Genome-wide analysis of DNA synthesis by BrdU immunoprecipitation on tiling microarrays (BrdU-IP-chip) in Saccharomyces cerevisiae.
Genome-wide analysis of DNA synthesis by BrdU immunoprecipitation on tiling microarrays (BrdU-IP-chip) in Saccharomyces cerevisiae.
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DOI：
10.1101/pdb.prot5385
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发表时间：
2010-02
影响因子：
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通讯作者：
C. Viggiani;S. Knott;O. Aparicio
中科院分区：
文献类型：
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作者：
C. Viggiani;S. Knott;O. Aparicio
将胸苷类似物（例如 5-溴-2'-脱氧尿苷 (BrdU)）掺入新合成的 DNA 中，是分析 DNA 复制、修复和 DNA 代谢其他方面的强大工具。在酿酒酵母中，已经开发出多种检测方法来鉴定掺入 BrdU 的染色体 DNA。在这里，我们描述了一种将 BrdU 免疫沉淀与 Roche NimbleGen 提供的 DNA 微阵列（BrdU-IP 芯片）结合起来的方法，以实现 BrdU 标记的染色体 DNA 的全基因组鉴定。在此过程中，酵母细胞经过工程改造，可以从生长培养基中吸收 BrdU 并将其掺入复制 DNA，并在实验条件下在 BrdU 存在的情况下生长。收获这些细胞，分离基因组 DNA 并随机剪切，然后对 BrdU 标记的 DNA 进行免疫沉淀。这种免疫沉淀的 DNA 经过聚合酶链式反应 (PCR) 扩增，用荧光团标记，并与参考样品（通常是不进行免疫沉淀的总基因组 DNA，但用不同的荧光团扩增和标记）一起杂交到 DNA 微阵列上。然后对数据进行归一化，并识别 BrdU 掺入的染色体区域（BrdU 峰）。可以量化 BrdU 峰高，并与不同基因组位置或不同实验条件（例如，不同突变体或时间点）下的 BrdU 峰高进行比较。 BrdU-IP 芯片具有许多潜在的应用，并且已用于识别复制起点、对菌株之间的起点激发进行定量比较以及检查复制叉进展。
The incorporation of thymidine analogs, such as 5-bromo-2′-deoxyuridine (BrdU), into newly synthesized DNA is a powerful tool for analysis of DNA replication, repair, and other aspects of DNA metabolism. In Saccharomyces cerevisiae, several assays have been developed to identify chromosomal DNA that has incorporated BrdU. Here we describe an approach that couples BrdU immunoprecipitation with DNA microarrays (BrdU-IP-chip) supplied by Roche NimbleGen to enable the genome-wide identification of BrdU-labeled chromosomal DNA. In this procedure, yeast cells that have been engineered to assimilate BrdU from the growth medium and incorporate it into replicating DNA are grown in the presence of BrdU under experimental conditions. These cells are harvested, the genomic DNA is isolated and randomly sheared, and the BrdU-labeled DNA is then immunoprecipitated. This immunoprecipitated DNA is polymerase chain reaction (PCR)-amplified, labeled with a fluorophore, and cohybridized along with a reference sample (typically total genomic DNA not subject to immunoprecipitation, but amplified and labeled with a different fluorophore) onto DNA microarrays. The data are then normalized, and chromosomal regions of BrdU incorporation (BrdU peaks) are identified. BrdU peak heights can be quantified and compared with BrdU peak heights at different genomic locations or under different experimental conditions (e.g., different mutants or time points). BrdU-IP-chip has many potential applications and has already been used to identify replication origins, make quantitative comparisons of origin firing between strains, and examine replication fork progression.