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A method for the ultrasensitive simultaneous detection of multiple DNA glycosylases using intrinsically fluorescent nucleotides

A glycosylase and nucleotide sequence technology, applied in the field of DNA glycosylase detection, can solve the problems of complex design and high cost, and achieve good specificity, high sensitivity, and high sensitivity

Active Publication Date: 2020-05-05
SHANDONG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, fluorescent methods rely on external labeling with fluorophores and quenchers for homogeneous assays, involving complex design and cost
Furthermore, currently reported fluorescent methods can only detect a single type of DNA glycosylase

Method used

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  • A method for the ultrasensitive simultaneous detection of multiple DNA glycosylases using intrinsically fluorescent nucleotides
  • A method for the ultrasensitive simultaneous detection of multiple DNA glycosylases using intrinsically fluorescent nucleotides
  • A method for the ultrasensitive simultaneous detection of multiple DNA glycosylases using intrinsically fluorescent nucleotides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1: Detection of HOGG1 and UDG

[0070] (1) Preparation of DNA stock solution: add 10 micromoles per liter of bifunctional DNA probe, 10 micromoles per liter of trigger probe 1 and 10 micromoles per liter of trigger probe 2 into a solution containing 50 millimoles per liter Sodium chloride (NaCl), 10 mmol per liter of Tris-HCl (pH 8.0) and 1 mmol per liter of ethylenediaminetetraacetic acid (EDTA) were incubated at 95°C for 5 minutes, then slowly cooled to room temperature to form a sandwich structure. 10 micromoles per liter of the 2-AP signal probe and 10 micromoles per liter of the P-dC signal probe were contained in 1.5 millimoles per liter of magnesium chloride (MgCl 2 ) and 10 millimolar Tris-HCl (pH 8.0) buffer, incubated at 95°C for 5 minutes, and then slowly cooled to room temperature to fold the 2-AP signal probe and the P-dC signal probe to form a hairpin structure . The obtained DNA stock solution was stored at -20°C for future use.

[0071] (2) F...

Embodiment 2

[0072] Embodiment 2: actual sample analysis

[0073] Human cervical cancer cells (HeLa) were used to detect hOGG1 and UDG.

[0074] 1. Test method:

[0075] (1) Cell culture and preparation of cell extracts: human cervical cancer cell line (HeLa) was placed in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin , at 37°C with 5% CO 2 cultivated in a moist atmosphere. Cell numbers were counted by a Countstar cell counter. Cell extracts were extracted using a nuclear extraction kit (ActiveMotif) according to the instructions.

[0076] (2) The hOGG1 and UDG in the cell extract were detected according to the method in Example 1.

[0077] 2. Test results:

[0078] For hOGG1, the normalized fluorescence intensity increases with the number of HeLa cells, linearly in the range of 5 to 10,000 cells ( Figure 7 A). The regression equation is F=0.3803+0.1473 log 10 N, the correlation coefficient is 0.9855, where F is the...

Embodiment 3

[0079] Example 3: Analysis of DNA Glycosylase Inhibitors

[0080] We chose 5-fluorouracil (5-FU) as the inhibitor for research. Some chemicals such as 5-FU and gentamicin can interact with DNA glycosylase and affect its activity. Among them, 5-FU is the most widely used chemotherapeutic drug for the treatment of various tumors.

[0081] Different concentrations of 5-fluorouracil (5-FU) were incubated with sandwich hybridization DNA substrate at 25°C for 15 minutes, then hOGG1 and UDG were added and incubated at 37°C for 60 minutes. Subsequent reaction procedures and fluorescence measurements followed the steps described above. According to the formula:

[0082]

[0083] To calculate the relative activity (RA) of DNA glycosylase. where F 0 is the fluorescence intensity in the absence of hOGG1 or UDG, F t is the fluorescence intensity in the presence of 32U per milliliter of hOGG1 or 50U per milliliter of UDG, and F i is the fluorescence intensity in the presence of hO...

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Abstract

The invention discloses a method for ultra-sensitively simultaneously detecting multiple DNA glycosylases by using intrinsic fluorescent nucleotide. The method is a fast and sensitive fluorescence method for simultaneously detecting multiple DNA glycosylases by adopting 2-aminopurine and pyrrole-deoxycytidine as fluorophores and a DNA molecule as an intrinsic quenching agent and combining excision enzyme-assisted circulating signal amplification; and extra fluorophore and quenching group are not needed for marking, so that the target of simple, intuitive and high-sensitivity detection of an actual sample is achieved, and above all, simultaneous detection of multiple DNA glycosylases is achieved.

Description

technical field [0001] The invention relates to the technical field of DNA glycosylase detection, in particular to a method for supersensitively detecting multiple DNA glycosylases simultaneously by using inherent fluorescent nucleotides. Background technique [0002] Endogenous DNA damage is the result of various internal and environmental factors, which can cause DNA mutations and replication errors, which further lead to cancer. Base excision repair (BER) is the main repair pathway to deal with endogenous DNA base damage caused by oxidation, alkylation and deamination. The BER pathway is initiated by DNA glycosylases, which catalyze the cleavage of damaged / mismatched bases and generate apurinic / apyrimidinic sites that act in the downstream BER repair process. [0003] The abnormal expression of DNA glycosylase is closely related to human diseases, and the accurate detection of DNA glycosylase is of great significance for understanding the process of DNA damage repair and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/682C12Q1/34C12N15/11
CPCC12Q1/34C12Q1/682C12Q2521/301C12Q2521/319C12Q2563/107
Inventor 张春阳唐波张艳李琛琛
Owner SHANDONG NORMAL UNIV
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