Matsumura Ichiro
Department of Biochemistry, Emory University School of Medicine, Rollins Research Center, Atlanta, GA.
Biotechniques. 2015 Sep 1;59(3):IV-XIII. doi: 10.2144/000114324. eCollection 2015 Sep.
The demand for cloned genes has increased incessantly over the past 32 years, but some who need recombinant plasmids struggle to produce them. While the pitfalls of traditional ligation-dependent cloning are non-trivial, most can be avoided with sufficient effort and attention to detail. Here, the chemical properties of enzymes and reagents used to clone genes into plasmids are reviewed to draw attention to the most pertinent details. In particular, the virtues of agarose gel electrophoresis monitoring, the nature of the interactions between DNA and silica, and challenges associated with thermostable DNA polymerases, restriction endonucleases, and T4 DNA ligase are explored. Common pitfalls associated with Escherichia coli transformation and DNA modifying enzymes are also described. A thorough understanding of established methods is essential for troubleshooting, implementing alternative approaches, and inventing new techniques in response to changes in technology and demand.
在过去32年里，对克隆基因的需求持续增长，但一些需要重组质粒的人在生产过程中却面临困难。虽然传统的依赖连接的克隆存在不少问题，但只要付出足够的努力并注重细节，大多数问题是可以避免的。在此，我们对用于将基因克隆到质粒中的酶和试剂的化学性质进行综述，以引起对最相关细节的关注。特别探讨了琼脂糖凝胶电泳监测的优点、DNA与二氧化硅之间相互作用的性质，以及与热稳定DNA聚合酶、限制性内切酶和T4 DNA连接酶相关的挑战。还描述了与大肠杆菌转化和DNA修饰酶相关的常见问题。透彻理解既定方法对于故障排除、采用替代方法以及根据技术和需求的变化发明新技术至关重要。
