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Method for detecting uracil-DNA glycosylase activity by using fluorescent probe

A technology of glycosylase activity and fluorescent probe, which is applied in the field of fluorescent probe detection of uracil-DNA glycosylase activity, can solve problems such as incorrectness and high risk of cross-contamination, and achieve high sensitivity

Pending Publication Date: 2021-05-07
诸暨加向生物科技有限公司
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Problems solved by technology

However, these methods are incorrect in assessing the activity of actual enzyme units and carry a high risk of cross-contamination when used in PCR laboratories

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  • Method for detecting uracil-DNA glycosylase activity by using fluorescent probe

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Embodiment Construction

[0022] like figure 1 A method for detecting uracil-DNA glycosylase activity with a fluorescent probe is shown, the method is to mix a polymerase chain reaction buffer solution and a fluorescent resonance energy transfer based method in a real-time polymerase chain reaction instrument Fluorescent DNA probe, uracil-DNA-glycosylase; take 10 µl of polymerase chain reaction buffer.

[0023] The reaction solution is incubated at 25°C-37°C for 30 minutes, at this time, the fluorescent DNA probe nucleic acid-labeled fluorescent dye based on fluorescence resonance energy transfer is excised. Collect pyrimidine-DNA glycosylase activity data (that is, Rn fluorescence value data) in the fluorescence measurement channel at a wavelength of 520 nm every 10 seconds, and then heat at 95°C for 2 minutes to inactivate uracil-DNA-glycosylase. Incubate at 25°C-37°C for 10 minutes to degrade fluorescent DNA probes based on fluorescence resonance energy transfer. Collect pyrimidine-DNA glycosylas...

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Abstract

The invention discloses a method for detecting uracil-DNA glycosylase activity by using a fluorescent probe. The method comprises the following steps of mixing a polymerase chain reaction buffer solution, the fluorescent DNA probe based on fluorescence resonance energy transfer and uracil-DNA-glycosylase in a real-time polymerase chain reaction instrument, culturing the reaction solution at 25-37 DEG C for 30 minutes, then heating at 95 DEG C for 2 minutes, and finally, culturing for 10 minutes at the temperature of 25-37 DEG C, and collecting pyrimidine-DNA glycosylase activity data at the position with the wavelength of 520 nm in a fluorescence measurement channel of the polymerase chain reaction instrument, wherein the fluorescent DNA probe based on fluorescence resonance energy transfer is FAM-GCCAACCdUGGCTCT-BHQ1, FAM is a fluorescence reporter group, and BHQ1 is a fluorescence quenching group; and the polymerase chain reaction buffer solution is prepared from the following components of Tris-HCl, KCl and MgCl2. The method has the technical advantages of simplicity and high efficiency.

Description

technical field [0001] The invention relates to a method for detecting uracil-DNA glycosylase activity with a fluorescent probe. Background technique [0002] Uracil-DNA-glycosylase is a key enzyme in the base excision repair process, which can remove uracil residues introduced into DNA by mismatch nucleic acid-labeled fluorescent dyes or cytosine deamination. Uracil-DNA-glycosylase hydrolyzes uracil glycosidic bonds in single- or double-stranded DNA, excising uracil and creating an abasic site in the DNA strand. These abasic sites can be hydrolyzed by endonucleases, heat or alkaline treatment. Due to its properties, uracil-DNA-glycosylase acts as the enzyme of choice to prevent carryover contamination in the polymerase chain reaction (PCR). To date, most methods for measuring uracil-DNA-glycosylase activity use radioisotope-labeled compounds, which are not suitable for establishing good quality control in general PCR laboratories. [0003] However, the main concern is to...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/686
CPCC12Q1/686
Inventor 乌拉迪斯拉夫
Owner 诸暨加向生物科技有限公司
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