Single-cell PCR amplification of thecate dinoflagellates: a case study of Tripos (Dinophyceae)

The relief of amplification inhibition in preserved phytoplankton samples is a major challenge in genetic studies relying on single-cell sequencing. The successful amplification of rDNA genes varies considerably depending on the organisms, fixatives, and time of analysis after collection. Among othe...

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Bibliographic Details
Published in:Journal of applied phycology 2018-04, Vol.30 (2), p.1117-1124
Main Authors: Hernández-Rosas, A., Meave del Castillo, M. E., Díaz-Larrea, J., Rodríguez, F.
Format: Article
Language:English
Subjects:
Amplification
Biomedical and Life Sciences
Case studies
Cell culture
Dinoflagellates
DNA
Ecology
Fixatives
Freshwater & Marine Ecology
Gene sequencing
Life Sciences
Microorganisms
Nucleotide sequence
PCR
Phytoplankton
Plant Physiology
Plant Sciences
Storage
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Summary:The relief of amplification inhibition in preserved phytoplankton samples is a major challenge in genetic studies relying on single-cell sequencing. The successful amplification of rDNA genes varies considerably depending on the organisms, fixatives, and time of analysis after collection. Among other fixatives, RNA later ® is found to be a suitable choice for PCR amplification after long-term storage. In this study, we tested the performance of RNA later ® samples applied to single-cell PCR amplification in isolates of the thecate dinoflagellate genus Tripos , with an amplification success over 70%. The single-cell protocol proposed in this study amplified an average of 650 pb of large subunit and small subunit rDNA fragments in RNA later ® preserved cells after 5 months. Furthermore, it was possible to obtain rDNA sequences from samples up to 8 months old. The approach described here could also be useful to amplify a wide range of thecate and non-thecate dinoflagellate taxa, especially those species difficult to maintain in culture.
ISSN:0921-8971
1573-5176
DOI:10.1007/s10811-017-1269-1