Cohn S M, Lieberman M W
J Biol Chem. 1984 Oct 25;259(20):12456-62.
We have developed an immunological method for isolation and identification of DNA sequences containing 5-bromo-2'-deoxyuridine (BrdUrd) incorporated during UV-induced excision-repair synthesis. DNA fragments containing BrdUrd incorporated during repair synthesis were incubated with goat anti-BrdUrd and rabbit anti-goat IgG, and the antibody-DNA complexes were separated from bulk DNA by nitrocellulose filter binding. With this method, 80% of DNA sequences containing BrdUrd-labeled excision-repair sites were recovered, contaminated with less than 1% of DNA fragments devoid of excision-repair sites. Recovery of DNA fragments containing repair sites was independent of size from 2 to 20 kilobases. We have used this method in conjunction with blot hybridization to demonstrate that repair synthesis occurs in human ribosomal gene sequences in cells treated with UV.
我们开发了一种免疫学方法，用于分离和鉴定在紫外线诱导的切除修复合成过程中掺入5-溴-2'-脱氧尿苷（BrdUrd）的DNA序列。将在修复合成过程中掺入BrdUrd的DNA片段与山羊抗BrdUrd和兔抗山羊IgG一起孵育，然后通过硝酸纤维素滤膜结合将抗体-DNA复合物与大量DNA分离。用这种方法，80%含有BrdUrd标记的切除修复位点的DNA序列被回收，污染的不含切除修复位点的DNA片段不到1%。含有修复位点的DNA片段的回收与2至20千碱基的大小无关。我们已将此方法与印迹杂交结合使用，以证明在用紫外线处理的细胞中，人类核糖体基因序列中发生了修复合成。
