Marx Andreas, Summerer Daniel, Sauter Katharina B M, Gloeckner Christian, Rudinger Nicolas Z
Department of Chemistry, Universitätsstrasse 10, D-78457 Konstanz, Germany.
Nucleic Acids Symp Ser (Oxf). 2007(51):81-2. doi: 10.1093/nass/nrm041.
DNA polymerases are involved in all DNA synthesis occurring in nature. Furthermore, DNA polymerases are the workhorses in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR), cDNA cloning, genome sequencing and nucleic acids based diagnostics. In order to identify DNA polymerase mutants with altered properties, we set up an efficient high through put setup to rapidly screen libraries of DNA polymerase mutants in automated parallel fashion and identified entities with significantly increased selectivity. Furthermore, our results indicate a rational to generally increase DNA polymerase selectivity as we demonstrate for several enzymes from different DNA polymerase families. Additionally, we show that the generation of a new DNA polymerase function is achievable through iterative screening of small libraries of DNA polymerase derived by randomization of the respective genes. We demonstrate that the identified mutants find immediate applications and provide the basis for the development of new means for diagnostic technologies.
DNA聚合酶参与自然界中发生的所有DNA合成过程。此外，DNA聚合酶是众多重要分子生物学核心技术的主力军，如无处不在的聚合酶链反应（PCR）、cDNA克隆、基因组测序和基于核酸的诊断技术。为了鉴定具有改变特性的DNA聚合酶突变体，我们建立了一种高效的高通量方法，以自动并行方式快速筛选DNA聚合酶突变体文库，并鉴定出选择性显著提高的实体。此外，我们的结果表明，正如我们对来自不同DNA聚合酶家族的几种酶所证明的那样，普遍提高DNA聚合酶选择性是合理的。此外，我们表明，通过对各自基因进行随机化产生的DNA聚合酶小文库进行迭代筛选，可以实现新的DNA聚合酶功能的产生。我们证明，所鉴定的突变体可立即得到应用，并为开发新的诊断技术手段提供基础。
