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Method for detecting UDG (Uracil Dna Glycosylase) activity based on sticky end-mediated strand displacement reaction combined with polymerization incising isothermal amplification technology

A sticky end and reaction technology, which is applied in recombinant DNA technology, biochemical equipment and methods, microbial measurement/testing, etc., can solve the problems of limited sensitivity, weakened response, difficulty in distinguishing a small amount of mismatches, etc.

Active Publication Date: 2016-12-21
SHANDONG UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

However, when the low-activity UDG can only remove 1 or 2 U bases in the probe to generate a small number of apurinic / apyrimidinic site (AP) sites, since AP sites are also a type of mismatch , and the direct hybridization mode is difficult to distinguish a small number of mismatches, so the signal probe or primer sequence is still blocked in the double-stranded DNA at this time, resulting in weakened response and limiting the improvement of sensitivity

Method used

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  • Method for detecting UDG (Uracil Dna Glycosylase) activity based on sticky end-mediated strand displacement reaction combined with polymerization incising isothermal amplification technology
  • Method for detecting UDG (Uracil Dna Glycosylase) activity based on sticky end-mediated strand displacement reaction combined with polymerization incising isothermal amplification technology
  • Method for detecting UDG (Uracil Dna Glycosylase) activity based on sticky end-mediated strand displacement reaction combined with polymerization incising isothermal amplification technology

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1 detects the activity of UDG

[0040] In order to obtain the corresponding hairpin structure, the hairpin probe TP containing sticky ends and the signal reporter probe RP were heated and denatured at 90 °C for 5 min, and the products were placed at 4 °C after the temperature was slowly lowered to room temperature. Save it for later use. Add 100nM-500nM recognition probe UP and different concentrations of UDG to 1×ThermoPol buffer solution (20mM Tris-HCl, pH 8.8, 10mM KCl, 10mM (NH 4 ) 2 SO 4 , 2mM MgSO 4 , 0.1% TritonX-100) to obtain a mixed solution with a final volume of 20 μL, and the solution was mixed and incubated at 37° C. for 20 min-60 min to complete the recognition reaction of UDG. Next, 150nM-1.80μM hairpin probe TP and 1.0μL 10×ThermoPol buffer solution (200mM Tris-HCl, pH 8.8, 100mM KCl, 100mM (NH4) 2 SO 4 , 20 mM MgSO 4 , 1% TritonX-100), to obtain a mixed solution with a final volume of 30 μL, and place the solution at 37° C. for 30 min...

Embodiment 2

[0041] Embodiment 2 The inventive method detects the UDG activity in the Hela cell lysate

[0042] Preparation of Hela cell lysate: Hela cell samples were pelleted by centrifugation (5min, 3000rpm, 4°C) and redispersed in lysis buffer (10mM Tris-Hcl, pH 7.0) by sonication on ice middle. The mixed solution was then centrifuged at 12,000 rpm for 30 min at 4°C to remove insoluble substances. The supernatant was collected and filtered through a 0.45 μm filter to generate a crude HeLa cell lysate.

[0043] 1.0 μL of HeLa cell lysate was used as the sample to be tested, wherein the concentration of the HeLa cell lysate was 1.0×10 per 1.0 μL of cell lysate 4 cells. Such as Figure 5 As shown, the buffer solution can only cause a very low fluorescence signal, while the HeLa cell lysate can cause a significantly enhanced fluorescence signal. In addition, when the UDG inhibitor UGI was added to the system containing HeLa cell lysate, the fluorescence intensity of the sensing system w...

Embodiment 3

[0044] Example 3 Feasibility verification of detecting UDG activity

[0045] The feasibility of this method for detecting UDG activity was verified by fluorescence emission spectroscopy. Such as figure 2 As shown in curve d in , when the signal reporter probe RP is not included in the sensing system, the sensing system exhibits a very low fluorescence signal. This indicates that the generation of fluorescent signal is inseparable from the presence of probe RP. Such as figure 2 As shown in curve c in , when the recognition probe UP is not included in the system, the fluorescence signal of the sensing system is also very low. This indicates that in this method, the recognition of UDG by the sensing system is inseparable from the participation of the UP probe. Such as figure 2 As shown in curve b in , the sensing system still exhibits a very low fluorescence signal when UDG is absent. This is because the probe UP can generate TSDR with the hairpin probe TP, so that the p...

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Abstract

The invention discloses a method for detecting UDG (Uracil Dna Glycosylase) activity based on sticky end-mediated strand displacement reaction combined with polymerization incising isothermal amplification technology. When UDG exists, two U basic groups in a single-stranded DNA probe are removed, and the single-stranded DNA probe containing AP mispairing is obtained. On the basis of a specific recognition capability of TSDR on a few mispairing, the single-stranded DNA probe containing AP mispairing and a hairpin probe containing a sticky end cannot be subjected to TSDR, so that a primer sequence in the single-stranded DNA probe can still exist freely. Later, the primer sequence is hybridized with a signal report probe, so that subsequent polymerization incising isothermal amplification reaction is triggered, the sensitive response to the removal of a few U basic groups is realized, and the sensitivity in detecting the UDG activity is improved. However, when the UDG does not exist, the single-stranded DNA probe containing AP mispairing and the hairpin probe containing the sticky end can be subjected to TSDR, and the primer sequence is closed, so that the subsequent isothermal amplification reaction cannot be triggered, and a negative signal is reduced. The method can be used for detecting the UDG activity as low as 0.000027U / mL.

Description

technical field [0001] The invention relates to a method for detecting UDG activity based on sticky end-mediated strand displacement reaction combined with polymerization nick isothermal amplification technology. Background technique [0002] Uracil-DNA glycosylase (UDG) can specifically recognize and remove uracil (U) damage in DNA, thereby triggering the base removal repair process of U damage (Stivers, J.T.; Jiang, Y.L., A mechanistic perspective on the chemistry of DNA repair glycosylases. Chem. Rev., 2003, 103, 2729-2759; Parikh, S.S.; Walcher, G.; Jones, G.D.; Slupphaug, G.; Krokan, H.E.; Blackburn, G.M.; Tainer , J.A., Uracil-DNA glycosylase-DNA substrate and product structures: conformational strain promotes catalytic efficiency by coupledstereoelectronic effects. PNAS., 2000, 97, 5083-5088.). In the human body, hundreds of U damages appear in the genome of each cell every day, part of which comes from the spontaneous hydrolytic deamination of cytosine (C), and the ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/34C12N15/11
CPCC12Q1/6844C12Q2531/119C12Q2521/531C12Q2521/307C12Q2525/301
Inventor 姜玮王磊吴玉姝
Owner SHANDONG UNIV
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