质粒
DNA
抄写(语言学)
核糖核酸
解旋酶
生物
分子生物学
体外
基因
核酸
电泳迁移率测定
计算生物学
细胞生物学
基因表达
化学
遗传学
语言学
哲学
作者
Lei Shen,Yanzhong Yang
出处
期刊:Methods in molecular biology
日期:2023-01-01
卷期号:: 265-278
标识
DOI:10.1007/978-1-0716-3191-1_19
摘要
R-loops are three-stranded nucleic acid structures that consist of a DNA-RNA hybrid and a displaced single-stranded DNA. Since it was first reported by Ronald Davis and colleagues over 40 years ago, the study of R-loops has become an increasingly expanded area of research. Numerous factors have been identified to modulate the dynamic formation and resolution of R-loops, which are critical for proper controls of gene expression and genome stability. Along the lines of these discoveries, various biochemical and cellular assays have been developed to detect R-loop changes in vitro and in vivo. In this chapter, we describe a protocol for measuring R-loop formation using a plasmid-based in vitro transcription assay. The R-loop formed is then detected and quantified by using gel mobility, antibody staining, and DNA-RNA immunoprecipitation (DRIP)-qPCR assays. Unlike the helicase assay that uses short R-loop substrates, this assay system introduces DNA topology and active transcription as additional variables that impact R-loop formation, thus, more closely recapitulating in vivo situations. Furthermore, this method can be adopted for investigation of cis-elements and trans-acting factors that influence R-loop formation.
科研通智能强力驱动
Strongly Powered by AbleSci AI