Quantitative detection method of T4 polynucleotide kinase

A quantitative detection method and polynucleotide technology, which are applied in measurement devices, material analysis by electromagnetic means, instruments, etc., can solve the problems of cumbersome and time-consuming experimental steps, harmful isotope labeling, low selectivity and sensitivity, etc. Achieve high sensitivity, fast cost and good selectivity

Active Publication Date: 2019-11-22
SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI
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Problems solved by technology

However, these methods are cumbersome and time-consuming and require special equipment, or have low selectivity and sensit

Method used

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  • Quantitative detection method of T4 polynucleotide kinase
  • Quantitative detection method of T4 polynucleotide kinase
  • Quantitative detection method of T4 polynucleotide kinase

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Embodiment 1

[0027] 1) DNA probe design:

[0028] The sequence of probe a is: 5'-GCAAGAATTTCGACATGGCGTG-SH-3';

[0029] The sequence of probe b is: 5'-CACGCCATGTCGAAATTCTTGCGTGCCTAT-3';

[0030] Probe c sequence: 5’-NH 2 -TTTATAGGCAC-3’.

[0031] 2) Prepare silver nanoparticles with a diameter of about 5 nm: first prepare a silver nitrate solution and sodium citrate solution with a concentration of 0.25 mM; then prepare a sodium borohydride solution with a concentration of 10 mM; then combine 100 mL of silver nitrate / sodium citrate solution with 3 mL The sodium borohydride solution was mixed, and the reaction was stirred at room temperature; after 30 minutes, the stirring was stopped, and the mixed reaction solution was placed in the dark overnight; the yellow silver nanoparticles were purified by centrifugation at a rotation speed of 12000 g and a time of 30 minutes.

[0032] 3) Modify probe a on the surface of the gold electrode

[0033] Gold electrode pretreatment: soak the gold electrode in pira...

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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...

Claims

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Application Information

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IPC IPC(8): G01N27/327
CPCG01N27/3278G01N27/3277
Inventor 缪鹏柴华杨大威陈锡峰
Owner SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI
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