| Identification of Insertion Site by RESDA-PCR in Chlamydomonas Mutants Generated by AphVIII Random Insertional Mutagenesis
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Author:
Date:
2018-02-05
[Abstract] Chlamydomonas reinhardtii is frequently used as a model organism to study fundamental processes in photosynthesis, metabolism, and flagellar biology. Versatile tool boxes have been developed for this alga (Fuhrmann et al., 1999; Schroda et al., 2000; Schroda, 2006). Among them, forward genetic approach has been intensively used, mostly because of the high efficiency in the generation of hundreds of thousands of mutants by random insertional mutagenesis and the haploid nature therefore phenotypic analysis can be done in the first generation (Cagnon et al., 2013; Tunçay et al., 2013). A major bottleneck in the application of high throughput methods in a forward genetic approach is the identification of the genetic lesion(s) responsible for the ...
[摘要] 莱茵衣藻（Chlamydomonas reinhardtii）是光合作用，代谢和鞭毛生物学的基础研究者。已经为这种藻类开发了多功能的工具箱（Fuhrmann等人，1999; Schroda等人，2000; Schroda，2006）。其中，正向遗传方法已经被广泛使用，主要是因为通过随机插入诱变产生了数十万个突变体的高效率，并且可以在第一代进行单倍体性质表型分析（Cagnon等人 2013，Tunçay et。 2013）。在正向遗传方法中应用高通量方法的主要瓶颈是鉴定与观察到的表型相关的遗传损伤。在该协议中，我们详细描述了最初在（González-Ballester等人，2005）中报道的限制性定点扩增PCR（RESDA-PCR）的改进版本。优化包括引物组合的优化，DNA聚合酶的选择，PCR循环参数的优化以及PCR产物直接测序的应用。这些修改使得获得特定的PCR产物变得更加容易，并且加速了亚克隆步骤以更快地获得测序数据。
【背景】除了限制酶位点定向扩增PCR（RESDA-PCR）（González-Ballester等人，2005）之外，还发现了其它几种分子技术。 包括Genome ...
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| Single-molecule Analysis of DNA Replication Dynamics in Budding Yeast and Human Cells by DNA Combing
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Author:
Date:
2017-06-05
[Abstract] The DNA combing method allows the analysis of DNA replication at the level of individual DNA molecules stretched along silane-coated glass coverslips. Before DNA extraction, ongoing DNA synthesis is labeled with halogenated analogues of thymidine. Replication tracks are visualized by immunofluorescence using specific antibodies. Unlike biochemical and NGS-based methods, DNA combing provides unique information on cell-to-cell variations in DNA replication profiles, including initiation and elongation. Finally, this assay can be used to monitor the effect of DNA lesions on fork progression, arrest and restart.
[摘要] DNA梳理方法允许在沿着硅烷涂覆的玻璃盖玻片拉伸的单个DNA分子的水平上分析DNA复制。在DNA提取前，进行的DNA合成用胸苷的卤化类似物标记。使用特异性抗体通过免疫荧光可视化复制轨迹。与生物化学和基于NGS的方法不同，DNA梳理提供了DNA复制谱中细胞间细胞变化的独特信息，包括引发和延长。最后，该测定可用于监测DNA损伤对叉进展，停止和重新启动的影响。
背景 在称为复制起点的真核染色体上的数千个位点处启动DNA合成。原始激活遵循由检查点激酶和染色质的表观遗传修饰（Prioleau和MacAlpine，2016）控制的定义良好的复制计时程序。复制叉在正常S阶段经常停顿。叉停止是由多个事件引起的，例如DNA损伤，紧密结合的蛋白质复合物和高表达基因的转录（Tourriere和Pasero 2007; Zeman and Cimprich，2013）。真核生物已经制定了不同的策略来应对这种复制压力，包括修复机制来重新启动捕获的叉子和激活休眠复制起源以抢救终末抓捕的叉。
＆NBSP; ...
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| RNA-binding Protein Immunoprecipitation (RIP) to Examine AUF1 Binding to Senescence-Associated Secretory Phenotype (SASP) Factor mRNA
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Author:
Date:
2015-05-20
[Abstract] Immunoprecipitation and subsequent isolation of nucleic acids allows for the investigation of protein:nucleic acid interactions. RNA-binding protein immunoprecipitation (RIP) is used for the analysis of protein interactions with mRNA. Combining RIP with quantitative real-time PCR (qRT-PCR) further enhances the RIP technique by allowing for the quantitative assessment of RNA-binding protein interactions with their target mRNAs, and how these interactions change in different cellular settings. Here, we describe the immunoprecipitation of the RNA-binding protein AUF1 with several different factors associated with the senescence-associated secretory phenotype (SASP) (Alspach and Stewart, 2013), specifically IL6 and IL8. This protocol was originally published in Alspach et al. (2014).
[摘要] 免疫沉淀和随后的核酸分离允许研究蛋白质：核酸相互作用。 RNA结合蛋白免疫沉淀（RIP）用于分析蛋白质与mRNA的相互作用。 结合RIP与定量实时PCR（qRT-PCR）通过允许RNA结合蛋白与其靶mRNA的相互作用的定量评估以及这些相互作用在不同细胞设置中如何变化来进一步增强RIP技术。 在这里，我们描述RNA结合蛋白AUF1与几种不同的因素与衰老相关的分泌表型（SASP）（Alspach和斯图尔特，2013年），特别是IL6和IL8相关的免疫沉淀。 该协议最初发表在Alspach等人（2014）。
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