PCR amplification of microalgal DNA for sequencing and species identification: studies on fixatives and algal growth stages

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.