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A method for the detection of thymidine-DNA glycosylase activity based on a dual-signal amplification strategy mediated by cyclic enzyme repair

A double-signal amplification and thymine technology, applied in the field of biological analysis, can solve the problem of limited sensitivity improvement

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

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Problems solved by technology

Usually in fluorescence analysis, a pair of fluorescent and quencher molecules are designed that are very close to each other. When thymine DNA glycosylase (TDG) exists, the quencher molecule is released, causing the fluorescent molecule to fluoresce, and finally through the detection of fluorescence Quantitative analysis of the signal to the enzyme activity; in the electrochemical analysis method, it has been reported that the three-step signal amplification of the target detection substance can be achieved with the assistance of the DNA-quantum dots (QDs) polymer superlattice structure, but the above methods are all There is a problem that the sensitivity improvement is limited

Method used

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  • A method for the detection of thymidine-DNA glycosylase activity based on a dual-signal amplification strategy mediated by cyclic enzyme repair
  • A method for the detection of thymidine-DNA glycosylase activity based on a dual-signal amplification strategy mediated by cyclic enzyme repair
  • A method for the detection of thymidine-DNA glycosylase activity based on a dual-signal amplification strategy mediated by cyclic enzyme repair

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Embodiment 1

[0061] Preparation of circular template DNA: Dilute the ligation probe and linear padlock probe with 1× Tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetic acid (Tris-EDTA) buffer to 10 micromoles per liter, and in Denature at 95°C for 5 minutes. Then 2 microliters of ligation probe and linear padlock probe were added to 20 microliters of ligation buffer, including 1×T4 ligase buffer (6.6 mmol per liter of magnesium chloride, 10 mmol per liter of Dithiothreitol, 0.1 mmol per liter of adenosine triphosphate, 66 mmol per liter of tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) (pH 7.6)), 50 units of T4DNA ligase, in Incubate overnight at 16°C. After the ligation reaction, transfer 10 µl of the ligation product to 10 µl of digestion buffer, including 1 mmol / L of dithiothreitol, 6.7 mmol / L of MgCl, 67 mmol / L of glycine - Potassium hydroxide (pH 9.5), 10 units of exonuclease I and 20 units of exonuclease III, incubated at 37°C for 2 hours, then inactivated at ...

Embodiment 2

[0063] Cell lysis buffer preparation: 10 mmol per liter of tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) (pH8.0), 150 mmol per liter of sodium chloride, 1% (mass / volume) Ethylphenyl polyethylene glycol (NP-40), 0.25 mmol per liter of sodium deoxycholate, 1% (mass / volume) glycerol, 0.1 mmol per liter of 4-(2-aminoethyl ) Benzenesulfonyl fluoride hydrochloride.

[0064] Cell extract preparation: Human cervical cancer cell (HeLa) and human breast cancer cell (MCF-7) culture medium is Dulbecco's modified Eagle's-Egg with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin Dill medium (DMEM), placed in an incubator containing 5% carbon dioxide, 37 degrees Celsius for cultivation. When the cells grow to the logarithmic growth phase, they are digested with trypsin and washed with ice phosphate buffer (137 mmol sodium chloride solution, 2.7 mmol potassium chloride solution, 10 mmol phosphate buffer saline, pH 7.4) Wash twice, then centrifuge at 4°C and 800 rpm...

Embodiment 3

[0070] 3.1 Experimental verification of the principle

[0071] In order to verify the feasibility of this scheme, we detected and analyzed the reaction products, and used SYBR Gold as an indicator for 1% agarose gel electrophoresis for verification analysis. From figure 2 (A) It can be seen that when there is thymine DNA glycosylase (TDG), the characteristic band of the reaction product can be seen, and when no thymine DNA glycosylase (TDG) is added, no characteristic band appears. This is because thymine DNA glycosylase (TDG) excises mismatched thymines and initiates subsequent uracil excision-mediated circular rolling circle exponential amplification. In order to prove the dual signal amplification reaction mediated by enzyme repair, we added the signal probe and detected the reaction process with real-time fluorescence. Such as figure 2 As shown in (B), in the presence of thymine DNA glycosylase (TDG), the fluorescence intensity increased rapidly in the first 10 minute...

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Abstract

The invention discloses a method for detecting thymine DNA glycosylase activity based on a cyclophorase-remediation mediated double-signal amplification strategy. A fluorescent method for detecting the thymine DNA glycosylase activity in real time can be achieved through circular rolling circle index amplification triggered by thymine DNA glycosylase and assisted by enzyme remediation and circular cutting catalyzed and mediated by endonuclease IV. As efficiency of circular rolling circle amplification assisted by uracil excision and efficiency of circular cutting based on endonuclease IV are both high and index amplification mediated by uracil can greatly inhibit non-specific amplification, the method has very high detection flexibility; detection shows that lower detection limit of the method to thymine DNA glycosylase is 5.6*10<-7>U / mu L; meanwhile, the method disclosed by the invention utilizes self repair characteristics of the thymine DNA glycosylase and uracil DNA glycosylase to enable the whole repairing reaction to be performed strictly according to a natural repairing mechanism, so that the method disclosed by the scheme of the invention has very high specificity.

Description

technical field [0001] The invention belongs to the technical field of biological analysis, and in particular relates to a method for detecting the activity of thymine DNA glycosylase based on a double-signal amplification strategy mediated by cyclic enzyme repair. Background technique [0002] Thymine DNA glycosylase (TDG) is a very important initiation enzyme in the base excision repair (BER) pathway in vivo. In damaged genomic DNA, it can specifically recognize mismatched base pairs of guanine / thymine (G / T) and guanine / uracil (G / U), and excise the mismatched base thymine ( T) and uracil (U). Thymine DNA glycosylase (TDG) also plays an important role in the process of DNA demethylation. Through the deamination-base excision repair reaction, thymine DNA glycosylase (TDG) can efficiently remove 5-methylcytosine (5mC) oxidized by ten-eleven translocation (TETs) enzymes The resulting 5-formylcytosine (5fC) and 5-carboxycytosine (5caC). In addition, thymine DNA glycosylase ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6844C12Q1/34
CPCC12Q1/6844C12Q2521/301C12Q2521/531C12Q2531/125C12Q2563/107
Inventor 张春阳王黎娟王子月
Owner SHANDONG NORMAL UNIV
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