Demidov V V, Bukanov N O, Frank-Kamenetskii D
Boston University, Massachusetts 02215, USA.
Curr Issues Mol Biol. 2000 Jan;2(1):31-5.
This article describes the sequence-specific isolation and purification of intact double-stranded DNA (dsDNA) by oligonucleotide/PNA-assisted affinity capture (OPAC). The OPAC assay is based on selective tagging of a DNA duplex by biotinylated oligodeoxyribonucleotide (ODN) through formation of a so-called PD-loop. The PD-loop is assembled with the aid of a pair of PNA "openers", which allow sequence-specific targeting with a Watson-Crick complementary ODN probe in the exposed region of the dsDNA. The protocol involves three steps. First, two cationic bis-PNAs locally pry the DNA duplex apart at a predetermined site. Then, the exposed DNA single strand is targeted by a complementary biotinylated ODN to selectively form a stable PD-loop complex. Finally, the capture of dsDNA is performed using streptavidin covered magnetic beads. The OPAC procedure has many advantages in the isolation of highly purified native DNA over other affinity capture and amplification techniques.
本文介绍了通过寡核苷酸/肽核酸辅助亲和捕获（OPAC）对完整双链DNA（dsDNA）进行序列特异性分离和纯化的方法。OPAC分析基于生物素化的寡脱氧核糖核苷酸（ODN）通过形成所谓的PD环对DNA双链体进行选择性标记。PD环借助一对肽核酸“开启器”组装而成，这使得在dsDNA的暴露区域能够用与沃森-克里克互补的ODN探针进行序列特异性靶向。该方案包括三个步骤。首先，两个阳离子双肽核酸在预定位点局部撬开DNA双链体。然后，通过互补的生物素化ODN靶向暴露的DNA单链，以选择性地形成稳定的PD环复合物。最后，使用链霉亲和素包被的磁珠进行dsDNA的捕获。与其他亲和捕获和扩增技术相比，OPAC程序在分离高度纯化的天然DNA方面具有许多优势。
