Haber C, Wirtz D
Department of Chemical Engineering, Johns Hopkins University, Baltimore, Maryland 21218 USA.
Biophys J. 2000 Sep;79(3):1530-6. doi: 10.1016/S0006-3495(00)76404-6.
Recombinant DNA technology, which is based on the assembly of DNA fragments, forms the backbone of biological and biomedical research. Here we demonstrate that a uniform shear flow can induce and control the assembly of lambda-phage DNA molecules: increasing shear rates form integral DNA multimers of increasing molecular weight. Spontaneous assembly and grouping of end-blunted lambda-phage DNA molecules are negligible. It is suggested that shear-induced DNA assembly is caused by increasing the probability of contact between molecules and by stretching the molecules, which exposes the cohesive ends of the otherwise undeformed lambda-phage DNA molecules. We apply this principle to enhance the kinetics and extent of DNA concatenation in the presence of ligase. This novel approach to controlled DNA assembly could form the basis for improved approaches to gene-chip and recombinant DNA technologies and provide new insight into the rheology of associating polymers.
基于DNA片段组装的重组DNA技术构成了生物学和生物医学研究的核心。在此我们证明,均匀剪切流能够诱导并控制λ噬菌体DNA分子的组装:剪切速率增加会形成分子量不断增大的完整DNA多聚体。平头λ噬菌体DNA分子的自发组装和聚集可以忽略不计。有人提出,剪切诱导的DNA组装是由于增加了分子间接触的概率以及拉伸了分子,从而暴露出原本未变形的λ噬菌体DNA分子的粘性末端。我们应用这一原理来提高连接酶存在时DNA连接的动力学和程度。这种控制DNA组装的新方法可能为改进基因芯片和重组DNA技术的方法奠定基础,并为缔合聚合物的流变学提供新的见解。