在里氏木霉中的体内足迹分析。

In Vivo Footprinting Analysis in Trichoderma reesei.

机构信息

ACIB GmbH, c/o BOKU, Vienna, Austria.

出版信息

Methods Mol Biol. 2021;2234:177-189. doi: 10.1007/978-1-0716-1048-0_15.

Abstract

The in vivo footprinting method identifies protein-targeted DNA regions under different conditions such as carbon sources. Dimethyl sulfate (DMS) generates methylated purine bases at DNA sites which are not bound by proteins or transcription factors. The DNA is cleaved by HCl, and the resulting DNA fragments are 5'-end [6-FAM]-labeled by a linker-mediated PCR (LM-PCR). Fluorescent fragments are separated and analyzed on a capillary sequencer, followed by automated data analysis using the software tool ivFAST.

摘要

体内足迹法可鉴定不同条件下（如碳源）的蛋白质靶向 DNA 区域。硫酸二甲酯 (DMS) 在未与蛋白质或转录因子结合的 DNA 位点上生成甲基化嘌呤碱基。DNA 用 HCl 切割，所得 DNA 片段通过连接介导的 PCR（LM-PCR）在 5'端用[6-FAM]标记。荧光片段在毛细管测序仪上分离和分析，然后使用软件工具 ivFAST 进行自动数据分析。