Three-Enzyme Cascade Bioreactor for Rapid Digestion of Genomic DNA into Single Nucleosides

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• 作者：Junfa Yin ; Tian Xu ; Ning Zhang ; Hailin Wang
• 刊名：Analytical Chemistry
• 出版年：2016
• 出版时间：August 2, 2016
• 年：2016
• 卷：88
• 期：15
• 页码：7730-7737
• 全文大小：439K
• 年卷期：0
• ISSN：1520-6882

文摘

Structure-based DNA modification analysis provides accurate and important information on genomic DNA changes from epigenetic modifications to various DNA lesions. However, genomic DNA strands are often required to be efficiently digested into single nucleosides. It is an arduous task because of the involvement of multiple enzymes with different catalytic acitivities. Here we constructed a three-enzyme cascade capillary monolithic bioreactor that consists of immobilized deoxyribonuclease I (DNase I), snake venom phosphodiesterase (SVP), and alkaline phosphatase (ALPase). By the use of this cascade capillary bioreactor, genomic DNA can be efficiently digested into single nucleosides with an increasing rate of ∼20 folds. The improvement is mainly attributed to dramatically increase enzymatic capacity and activity. With a designed macro-porous structure, genomic DNA of 5–30 Kb (∼1.6–10 million Daltons) can be directly passed through the bioreactor simply by hand pushing or a low-pressure microinjection pump. By coupling with liquid chromatography-tandem mass spectrometry (LC-MS/MS), we further developed a sensitive assay for detection of an oxidative stress biomarker 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) in DNA. The proposed three-enzyme cascade bioreactor is also potentially applicable for fast identification and quantitative detection of other lesions and modifications in genomic DNA.