| Summary: | 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. Keywords: Assembly efficiency, DNA assembly, qPCR, Secondary structure, Transformation
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