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A Colorimetric Assay for Human 8-Oxoguanine DNA Glycosylase Activity

A technology for glycosylase activity and colorimetric analysis, which is applied in the direction of material analysis by observing the influence of chemical indicators, and analysis by making materials undergo chemical reactions, etc. Time and other problems, to achieve the effect of high sensitivity, strong practicability and simple process

Active Publication Date: 2016-08-24
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The present invention solves the deficiencies of the existing hOGG1 activity analysis methods such as cumbersome and time-consuming, complicated labeling process, high cost and poor practicability, and provides a simple, fast, economical and practical quantitative analysis method of hOGG1 activity

Method used

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  • A Colorimetric Assay for Human 8-Oxoguanine DNA Glycosylase Activity
  • A Colorimetric Assay for Human 8-Oxoguanine DNA Glycosylase Activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Preparation of graphene / gold nanoparticles

[0024] Graphene / gold nanoparticles were synthesized by a hydrothermal method. Mix 0.01~1g / L graphite oxide (GO) solution, 0.2~20.0mM chloroauric acid (HAuCl 4 ) solution and 0.002-0.2M sodium hydroxide (NaOH) solution were mixed evenly. After ultrasonication for 2-12 hours, transfer to a polytetrafluoro-based hydrothermal reaction kettle, react at 175-185°C for 8-12 hours, and cool naturally to room temperature for later use.

Embodiment 2

[0026] Determination of hOGG1 content in the configuration sample:

[0027] (1) Preparation of graphene / nano gold

[0028] Graphene / gold nanoparticles were synthesized by a hydrothermal method. 0.01g / LGO solution, 0.2mMHAuCl 4 solution, 0.004M NaOH solution mixed evenly. After ultrasonication for 2 hours, it was transferred to a hydrothermal reaction kettle with a polytetrafluoroethylene substrate, reacted at 175°C for 12 hours, and cooled naturally to room temperature for later use.

[0029] (2) Hybridization of S1 and S2

[0030] Add S2 at a concentration of 0.1 μM to 0.1 μM S1, react at 85°C for 2 min, and at 10°C for 1 min, so that the temperature is alternately cycled for 8 times.

[0031] (3) Analysis method

[0032] Add 0~0.2U / μL hOGG1, 0.01mg / mL BSA and 0.01M NaBH to the double-stranded DNA solution obtained in step (2) in sequence 3 CN, react at 35°C for 5h, and heat at 80°C for 10min to terminate the reaction. Then add 0.05U / μLExo I and 0.2U / μLExo III to the m...

Embodiment 3

[0041] Determination of hOGG1 content in the configuration sample:

[0042] (1) Preparation of graphene / nano gold

[0043] Graphene / gold nanoparticles were synthesized by a hydrothermal method. 0.1g / LGO solution, 2.0mMHAuCl 4 solution, 0.02M NaOH solution mixed evenly. After ultrasonication for 6 hours, it was transferred to a polytetrafluoro-based hydrothermal reactor, reacted at 180°C for 10 hours, and cooled naturally to room temperature for later use.

[0044] (2) Hybridization of S1 and S2

[0045] Add S2 at a concentration of 1.0 μM to 0.9 μM S1, react at a temperature of 95° C. for 1 min, and react at a temperature of 25° C. for 2 min, so that the temperature is alternately cycled for 10 times.

[0046] (3) Add 0~0.23U / μL hOGG1, 0.1mg / mL BSA and 0.1M NaBH to the double-stranded DNA solution obtained in step (2) in sequence 3 CN, react at 37°C for 2h, and heat at 90°C for 5min to terminate the reaction. Then add 0.3U / μLExo I and 1.5U / μLExo III to the mixture, let s...

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Abstract

The invention belongs to the biological field, and relates to a colorimetric analysis method for human 8-oxoguanine DNA glycosylase activity. Add 0.1-5.0 μM S2 to 0.1-5.0 μM S1, react at 85-100°C for 0.5-2min, 10-30°C for 1-5min, alternately react 8-15 times, then add hOGG1, BSA and NaBH 3 CN, react at 35-40°C for 0.5-5h, heat at 80-100°C for 1-10min. Add ExoI and ExoIII, let stand at 35-40°C for 10-60min, add graphene / nano-gold, and mix well. Add color developer and H in sequence 2 o 2 , for absorbance measurements. Compared with the traditional inorganic nanometer material analysis method, the colorimetric analysis method of the invention has lower cost, simple operation and strong practicability.

Description

technical field [0001] The invention belongs to the field of biology and relates to a colorimetric analysis method for human 8-oxoguanine DNA glycosylase activity. Background technique [0002] Among genes, 8-oxoguanine is one of the most common DNA oxidative damages due to reactive oxygen species. During DNA replication, this damage can prevent the DNA chain from elongating, resulting in gene mutations caused by mismatches, posing a huge threat to gene stability. Because of its high content and strong mutagenic ability in the body, it has now been regarded as a marker of DNA oxidative damage in the body. [0003] Human 8-oxoguanine glycosylase (hOGG1) is an important repair enzyme in the repair of damaged bases in the human body, which can recognize and excise 8-oxoguanine . In addition, hOGG1 also has the hydrolysis function of the AP site of AP (apurinic / apyrimidinic) endonuclease. Under the action of this enzyme, the site of the damaged base can be completely excised ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/78C12Q1/68
Inventor 赵慧敏袁芳刘猛全燮张耀斌
Owner DALIAN UNIV OF TECH
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