Abstract
A simple and effective protocol is described for multiplex polymerase chain reaction (PCR) amplification of single cells of Karenia brevis. The protocol requires minimum processing, avoids additions that might dilute target DNA template, and can be used on cells preserved in Lugol’s iodine preservative. Destaining of Lugol’s-preserved cells with sodium thiosulfate allowed successful amplification of single-copy, nuclear-encoded microsatellites in single cells of K. brevis that have been preserved for up to 6 years.
This is a preview of subscription content, log in via an institution to check access.
References
Doyle JJ, Doyle JL (1990) A rapid total DNA isolation procedure for fresh plant tissue. Focus 12, 13–15
Hutchison CA, Hamilton OS, Pfannkoch C, Venter JC (2005) Cell-free cloning using φ29 DNA polymerase. Proc Natl Acad Sci USA 102, 17332–17336
Iglesias-Rodriguez MD, Schofield OM, Batley J, Medlin LK, Hayes PK (2006) Intraspecific genetic diversity in the marine coccolithophore Emiliania huxleyi (Prymnesiophyceae): the use of microsatellite analysis in marine phytoplankton population studies. J Phycol 42, 526–536
Kai AKL, Cheung YK, Yeung PKK, Wong JTY (2006) Development of single-cell PCR methods for the Raphidophyceae. Harmful Algae 5, 649–657
Ki J-S, Jang GY, Han M (2005) Integrated method for single-cell DNA extraction, PCR amplification, and sequencing of ribosomal DNA from harmful dinoflagellates Cochlodinium polykrikoides and Alexandrium catenella. Mar Biotechnol 6, 587–593
Loret P, Tengs T, Villareal TA, Singler H, Richardson B, McGuire P, Morton S, Busman M, Campbell L (2002) No difference found in ribosomal DNA sequences from physiologically diverse clones of Karenia brevis (Dinophyceae) from the Gulf of Mexico. J Plankton Res 24, 735–739
Nagai S, Sekino M, Matsuyama Y, Itakura S (2006) Development of microsatellite markers in the toxic dinoflagellate Alexandrium catenella (Dinophyceae). Mol Ecol Notes 6, 120–122
Nagai S, Lian C, Yamaguchi S, Hamaguchi M, Matsuyama Y, Itakura S, Shimada H, Kaga S, Yamauchi H, Sonda Y, Nishikawa T, Kim CH, Hogetsu T (2007) Microsatellite markers reveal population genetic structure of the toxic dinoflagellate Alexandrium tamarense (Dinophyceae) in Japanese coastal waters. J Phycol 43, 43–54
Raghunathan A, Ferguson, Jr. HR, Bornarth CJ, Song W, Driscoll M, Lasken RS (2005) Genomic DNA Amplification from a Single Bacterium. Environ Microbiol 71, 3342–3347
Renshaw M, Soltysiak K, Arreola D, Loret P, Patton JC, Gold JR, Campbell L (2006) Microsatellite DNA markers for population genetic studies in the dinoflagellate Karenia brevis. Mol Ecol Notes 6, 1157–1159
Richlen ML, Barber PH (2005) A technique for the rapid extraction of microalgal DNA from single live and preserved cells. Mol Ecol Notes 5, 688–691
Rynearson TA, Armbrust EV (2004) Genetic differentiation among populations of the planktonic marine diatom Ditylum brightwellii (Bacillariophyceae). J Phycol 40, 34–43
Sebastián CR, O’Ryan C (2001) Single-cell sequencing of dinoflagellate (Dinophyceae) nuclear ribosomal genes. Mol Ecol Notes 1, 329–331
Steidinger KA, Vargo GA, Tester PA, Tomas CR (1998) “Bloom dynamics and physiology of Gymnodinium breve with emphasis on the Gulf of Mexico”. In: NATO ASI Series: Physiological Ecology of Harmful Algal Blooms, Vol. G 41. Anderson DM, Cembella AD, Hallegraeff GM, eds. (Berlin: Springer-Verlag), pp 133–153
Tittel J, Bissinger V, Zippel B, Gaedke U, Bell E, Lorke A (2003) Mixotrophs combine resource use to outcompete specialists: implications for aquatic food webs. Proc Natl Acad Sci USA 100, 12776–2781
Acknowledgments
This work was supported by grants to L.C. and J.R.G. from the EPA’s Science to Achieve Results (STAR) Program (Award R-83041301-0) and from the National Oceanic and Atmospheric Administration Coastal Ocean Program (Award NA06NOS4780244) and by the Texas Agricultural Experiment Station (Project H-6703). We thank T.J. Frommlet for technical advice. This article is Contribution Number 154 of the Center for Biosystematics and Biodiversity at Texas A&M University and ECOHAB Contribution Number 240.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Henrichs, D.W., Renshaw, M.A., Santamaria, C.A. et al. PCR Amplification of Microsatellites from Single Cells of Karenia brevis Preserved in Lugol’s Iodine Solution. Mar Biotechnol 10, 122–127 (2008). https://doi.org/10.1007/s10126-007-9044-y
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10126-007-9044-y