Lynn Denis H, Pinheiro Marcel
Department of Integrative Biology, University of Guelph, Ontario, Canada.
J Eukaryot Microbiol. 2009 Sep-Oct;56(5):406-12. doi: 10.1111/j.1550-7408.2009.00439.x.
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.
我们调查了多种使用单细胞聚合酶链反应（SC-PCR）来检测各种单细胞原生生物基因序列的研究。真核生物所有超群的代表都已接受SC-PCR检测，其中纤毛虫和甲藻是最常检测的对象。SC-PCR的实施方式有两种，一种是直接扩增单个裂解细胞，另一种是先提取DNA，然后对DNA提取物进行扩增。细胞裂解方法包括加热、冷冻、机械破碎和酶消化。用乙醇、甲醇和卢戈氏液固定或保存的细胞也已成功使用。初始PCR之后可能会进行半巢式或巢式PCR，其产物随后直接测序或克隆后再测序。这些方法并不复杂。这应该会鼓励原生生物学家在描述新的或修订的分类单元时使用SC-PCR，特别是罕见的和不可培养的种类，而且这也能够探究与生活史阶段相关的基因表达。
