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Method for detecting thymine DNA glycosylase activity based on cyclophorase-remediation mediated double-signal amplification strategy

A dual-signal amplification, thymine technology, applied in the field of biological analysis, can solve problems such as limited sensitivity improvement

Active Publication Date: 2017-08-01
SHANDONG NORMAL UNIV
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  • Summary
  • 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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  • Method for detecting thymine DNA glycosylase activity based on cyclophorase-remediation mediated double-signal amplification strategy
  • Method for detecting thymine DNA glycosylase activity based on cyclophorase-remediation mediated double-signal amplification strategy
  • Method for detecting thymine DNA glycosylase activity based on cyclophorase-remediation mediated double-signal amplification strategy

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Experimental program
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Effect test

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 Denaturation at 95 degrees Celsius for 5 minutes. Then add 2 microliters of ligation probe and linear lock probe to 20 microliters of ligation buffer, including 1×T4 ligase buffer (6.6 millimoles per liter of magnesium chloride, 10 millimoles per liter of magnesium chloride). Dithiothreitol, 0.1 millimole per liter of adenosine triphosphate, 66 millimole per liter of tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) (pH 7.6)), 50 units of T4DNA ligase, in Incubate overnight at 16 degrees Celsius. After the ligation reaction, transfer 10 microliters of the ligation product to 10 microliters of digestion buffer, including 1 mmol per liter of dithiothreitol, 6.7 mmol per liter of magnesium chloride, and 67 mmol per liter of glycine -Potassium hydroxide ...

Embodiment 2

[0063] Cell lysis buffer preparation: 10 millimoles per liter of tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) (pH 8.0), 150 millimoles per liter of sodium chloride, 1% (mass / volume) Ethyl phenyl polyethylene glycol (NP-40), 0.25 millimoles per liter of sodium deoxycholate, 1% (mass / volume) glycerol, 0.1 millimoles per liter of 4-(2-aminoethyl) ) Benzenesulfonyl fluoride hydrochloride.

[0064] Cell extract preparation: The culture medium for human cervical cancer cells (HeLa) and human breast cancer cells (MCF-7) is Dulbec's modified Eagle with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin DMEM, placed in an incubator containing 5% carbon dioxide and 37 degrees Celsius for cultivation. When the cells grow to the logarithmic growth phase, they are digested with trypsin, and ice phosphate buffer (137 mmol sodium chloride solution, 2.7 mmol potassium chloride solution, 10 mmol phosphate buffer, pH 7.4) Wash twice, then centrifuge at 800 rpm at 4°C. T...

Embodiment 3

[0070] 3.1 Experimental verification of the principle

[0071] In order to verify the feasibility of this scheme, we tested and analyzed the reaction products, using 1% agarose gel electrophoresis with SYBR Gold as an indicator for verification and analysis. From figure 2 (A) It can be seen that when thymine DNA glycosylase (TDG) is present, characteristic bands of the reaction product can be seen, and when thymine DNA glycosylase (TDG) is not added, no characteristic bands appear. This is because thymine DNA glycosylase (TDG) can excise mismatched thymines and initiate subsequent uracil excision-mediated circular rolling circle exponential amplification. In order to prove the double-signal amplification reaction mediated by enzyme repair, we added a signal probe to detect the reaction process in real time with fluorescence. Such as figure 2 As shown in (B), in the presence of thymine DNA glycosylase (TDG), the fluorescence intensity increased rapidly 10 minutes before the rea...

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

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/34
CPCC12Q1/6844C12Q2521/301C12Q2521/531C12Q2531/125C12Q2563/107
Inventor 张春阳王黎娟王子月
Owner SHANDONG NORMAL UNIV
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