| 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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| Fluorescence in situ Hybridization to the Polytene Chromosomes of Anopheles Mosquitoes
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Author:
Date:
2013-08-20
[Abstract] Fluorescence in situ hybridization (FISH) is a method that uses a fluorescently labeled DNA probe for mapping the position of a genetic element on chromosomes. A DNA probe is prepared by incorporating Cy-3 or Cy-5 labeled nucleotides into DNA by nick-translation or a random primed labeling method. This protocol was used to map genes (Sharakhova et al., 2010) and microsatellite markers (Kamali et al., 2011; Peery et al., 2011) on polytene chromosomes from ovarian nurse cells and salivary glands of malaria mosquitoes. Detailed physical genome mapping performed on polytene chromosomes has the potential to link DNA sequences to specific chromosomal structures such as heterochromatin (Sharakhova et al., 2010). This method also allows comparative ...
[摘要] 荧光原位杂交（FISH）是一种使用荧光标记的DNA探针来绘制遗传元件在染色体上的位置的方法。 通过缺口翻译或随机引物标记法将Cy-3或Cy-5标记的核苷酸掺入DNA中来制备DNA探针。 该方案用于将基因（Sharakhova等人，2010）和微卫星标记物（Kamali等人，2011; Peery等人， >，2011）对来自卵巢保护细胞和疟疾蚊子的唾液腺的多角体染色体。 在多聚染色体上进行的详细的物理基因组作图具有将DNA序列连接到特异性染色体结构（例如异染色质）的潜力（Sharakhova等人，2010）。 该方法还允许比较细胞遗传学研究（Sharakhova等人，2011; Xia等人，2010）和物种系统发育的重建（Kamali等人， ，2012）。
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