An alternative and convenient strategy for generation of substantial quantities of singly 5′-32p-end-labeled double-stranded DNA for binding studies: Development of a protocol for examination of functional features of (+)-CC-1065 and the duocarmycins that contribute to their sequence-selective DNA alkylation properties
作者:Dale L Boger、Stephen A Munk、Hamideh Zarrinmayeh、Takayoshi Ishizaki、Joan Haught、M Bina
DOI:10.1016/s0040-4020(01)81798-1
日期:1991.1
label is introduced onto the free 5′-hydroxyl group of a chemically synthesized universal primer [5′-32P-d(GTAAAACGACGGCCAGT)-3′] which is used to initiate DNA synthesis on M13-derived single-stranded DNA templates. Following DNA synthesis, a restriction enzyme cleavage reaction produces a uniform length duplex suitable for agent binding studies. The strategy further permits the use of the Sanger dideoxynucleotide
基于M133克隆技术,描述了一种可替代的策略的开发,该策略可确保大量大量的5'- 32 P-末端标记的双链DNA适于结合研究,并提供了可补充数量的5'- 32 P的生产优势-末端标记的均一长度的双链DNA,无需进行DNA分离(限制性片段),去磷酸化和冗长的制备性凝胶电泳程序。将32 P标签引入化学合成的通用引物[5'- 32Pd(GTAAAACGACGGCCAGT)-3']用于在M13衍生的单链DNA模板上启动DNA合成。DNA合成后,限制性内切酶裂解反应产生了适合试剂结合研究的均匀长度的双链体。该策略进一步允许使用Sanger双脱氧核苷酸测序技术直接和明确鉴定由末端标记的DNA上的试剂引入的切割位点。审查了该程序在检查(+)-CC-1065(1)和一系列合成类似物的DNA烷基化特性中的用途。通过这些研究,详细描述了(+)-CC-1065烷基化选择性的精确定义。拥有母体1,2,7,7a-四氢环丙[[1