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Quantitative detection method of t4 polynucleotide kinase

A quantitative detection method and polynucleotide technology, applied in the field of T4 polynucleotide kinase detection, can solve the problems of low selectivity and sensitivity, cumbersome and time-consuming experimental steps, harmful isotope labeling, etc., and achieve selectivity Good, high sensitivity, fast cost effect

Active Publication Date: 2022-03-08
SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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 sensitivity, or require isotope labeling, which is harmful to the human body, thus limiting the wide application of these methods

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 probe A sequence is: 5'-gcaagaatttcgacatgcgTG-SH-3 ';

[0029] The probe B sequence is: 5'-caccccatgtcgaAattctTgcgTGCCTAT-3 ';

[0030] Probe C sequence is: 5'-NH 2 -Tttataggcac-3 '.

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

[0032] 3) Modify the probe A in the surface of the gold electrode

[0033]Gold electrode pretreatment: soak the gold electrode in the tiger fish solut...

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Abstract

The invention discloses a quantitative detection method for T4 polynucleotide kinase, which comprises the steps of: first designing a sulfhydrylated single-stranded DNA probe a, a probe b, and a probe c modified with an amino group at the 5' end, and the probe a is passed through The gold-sulfhydryl bond is immobilized on the surface of the gold electrode; the hydroxyl group at the 5' end of probe a can be catalyzed by T4 polynucleotide kinase to phosphorylate, DNA probe a then hybridizes with probe b and probe c, and the 3 of probe c The 'end and the phosphate group at the 5' end of probe a undergo a ligation reaction under the action of T4 DNA ligase; after heat denaturation treatment, probe c remains on the electrode surface, and the amino group at the end of probe c can further capture silver nanoparticles particles, and finally obtain the quantitative detection result of T4 polynucleotide kinase by detecting the stripping voltammetry signal of silver nanoparticles. The invention has the characteristics of high sensitivity, good selectivity, rapidity and low cost, and the detection limit is 0.01U / mL.

Description

Technical field [0001] The present invention relates to the field of T4 polynucleotide kinase detection, and more particularly to a quantitative detection method of T4 polynucleotide kinase. Background technique [0002] Kinase is a class of enzymes that transfer the phosphate groups in high energy donor molecules to a particular target substrate, which is closely related to DNA replication, DNA recombinant, DNA repair and other normal cell physiological activities. The abnormal expression of kinase can lead to a variety of human diseases such as Brenian syndrome, Walner syndrome, pigmentation syndrome. T4 polynucleotide kinase is an important member of the kinase family, which can catalyze the gamma phosphoric acid 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 DNA injury repair. Therefore, the detection technique for developing T4 polynucleotide kinase helps...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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