A survey of polymerase chain reaction (PCR) amplification studies of unicellular protists using single-cell PCR.
We surveyed a variety of studies that have used single-cell polymerase chain reaction (SC-PCR) to examine the gene sequences of a diversity of unicellular protists. Representatives of all the Super-Groups of eukaryotes have been subjected to SC-PCR with ciliates and dinoflagellates being most commonly examined. The SC-PCR was carried out either by directly amplifying a single lysed cell or by first extracting DNA and following this with amplification of the DNA extract. Cell lysis methods included heating, freezing, mechanical rupture, and enzyme digestion. Cells fixed or preserved with ethanol, methanol, and Lugol's have also been used successfully. Heminested or seminested PCR might follow the initial PCR, whose products were then directly sequenced or cloned and then sequenced. The methods are not complicated. This should encourage protistologists to use SC-PCR in the description of new or revised taxa, especially rare and unculturable forms, and it should also enable the probing of gene expression in relation to life history stages.
Lynn
Denis H
DH
Department of Integrative Biology, University of Guelph, Ontario, Canada. ddr@uoguelph.ca
Pinheiro
Marcel
M
D000818:Animals; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D056890:Eukaryota / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA
D016054:DNA, Protozoan