一种经设计突变的 Pfu DNA 聚合酶，用于高级尿嘧啶切除 DNA 工程。
A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering.
机构信息
Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, SE-10691 Stockholm, Sweden.
出版信息
BMC Biotechnol. 2010 Mar 16;10:21. doi: 10.1186/1472-6750-10-21.
BACKGROUND
The combined use of restriction enzymes with PCR has revolutionized molecular cloning, but is inherently restricted by the content of the manipulated DNA sequences. Uracil-excision based cloning is ligase and sequence independent and allows seamless fusion of multiple DNA sequences in simple one-tube reactions, with higher accuracy than overlapping PCR.
RESULTS
Here, the addition of a highly efficient DNA polymerase and a low-background-, large-insertion- compatible site-directed mutagenesis protocol is described, largely expanding the versatility of uracil-excision DNA engineering.
CONCLUSIONS
The different uracil-excision based molecular tools that have been developed in an open-source fashion, constitute a comprehensive, yet simple and inexpensive toolkit for any need in molecular cloning.
背景
限制酶与 PCR 的联合使用彻底改变了分子克隆技术，但本质上受到所操作 DNA 序列内容的限制。基于尿嘧啶切除的克隆是连接酶和序列无关的，允许在简单的单管反应中无缝融合多个 DNA 序列，其准确性高于重叠 PCR。
结果
本文描述了一种高效 DNA 聚合酶的添加和一种低背景、大插入兼容的定点诱变协议，这在很大程度上扩展了尿嘧啶切除 DNA 工程的多功能性。
结论
以开源方式开发的不同基于尿嘧啶切除的分子工具构成了一个全面的、简单且廉价的分子克隆工具包，可满足任何需求。
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