Enzyme activity determination method of DNA ligase

Abstract

The invention provides an enzyme activity determination method of DNA ligase. The enzyme activity determination method comprises the following steps: (1) providing an RNA single strand, and a receptor DNA single strand and a donor DNA single strand which are complementary with the RNA single strand; (2) annealing to obtain a DNA / RNA hybrid double strand, enabling a 3'terminal hydroxyl group of a receptor DNA single strand and a 5 'terminal phosphate group of a donor DNA single strand to be adjacent but have a notch, and enabling a fluorophore of the hybrid double strand to generate fluorescence; (3) treating the DNA / RNA heterozygous double strand with DNA ligase to be detected to obtain a complete DNA / RNA heterozygous double strand connected at the notched part, and keeping fluorescence of a fluorophore of the heterozygous double strand; and (4) digesting the complete DNA / RNA hybrid double strand directly or after heating denaturation with ribonuclease, leaving a DNA ligation product, quantifying according to the fluorescence intensity of the DNA ligation product, and determining the enzyme activity of the DNA ligation enzyme to be detected. The method is accurate in quantification, simple, convenient and rapid in operation process, and does not involve the application of radioactive substances. Meanwhile, the invention also optimizes and designs a specific detection sequence which is suitable for enzyme activity determination and has high sensitivity.

Description

technical field [0001] The invention belongs to the technical field of biochemistry, and in particular relates to a method for measuring the activity of DNA ligase. Background technique [0002] DNA ligase was discovered in Escherichia coli cells in 1967. It is an important enzyme in organisms. The reaction it catalyzes plays an important role in the process of DNA replication and repair. DNA ligases are divided into two categories: one is ATP-dependent DNA ligases, which use the energy of ATP to catalyze the formation of phosphodiester bonds between two nucleotide chains; the other is dependent on nicotinamide adenine dinuclear NAD + ) DNA ligase that utilizes NAD + The energy catalyzes the formation of a phosphodiester bond between two nucleotide chains. [0003] PBCV-1 DNA Ligase (PBCV-1 DNA Ligase), also known as SplintR @ Ligase, or Chorellavirus DNA ligase, is an ATP-dependent DNA ligase that efficiently catalyzes the joining of two adjacent smaller DNA single stra...

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Problems solved by technology

However, the capillary electrophoresis instrument is expensive, not a common laboratory equipment, complex operation, high requirements for operators (due to the small amount of sample injection, thus the preparation ability is poor; due to the small diameter of the capillary, the optical path is too short, and some detection methods ( Such as UV absorption spectrometry), the sensitivity is low; electroosmosis will vary with the sample composition, thereby affecting the separation reproducibility)

Gel electrophoresis (Tong et al (2000), Ligation reaction specificities of an NAD+-dependent DNA ligase from the hyperthermophile Aquifex aeolicus. Nucleic Acids Res. ) and fluorescent dye staining method detection (Xu Xiaoyu et al., CN104278090B, a DNA ligase activity assay method, 2016); but gel electrophoresis pattern analysis has the disadvantages of cumbersome operation, long detection time, and inaccurate quantitative analysis.

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