
DNA组装效率的无偏差快速检验——基于qPCR而不依赖于转化的方法
Xiaoyan Ma, Xinxin Liang, Yi-Xin Huo
工程(英文) ›› 2019, Vol. 5 ›› Issue (4) : 803-810.
DNA组装效率的无偏差快速检验——基于qPCR而不依赖于转化的方法
Developing a Transformation-Independent and Unbiased qPCR Assay to Rapidly Evaluate the Determinants of DNA Assembly Efficiency
合成生物学正朝着大规模、复杂化的方向发展,这在很大程度上依赖于基因模块的高效组装。传统评估DNA组装效率(AE)的方法需要进行转化,整个过程耗时长达10 h,而且容易受到各种因素的干扰。为快速、可靠地测定组装效率,本研究建立了一种基于qPCR的不依赖于转化的测定方法,用连接上的片段占初始加入片段的比例表征组装效率,3 h即可完成测定。利用该方法测定了酶切连接、Golden Gate组装以及Gibson组装法的双片段或多片段组装效率,所得结果与菌落计数法表征的组装效率呈显著正相关。该方法消除了转化过程的随机性,降低了测定偏差,优于传统的菌落计数法。随后,用此方法研究了DNA片段末端的二级结构对组装效率的影响。结果显示,所有依赖于末端序列互补的组装技术,其组装效率主要受重叠序列整体性质的影响,而发夹结构可显著降低组装效率。这种基于qPCR的测定方法将促进DNA组装技术的发展,并有助于对组装效率影响因素的评估。
Synthetic biology is moving in the direction of larger and more sophisticated design, which depends heavily on the efficient assembly of genetic modules. Conventional evaluation of the DNA assembly efficiency (AE) requires transformation, and the whole process requires up to 10 h and is susceptible to various interferences. To achieve rapid and reliable determination of the AE, an alternative transformation-independent method was established using a modified quantitative polymerase chain reaction (qPCR) assay. The AE is represented by the proportion of the ligated fragment, which can be determined within 3 h. This qPCR-based measurement was tested by the commonly used restriction ligation, Golden Gate assembly, and Gibson assembly for the assembly of two or more DNA pieces; the results correlated significantly with the AEs represented by the counting of the colony-forming units (CFUs). This method outperformed the CFU-based measurement by reducing the measuring bias and the random deviations that stem from the transformation process. The method was then employed to investigate the effects of terminal secondary structures on DNA assembly. The results revealed the major effects of the overall properties of the overlap sequence and the negative effects of hairpin structures on the AE, which are relevant for all assembly techniques that rely on homologous annealing of the terminal sequences. The qPCR-based approach presented here should facilitate the development of DNA assembly techniques and the diagnosis of inefficient assemblies.
组装效率 / DNA组装 / qPCR / 二级结构 / 转化
Assembly efficiency / DNA assembly / qPCR / Secondary structure / Transformation
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