Method for rapidly assaying activity of DNA polymerase

Abstract

The invention discloses a method for rapidly assaying activity of DNA polymerase. The method comprises the steps: (1) drawing a standard curve and calculating an equation of linear regression: mixingtwo kinds of oligonucleotide probes H1 and H2 by different proportions, measuring fluorescence values, drawing the standard curve, and calculating the equation of linear regression; (2) carrying out aDNA polymerase activity detecting reaction under isothermal conditions: employing a working solution only containing H1 obtained in the step (1) as a substrate, adding a dNTP mixed solution and magnesium ions, and preparing a DNA polymerase activity detecting reaction system by employing a reaction buffer corresponding to the DNA polymerase; (3) determining a fluorescence value of a product afterstopping the reaction: forcefully stopping the reaction after an isothermal reaction, cooling the temperature of a system to room temperature, then, assaying the content of the product, and recordinga fluorescence added value in 30min; and (4) calculating enzymatic activity: substituting the fluorescence added value into the equation of linear regression corresponding to the standard curve obtained in the step (1) to obtain the content of the product in the system, and calculating the activity of raw enzyme liquid. Independent of isotopes, the operation is simple, and the accuracy and repeatability are good.

Description

technical field [0001] The invention relates to the technical fields of biochemistry and molecular biology, in particular to a method for measuring DNA polymerase activity. Background technique [0002] DNA polymerase is one of the most important tool enzymes in modern genetic engineering, and activity determination is an essential step in the development and process of DNA polymerase. At present, the methods for measuring DNA polymerase activity mainly include: (1) isotope labeling method, using isotope-labeled dNTP, the enzyme amount required to incorporate 10nmol dNTP into acid-insoluble matter at the optimum reaction temperature within 30 minutes is defined as 1 Activity unit (U), this method is the most accurate, and it is also the definition standard of DNA polymerase activity agreed by domestic and foreign mainstream manufacturers. However, limited by isotope operation qualifications and radioactive protection conditions, it is difficult to achieve in ordinary labora...

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Problems solved by technology

[0004] Aiming at the defects of complicated operation or low accuracy of the existing DNA polymerase activity determination methods, the purpose of the present invention is to provide a method with higher accuracy and higher repeatability by redesigning the standard curve drawing and DNA double-strand product quantity determination methods. A good DNA polymerase activity assay method to promote the development and transformation of biological tool enzymes

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