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Sanger Sequencing

Sanger Sequencing

DNA sequencing is a fundamental technique that is routinely used in the biological sciences. This method can be applied to a range of questions at different scales - from the sequencing of a cloned DNA fragment or the study of a mutation in a gene up to whole-genome sequencing. However, despite the widespread use of sequencing today, it was not until 1977 that Fredrick Sanger and his collaborators developed the chain-termination method to decode DNA sequences. It relies on the separation of a...

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Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Published on: June 3, 2017

CHEX-seq检测到具有催化潜力的单细胞基因组单链DNA.

Youtao Lu¹, Jaehee Lee², Jifen Li²

¹Department of Biology, School of Arts and Sciences, University of Pennsylvania, Philadelphia, PA, 19104, USA.

Nature communications

|November 14, 2023

概括

研究人员开发了CHEX-seq来绘制细胞内单链DNA (ssDNA) 的地图. 这项技术揭示了全基因组的ssDNA景观,并意外地发现了基因组ssDNA中的催化活性.

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科学领域:

分子生物学分子生物学
基因组学就是基因组学.
细胞生物学细胞生物学

背景情况:

基因组DNA (gDNA) 在转录,修复和复制等必要的细胞过程中,在单链和双链形式之间动态过渡.
了解单链DNA (ssDNA) 的分布和功能对于细胞平衡至关重要.

研究的目的:

引入和验证一种新的方法,CHEX-seq,用于识别和绘制单个细胞内的ssDNA.
为了研究全基因组的ssDNA景观及其动态调节.
探索基因组ssDNA的潜在功能作用.

主要方法:

CHEX-seq使用3'-终端受阻,光可激活的探针来启动从ssDNA中合成互补DNA.
然后将合成的互补DNA进行测序,以生成ssDNA的全基因组图.
该方法在人类K562细胞中进行了基准测试,并应用于小鼠和人类脑细胞,包括局部定位的神经元.

主要成果:

CHEX-seq成功地在现场绘制了全基因组的单链染色质景观.
观察到ssDNA的丰富性在响应细胞干扰时被动态调节.
在核和线粒体DNA中确定了单链区域.
令人惊的是,小鼠和人类gDNA中的特定ssDNA位点在试验室中显示出氨酸化催化活性.

结论:

CHEX-seq是一个强大的工具,用于以单细胞分辨率可视化ssDNA景观.
基因组ssDNA是动态调节的,存在于核和线粒体DNA中.
基因组ssDNA可能具有内在的催化功能,这表明内源DNA具有新的酶作用.