A quantitative fluorescence-based steady-state assay ofDNA polymerase

Max D. Driscoll; Julius Rentergent; Sam Hay

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A quantitative fluorescence-based steady-state assay ofDNA polymerase

机译：基于定量荧光的DNA聚合酶稳态分析

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Fluorescent dyes that bind DNA have been demonstrated as a useful alternativeto radionucleotides for the quantification of DNA and the in vitromeasurement of the activity of DNA polymerases and nucleases. However,this approach is generally used in a semi-quantitative way to determine relativerates of reaction. In this report, we demonstrate a method for thesimultaneous quantification of DNA in both its single-strand and doublestrandforms using the dye PicoGreen. This approach is used in a steadystateassay of DNA polymerase Klenow fragment exo, where wedetermine kcat and Km values for the DNA polymerase that are in excellentagreement with literature values.

机译：已证明结合DNA的荧光染料可作为放射性核苷酸的有用替代品，用于定量DNA和体外测量DNA聚合酶和核酸酶的活性。但是，该方法通常以半定量方式用于确定反应的相对速率。在本报告中，我们演示了使用染料PicoGreen同时定量其单链和双链DNA的方法。此方法用于DNA聚合酶Klenow片段exo的稳态分析，其中确定DNA聚合酶的kcat和Km值与文献值非常吻合。

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《The FEBS journal》 |2014年第8期|共9页
作者
Max D. Driscoll; Julius Rentergent; Sam Hay;
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正文语种 eng
中图分类生物化学;
关键词
DNA quantification; enzyme kinetics; fluorescence; Michaelis–Menten; PicoGreen; polymerase; steady-state;

机译：DNA定量;酶动力学;荧光;Michaelis–Menten;PicoGreen;聚合酶;稳态;

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1. A quantitative fluorescence-based steady-state assay ofDNA polymerase [J] . Max D. Driscoll, Julius Rentergent, Sam Hay The FEBS journal . 2014,第8期

机译：基于定量荧光的DNA聚合酶稳态分析
2. Specificity of a quantitative real-time polymerase chain reaction assay for the detection of invasive pneumococcal disease: identifying streptococcus pneumoniae using quantitative polymerase chain reaction. [J] . Kee C, Fatovich DM, Palladino Chest: The Journal of Circulation, Respiration and Related Systems . 2010,第1期

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3. Specificity of a Quantitative Real-Time Polymerase Chain Reaction Assay for the Detection of Invasive Pneumococcal Disease: Identifying Streptococcus pneumoniae Using Quantitative Polymerase Chain Reaction [J] . Cordelia Kee, Daniel M. Fatovich, Grant W. Waterer, Chest . 2010,第1期

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4. Correlation of semi-quantitative reverse transcription polymerase chain reaction assay results for porcine epidemic diarrhea virus and the presence of positive immunohistochemistry [C] . Alyssa Taplett American Association of Swine Veterinarians Annual Meeting . 2015

机译：半定量逆转录聚合酶链反应测定结果对猪流行性腹泻病毒的相关性以及阳性免疫组织化学的存在
5. Development, validation, and application of a quantitative polymerase chain reaction assay to assess Hematodinium perezi prevalence in environmental samples. [D] . Hanif, Ammar W. 2012

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6. Fluorescence-Based Assay for Phenotypic Characterization of Human Cytomegalovirus Polymerase Mutations Regarding Drug Susceptibility and Viral Replicative Fitness [O] . Meike Chevillotte, Axel Schubert, Thomas Mertens, 2009

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7. A quantitative fluorescence-based steady-state assay of DNA polymerase [O] . Driscoll, Max D, Rentergent, Julius, Hay, Sam 2014

机译：基于定量荧光的DNa聚合酶稳态测定
8. Single Nucleotide Polymorphisms (SNP)-specific Quantitative Real Time Polymerase Chain Reaction (PCR) Assay for Analyzing Competition and Emergence of the Military Hypersporulating Strains of Bacillus Atrophaeous var. Globigii. [R] . Zhelev, D. V., Dupuis, C., Ren, S., 2012

机译：单核苷酸多态性（sNp）特异性定量聚合酶链反应（pCR）分析用于分析军用过度表达的萎缩芽孢杆菌的竞争和出现。 Globigii。

A quantitative fluorescence-based steady-state assay ofDNA polymerase

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