Instituto de Biología Molecular Eladio Viñuela and Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain.
Proc Natl Acad Sci U S A. 2010 Sep 21;107(38):16506-11. doi: 10.1073/pnas.1011428107. Epub 2010 Sep 7.
Bacteriophage ϕ29 DNA polymerase is a unique enzyme endowed with two distinctive properties, high processivity and faithful polymerization coupled to strand displacement, that have led to the development of protocols to achieve isothermal amplification of limiting amounts of both circular plasmids and genomic DNA. To enhance the amplification efficiency of ϕ29 DNA polymerase, we have constructed chimerical DNA polymerases by fusing DNA binding domains to the C terminus of the polymerase. The results show that the addition of Helix-hairpin-Helix [(HhH)(2)] domains increases DNA binding of the hybrid polymerases without hindering their replication rate. In addition, the chimerical DNA polymerases display an improved and faithful multiply primed DNA amplification proficiency on both circular plasmids and genomic DNA and are unique ϕ29 DNA polymerase variants with enhanced amplification performance. The reported chimerical DNA polymerases will contribute to make ϕ29 DNA polymerase-based amplification technologies one of the most powerful tools for genomics.
φ29 噬菌体 DNA 聚合酶是一种独特的酶，具有两个显著的特性，即高连续性和与链置换相结合的忠实聚合，这使得开发用于等温扩增有限量环状质粒和基因组 DNA 的方案成为可能。为了提高 φ29 DNA 聚合酶的扩增效率，我们通过将 DNA 结合结构域融合到聚合酶的 C 末端构建了嵌合 DNA 聚合酶。结果表明，添加螺旋-发夹-螺旋 [(HhH)(2)] 结构域可增加杂交聚合酶的 DNA 结合能力，而不会阻碍其复制速率。此外，嵌合 DNA 聚合酶在环状质粒和基因组 DNA 上显示出改进的、忠实的多次引物 DNA 扩增能力，是具有增强扩增性能的独特 φ29 DNA 聚合酶变体。报道的嵌合 DNA 聚合酶将有助于使基于 φ29 DNA 聚合酶的扩增技术成为基因组学中最强大的工具之一。
