Wilson W W, Hoffman R M
Department of Pediatrics, University of California, San Diego, La Jolla 92093.
Anal Biochem. 1990 Dec;191(2):370-5. doi: 10.1016/0003-2697(90)90234-z.
Conditions were determined for the methylation of intact yeast chromosomes by EcoRI, HhaI, and MspI bacterial methylases using an endonuclease protection assay while the chromosomes were embedded in agarose plugs suitable for transverse-field electrophoresis. Parameters were also established for the methylation of human chromosomes by EcoRI methylase. Methylation of embedded chromosomes by EcoRI methylase required prewashes with EDTA. EcoRI, HhaI, and MspI methylases showed optimal activity when nonacetylated bovine serum albumin, high levels of S-adenosylmethionine, and high levels of methylase were used. The use of bacterial methylases for methylation of embedded chromosomes will allow investigators to normalize variations in cellular DNA methylation prior to restriction and create new and rare endonuclease recognition sites which will facilitate the detection of chromosomal alterations and deletions.
利用内切核酸酶保护分析法，在完整酵母染色体包埋于适合横向场电泳的琼脂糖凝胶块中时，确定了EcoRI、HhaI和MspI细菌甲基化酶对其进行甲基化的条件。还建立了EcoRI甲基化酶对人类染色体进行甲基化的参数。EcoRI甲基化酶对包埋染色体的甲基化需要用EDTA进行预洗涤。当使用未乙酰化的牛血清白蛋白、高水平的S-腺苷甲硫氨酸和高水平的甲基化酶时，EcoRI、HhaI和MspI甲基化酶表现出最佳活性。使用细菌甲基化酶对包埋染色体进行甲基化，将使研究人员能够在酶切之前使细胞DNA甲基化的变化标准化，并产生新的和罕见的内切核酸酶识别位点，这将有助于检测染色体改变和缺失。
