生物
DNA复制
溴脱氧尿苷
免疫沉淀
DNA
计算生物学
DNA测序
遗传学
基因
细胞生长
作者
Joanna E. Haye-Bertolozzi,Oscar M. Aparicio
出处
期刊:Methods in molecular biology
日期:2017-10-17
卷期号:: 209-225
被引量:8
标识
DOI:10.1007/978-1-4939-7306-4_16
摘要
Incorporation into DNA of nucleoside analogs like 5-bromo-2′-deoxyuridine (BrdU) is a powerful tool for in vivo studies of DNA synthesis during replication and repair. Immunoprecipitation of BrdU-labeled DNA analyzed by DNA sequencing (BrdU-IP-seq) allows for genome-wide, sequence-specific tracking of replication origin and replication fork dynamics under different conditions, such as DNA damage and replication stress, and in mutant strains. We have recently developed a quantitative method for BrdU-IP-seq (qBrdU-seq) involving DNA barcoding to enable quantitative analysis of multiple experimental samples subjected to BrdU-IP-seq. After initial barcoding of multiple, individually BrdU-labeled genomic DNA samples, a pooling strategy is used for all subsequent steps including immunoprecipitation, amplification, and sequencing, which eliminates sample-to-sample variability in these steps. Parallel processing of an aliquot of the pooled input sample provides a direct control for the normalization of the data and yields results that allow quantitative comparisons of the experimental samples. Though developed for the analysis of S. cerevisiae, this method should be directly adaptable to other model systems.
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