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首页> 外文期刊>Diatom Research >A protocol for a single-cell PCR of diatoms from fixed samples: method validation using Ditylum brightwellii (T. West) Grunow
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A protocol for a single-cell PCR of diatoms from fixed samples: method validation using Ditylum brightwellii (T. West) Grunow

机译：固定样品中硅藻的单细胞PCR方案：使用Ditylum Brightwellii（T. West）Grunow进行方法验证

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摘要

Many major advances in macro-organismal biology, population genetics and evolutionary biology may be attributed to the development of rapid and inexpensive molecular methodology. Here, a simple protocol is presented for a polymerase chain reaction (PCR) of alcohol-preserved single diatom cells that facilitate studies requiring a large number of individuals, inclusive of archived and uncultivable specimens. Our approach integrates cell isolation and PCR amplification of two molecular markers, as well as post-PCR identification by scanning electron microscopy, thereby bridging the gap between classical and molecular diatom biology. The amplification of two genes was performed in a two-step protocol. By applying multiple sets of PCR primers to a single diatom cell, an internal fragment of the large subunit of the ribulose-1,5-bisphosphate carboxylase gene (RuBisCO, rbcL) was successfully amplified and sequenced, as well as the internal transcribed spacer (ITS) region in 20% of the same, preserved cells derived from natural phytoplankton. Multiple-gene recovery from a single-cell source requires further optimization, likely specific for the taxa of interest. Single marker recovery (from first-step amplification to sequencing) from natural, fixed single-cell samples was more successful with overall success rate of similar to 31% for ITS and 67% for rbcL, respectively. Admittedly it is 50-70% lower than rates for a culture-pelleted DNA source (68% for ITS and 100% for rbcL) using current standard methods of sequence recovery in diatom studies, but our method is much less labor- and cost-intensive. This approach was applied to the diatom Ditylum brightwellii to assess the quality of recovered sequences. The phred quality scores of our sequences derived from single-cell material were similar to phred scores of our own, published sequences obtained from cultures grown in our laboratory.
机译：宏观有机生物学，种群遗传学和进化生物学的许多重大进展可能归因于快速，廉价的分子方法的发展。在这里，提出了一种简单的协议，用于保存酒精的单个硅藻细胞的聚合酶链反应（PCR），这有助于需要大量个体的研究，包括已归档和无法培养的标本。我们的方法整合了细胞分离和两个分子标记物的PCR扩增，以及通过扫描电子显微镜进行PCR后的鉴定，从而弥合了经典和分子硅藻生物学之间的空白。两个基因的扩增以两步方案进行。通过将多套PCR引物应用于单个硅藻细胞，成功扩增并测序了核糖-1,5-双磷酸羧化酶基因（RuBisCO，rbcL）的大亚基的内部片段以及内部转录间隔子（ ITS）区域中的20％来自天然浮游植物的保存完好的相同细胞。从单细胞来源进行多基因恢复需要进一步优化，这可能是特定于感兴趣的分类单元的。从天然，固定的单细胞样品中回收单个标记物（从第一步扩增到测序）更为成功，ITS的总体成功率接近31％，rbcL的总体成功率接近67％。公认的是，使用硅藻研究中当前标准的序列回收方法，该方法比培养有球粒的DNA来源的速率低50-70％（ITS的68％和rbcL的100％），但我们的方法节省了劳力和成本，密集的。该方法应用于硅藻Ditylum Brightwellii，以评估回收序列的质量。我们从单细胞材料获得的序列的phred评分与我们从实验室培养的培养物中获得的已发表序列的phred评分相似。

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