Kim Yun Jae, Lee Hyun Sook, Kwon Suk-Tae, Lee Jung-Hyun, Kang Sung Gyun
Korea Institute of Ocean Science and Technology, Ansan, 426-744, Korea,
Biotechnol Lett. 2014 May;36(5):985-92. doi: 10.1007/s10529-013-1441-x. Epub 2013 Dec 29.
Mechanisms that allow replicative DNA polymerases to attain high processivity are often specific to a given polymerase and cannot be generalised to others. Amplification efficiency is lower in family B-type DNA polymerases than in family A-type (Taq) polymerases because of their strong 3'-5' exonuclease-activity. Here, we have red the exonuclease domain of the Thermococcus onnurineus NA1 (TNA1) DNA polymerase, especially Asn210 to Asp215 residues in Exo II motif (NXXXFD), to improve the processivity. N213D mutant protein had higher processivity and extension rate than the wild-type TNA1 DNA polymerase, retaining a lower mutation frequency than recombinant Taq DNA polymerase. Consequently, the N213D mutant could amplify target DNA up to 13.5 kb in length from human genomic DNA and 16.2 kb in length from human mitochondrial DNA while wild-type TNA1 amplified target DNA of 2.7 kb in length from human genomic DNA.
使复制性DNA聚合酶具有高持续合成能力的机制通常特定于某一种聚合酶，无法推广至其他聚合酶。由于B族DNA聚合酶具有较强的3'-5'核酸外切酶活性，其扩增效率低于A族（Taq）DNA聚合酶。在此，我们对嗜热栖热袍菌NA1（TNA1）DNA聚合酶的核酸外切酶结构域进行改造，特别是Exo II基序（NXXXFD）中的Asn210至Asp215残基，以提高持续合成能力。N213D突变蛋白比野生型TNA1 DNA聚合酶具有更高的持续合成能力和延伸速率，且突变频率低于重组Taq DNA聚合酶。因此，N213D突变体能从人类基因组DNA中扩增出长度达13.5 kb的靶DNA，从人类线粒体DNA中扩增出长度达16.2 kb的靶DNA，而野生型TNA1只能从人类基因组DNA中扩增出长度为2.7 kb的靶DNA。
