Nucleic acid metabolic enzyme activity detection method

Abstract

The invention relates to a nucleic acid metabolic enzyme activity detection method which comprises the following steps: S1, artificially synthesizing a single-stranded nucleic acid fragment and a primer complementary from the 3'end, and forming a template through annealing reaction; s2, reacting a template, a reaction buffer solution, a fluorescence modified nucleotide derivative or a fluorescence modified nucleotide derivative analogue and a nucleic acid metabolic enzyme to be measured, and purifying to obtain a primary purified product; s3, the primary purification product, a Taq reaction buffer solution, dNTP and Taq DNA polymerase are subjected to a reaction and purification, and a secondary purification product is obtained; s4, analyzing a secondary purification product by using a capillary electrophoresis apparatus; and S5, making a standard curve, and substituting the result in the step S4 into the standard curve to obtain a detection result. The method is quick to operate, accurate in activity detection result and high in sensitivity, and realizes high-throughput detection.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for detecting the activity of nucleic acid metabolizing enzymes. Background technique [0002] Nucleic acid metabolism, including DNA and RNA synthesis and degradation, is fundamental to all nucleic acid research and related life science fields of study. Standard activity assays for nucleic acid metabolizing enzymes involved in DNA replication and repair are by assays that measure DNA or RNA product synthesis or degradation of radioactive or fluorescently labeled nucleic acid substrates. For example, the method of isotope labeling and the fluorescent dye method combined with DNA (PicoGreen or EvaGreen) are widely used in DNA quantification, and then applied in the activity detection method of nucleic acid metabolizing enzymes to measure the synthesis activity or degradation activity of nucleic acid metabolizing enzymes. In order to better study the reaction intermediates or...

AI Technical Summary

Problems solved by technology

In order to better study the reaction intermediates or by-products, more comprehensively capture the specific steps and details of the reaction pathway, and fully understand the activity of nucleases, polyacrylamide gel electrophoresis (PAGE) is widely used in the analysis of substrates, intermediates In experimental studies such as the size distribution of nucleases, further characterization and standard determination of nucleases are carried out. However, compared with the aforementioned radioactive or fluorescent labeling methods, the analysis method of polyacrylamide gel electrophoresis has the disadvantages of long experiment time, low efficiency and low throughput. and other shortcomings, which limit the scope of nuclease analysis

Images