Quantitative detection method of T4 polynucleotide kinase

Abstract

The invention discloses a quantitative detection method of T4 polynucleotide kinase. The quantitative detection method comprises the steps of: designing a sulfhydrylated single-stranded DNA probe a, aprobe b and a probe c of which a 5'end is modified with amino, and fixing the probe a on the surface of a gold electrode through a gold-sulfhydryl bond, wherein the hydroxyl at the 5'end of the probea can be catalyzed and phosphorylated by T4 polynucleotide kinase, the DNA probe a is then hybridized with the probe b and the probe c, and the 3 'end of the probe c and a phosphate group at the 5' end of the probe a are subjected to a ligation reaction under the action of T4 DNA ligase; performing heating denaturating treatment, wherein the probe c is still retained on the surface of the electrode, the amino group at the tail end of the probe c can further capture the silver nanoparticles, and finally, the quantitative detection result of the T4 polynucleotide kinase is obtained by detectingthe stripping volt-ampere signal of the silver nanoparticles. The method has the characteristics of the high sensitivity, the good selectivity, the rapidness and the low cost, and the detection limitis 0.01 U / mL.

Description

Technical field [0001] The present invention relates to the field of T4 polynucleotide kinase detection, in particular to a quantitative detection method of T4 polynucleotide kinase. Background technique [0002] Kinases are a class of enzymes that catalyze the transfer of phosphate groups in high-energy donor molecules to specific target substrates. These enzymes are closely related to normal cellular physiological activities such as DNA replication, DNA recombination, and DNA repair. The abnormal expression of kinases can cause various human diseases such as Brun's syndrome, Werner syndrome, and pigmentation syndrome. T4 polynucleotide kinase is an important member of the kinase family, which can catalyze the transfer of the γ-position phosphate of ATP to the 5'hydroxyl end of the oligonucleotide chain or nucleic acid. T4 polynucleotide kinase plays an important role in intracellular DNA metabolism, especially in the repair of DNA damage. Therefore, the development of T4 poly...

AI Technical Summary

Problems solved by technology

However, these methods are cumbersome and time-consuming and require special equipment, or have low selectivity and sensitivity, or require isotope labeling, which is harmful to the human body, thus limiting the wide application of these methods

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