A useful strategy to construct DNA polymerases with different properties by using genetic resources from environmental DNA
A useful strategy to construct DNA polymerases with different properties by using genetic resources from environmental DNA
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DOI：
10.1266/ggs.84.3
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发表时间：
2009-02-01
影响因子：
1.1
通讯作者：
Ishino, Yoshizumi
中科院分区：
文献类型：
--
作者：
Matsukawa, Hiroaki;Yamagami, Takeshi;Ishino, Yoshizumi
DNA聚合酶根据模板DNA合成新的DNA链，在DNA复制和修复过程中使用脱氧核苷酸三磷酸，并且在DNA代谢中对维持基因组完整性至关重要。此外，这些酶广泛用于基因工程技术，包括双脱氧测序、PCR、DNA标记、诱变和其他体外实验。热稳定DNA聚合酶对于PCR和循环测序特别有用。我们提出了一个强有力的策略，利用环境DNA作为遗传资源，调查家庭B DNA聚合酶的结构与功能的关系。用从日本各地土壤样品中的DNA扩增的PCR片段取代了对应于P.furiosus DNA聚合酶I（PolBI）结构基因中DNA聚合反应活性中心的区域。在PolBI表达质粒内构建嵌合pol基因。由此产生的嵌合酶显示具有不同性质的DNA聚合酶活性。
DNA polymerases synthesize new DNA strands according to the template DNA, using deoxynucleotide triphosphates during DNA replication and repair, and are essential to maintain genome integrity in DNA metabolism. In addition, these enzymes are widely used for genetic engineering techniques, including dideoxy-sequencing, PCR, DNA labeling, mutagenesis, and other in vitro experiments. Thermostable DNA polymerases are especially useful for PCR and cycle-sequencing. We propose a powerful strategy using environmental DNA as a genetic resource to investigate the structure-function relationships of the family B DNA polymerases. The region corresponding to the active center of the DNA polymerizing reaction in the structural gene of P. furiosus DNA polymerase I (PolBI) was substituted by PCR fragments amplified from DNAs within soil samples from various locations in Japan. The chimeric pol genes were constructed within the PolBI expression plasmid. The chimeric enzymes thus produced revealed DNA polymerase activities with different properties.