Heteroduplex mobility assay-guided sequence discovery: Elucidation of the small subunit (18S) rDNA sequences of Pfiesteria piscicida and related dinoflaqellates from complex algal culture and environmental sample DNA pools

摘要

The newly described heterotrophic estuarine dinoflagellate Pffes- terfe piscforda has been linked with fish kills in field and laboratory settings, and with a novel clinical syndrome of impaired cognition and memory disturbance among humans after presumptive toxin exposure. As a result, there is a pressing need to better characterize the organism and these associations. Advances in Pffesteria re- search have been hampered. however. by the absence of genomic sequence data. We employed a sequencing strategy directed by heteroduplex mobility assay to detect Pffort6ria pisctada 18S rDNA "signature" sequences in complex pools of DNA and used those data as the basis for determination of the complete P piscich 18S rDNA sequence. Specific PCR assays for P pishads and other estuarine heterotrophic dinoflagellates were developed. permit- ting their detection in algal cultures and in estuarine water samples collected during fish kill and fish lesion events. These tools should enhance efforts to characterize these organisms and their ecolog- ical relationships. Heteroduplex mobility assay-directed sequence discovery is broadly applicable, and may be adapted for the detection of genomic sequence data of other novel or noncultur- able organisms in complex assemblages.