JILA, National Institute of Standards and Technology and University of Colorado, Boulder, CO 80309, USA and Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309, USA.
Nucleic Acids Res. 2013 Oct;41(19):e179. doi: 10.1093/nar/gkt699. Epub 2013 Aug 9.
Controlled twisting of individual, double-stranded DNA molecules provides a unique method to investigate the enzymes that alter DNA topology. Such twisting requires a single DNA molecule to be torsionally constrained. This constraint is achieved by anchoring the opposite ends of the DNA to two separate surfaces via multiple bonds. The traditional protocol for making such DNA involves a three-way ligation followed by gel purification, a laborious process that often leads to low yield both in the amount of DNA and the fraction of molecules that is torsionally constrained. We developed a simple ligation-free procedure for making torsionally constrained DNA via polymerase chain reaction (PCR). This PCR protocol used two 'megaprimers', 400-base-pair long double-stranded DNA that were labelled with either biotin or digoxigenin. We obtained a relatively high yield of gel-purified DNA (∼500 ng/100 µl of PCR reaction). The final construct in this PCR-based method contains only one labelled strand in contrast to the traditional construct in which both strands of the DNA are labelled. Nonetheless, we achieved a high yield (84%) of torsionally constrained DNA when measured using an optical-trap-based DNA-overstretching assay. This protocol significantly simplifies the application and adoption of torsionally constrained assays to a wide range of single-molecule systems.
对单个双链 DNA 分子进行受控扭曲为研究改变 DNA 拓扑结构的酶提供了一种独特的方法。这种扭曲需要将单个 DNA 分子扭转约束。这种约束是通过将 DNA 的两端通过多个键固定到两个单独的表面上来实现的。制作这种 DNA 的传统方案涉及三向连接，然后进行凝胶纯化，这是一个繁琐的过程，通常会导致 DNA 的产量和扭转约束的分子分数都很低。我们通过聚合酶链反应 (PCR) 开发了一种简单的无连接程序来制作扭转约束 DNA。该 PCR 方案使用两种“megaprimers”，即长 400 个碱基对的双链 DNA，它们分别用生物素或地高辛标记。我们获得了相对较高产量的凝胶纯化 DNA（∼500 ng/100 µl PCR 反应）。与传统的 DNA 双链标记的构建相比，这种基于 PCR 的方法中的最终构建物仅包含一条标记链。尽管如此，当使用基于光学陷阱的 DNA 过度拉伸测定法测量时，我们仍获得了扭转约束 DNA 的高产量（84%）。该方案显著简化了扭转约束测定法在各种单分子系统中的应用和采用。
