A simple high-resolution procedure to study DNA methylation and in vivo DNA-protein interactions on a single-copy gene level in higher eukaryotes.
A simple high-resolution procedure to study DNA methylation and in vivo DNA-protein interactions on a single-copy gene level in higher eukaryotes.
复制标题
一种简单的高分辨率程序，用于研究高等真核生物中单拷贝基因水平的 DNA 甲基化和体内 DNA-蛋白质相互作用。
DOI：
--
复制
发表时间：
1989
影响因子：
11.1
通讯作者：
Jean
中科院分区：
文献类型：
--
作者：
Hanspeter Saluz;Jean
我们描述了一种方法，允许研究胞嘧啶甲基化状态和体内蛋白质- dna相互作用在高等真核生物。这种强大的技术适用于任何感兴趣的基因在单拷贝水平。为了研究DNA甲基化，用限制性内切酶酶切的总未克隆基因组DNA进行胞嘧啶特异性联氨反应和化学裂解。感兴趣的DNA片段用Taq聚合酶和序列特异性放射性标记的合成引物线性扩增。扩增后，DNA片段在测序凝胶上分离，直接进行放射自显影。为了研究蛋白质-DNA在体内的相互作用，我们使用了类似的方法，除了我们感兴趣的DNA是从用硫酸二甲酯或紫外线处理的细胞中分离出来的。这项技术的分辨率是通过两个例子来证明的，这两个例子之前已经通过传统的基因组测序和“足迹”方法进行了研究。
We describe a method that permits the study of the state of cytosine methylation and of in vivo protein-DNA interactions in higher eukaryotes. This powerful technique is applicable to any gene of interest at the single-copy level. To study DNA methylation, the total uncloned genomic DNA, digested with a restriction endonuclease is subjected to a cytosine-specific hydrazine reaction and chemical cleavage. The DNA fragments of interest are linearly amplified with Taq polymerase and a sequence-specific radioactivity labeled synthetic primer. Following amplification, the DNA fragments are separated on a sequencing gel that is directly autoradiographed. To study protein-DNA interactions in vivo, we use a similar method, except that the DNA of interest is isolated from cells treated either with dimethyl sulfate or UV light. The resolution power of this technique is demonstrated by two examples, which have been studied previously by the conventional methods of genomic sequencing and "footprinting."