Hartley J A, Souhami R L, Berardini M D
Department of Oncology, University College London Medical School, UK.
J Chromatogr. 1993 Aug 25;618(1-2):277-88. doi: 10.1016/0378-4347(93)80038-6.
Several electrophoretic and chromatographic techniques, many of which have only been developed recently, provide sensitive methods for the detection and separation of DNA containing interstrand crosslinks such as those produced by many cancer chemotherapeutic drugs and photoactive psoralen derivatives. Most of the methods rely on the fact that the presence of such crosslinks prevent the complete denaturation of the two complimentary DNA strands by heat or alkali. A simple and highly sensitive neutral agarose gel electrophoresis method is particularly applicable to detailed time-course experiments of both total crosslink formation, and the "second-arm" of the crosslink reaction. This method separates denatured single-stranded from double-stranded DNA which has reannealed as a result of an interstrand crosslink. Polyacrylamide gel-based assays using denaturing gels are more suited to the separation of smaller crosslinked DNA fragments and, in particular, small oligonucleotides on high-percentage gels. In addition, they provide methods for the determination of the exact base position and sequence selectivity of crosslink formation. Sephadex chromatography and high-performance liquid chromatography can separate small crosslinked oligonucleotides from non-crosslinked duplexes, and the hydroxyapatite column chromatographic separation of single- and double-stranded cellular DNA can be used to quantitate the level of interstrand crosslinking present in the bulk of the genome. Finally, the analysis of damage by crosslinking agents, and its repair, at the level of specific genes can be achieved by hybridization with specific probes following membrane transfer from neutral agarose gels used to fractionate restricted and fully denatured genomic DNA from drug-treated cells.
几种电泳和色谱技术，其中许多是最近才开发出来的，为检测和分离含有链间交联的DNA提供了灵敏的方法，比如许多癌症化疗药物和光活性补骨脂素衍生物所产生的链间交联。大多数方法都基于这样一个事实，即这种交联的存在会阻止两条互补DNA链通过加热或碱处理而完全变性。一种简单且高度灵敏的中性琼脂糖凝胶电泳方法特别适用于全面交联形成以及交联反应“第二臂”的详细时间进程实验。这种方法可将由于链间交联而复性的双链DNA与变性的单链DNA分开。使用变性凝胶的基于聚丙烯酰胺凝胶的分析方法更适合分离较小的交联DNA片段，特别是在高百分比凝胶上分离小寡核苷酸。此外，它们还提供了确定交联形成的确切碱基位置和序列选择性的方法。葡聚糖凝胶色谱法和高效液相色谱法可以将小的交联寡核苷酸与非交联双链体分开，而单链和双链细胞DNA的羟基磷灰石柱色谱分离可用于定量基因组主体中存在的链间交联水平。最后，通过与特定探针杂交，可以在特定基因水平上分析交联剂造成的损伤及其修复情况，杂交是在将用于分离来自药物处理细胞的限制性和完全变性基因组DNA的中性琼脂糖凝胶进行膜转移之后进行的。
