment was collected along the southwest coast of India and the west coast of Sweden. DNA extract from sediment including DNA from dinoflagellate cysts could be obtained after repeated grinding with mortar and pestle under liquid nitrogen followed by microwave boiling. All sediment samples that contained any of the target species as confirmed by microscopy, were also positive for PCR. Field samples negative for any of the target species by microscopy, were also negative by PCR. Restriction enzyme digestion and/or DNA sequencing confirmed the specificity of all the PCR products from field samples. The yield of DNA from sediment extraction was low, and therefore nested PCR was necessary for accurate species-specific detection of the three species in most of the field samples.

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