Activity detection method of formamidopyrimidine-DNA (Deoxyribose Nucleic Acid) glycosylase

Abstract

The invention discloses an activity detection method of formamide pyrimidine-DNA (deoxyribonucleic acid) glycosylase, which comprises the following steps: providing a double-chain probe which comprises two complementary single-chain probes containing 8-oxoG (8-oxoG) modification sites; uniformly mixing the Fpg enzyme, the double-stranded probe, a buffer solution and water to form a mixed system; carrying out PCR (Polymerase Chain Reaction) on the mixed system, and cutting the double-stranded probe containing the 8-oxoG modification site by using Fpg enzyme to obtain a longer and shorter fragment sample; performing electrophoresis on the fragment sample by using non-denaturing polyacrylamide gel; the non-denaturing polyacrylamide gel obtained after electrophoresis is completed is subjected to vibration dyeing, and a dyeing result is photographed so as to detect the activity of the Fpg enzyme. Compared with the prior art, the method is relatively simple and easy to operate, the reagent cost is relatively low, and the experimental result is sensitive and easy to observe; no radioactive and cancerogenic substance pollution exists, and the method is environment-friendly.

Description

Technical Field

[0001] This invention belongs to the field of biochemical analysis technology, specifically relating to a method for detecting the activity of formamide pyrimidine-DNA glycosylation enzyme. Background Technology

[0002] DNA damage repair is crucial for maintaining the genome of living cells. DNA undergoes numerous reactions, such as base deamination, oxidation, and alkylation, the products of which are often cytotoxic or mutagenic. To mitigate the harmful effects of this base damage, organisms have evolved various repair processes specific to different types of DNA damage. As a key enzyme in the base excision repair pathway, formamidopyrimidine-DNA glycosylase (Fpg) primarily eliminates the damage caused by oxidative damage to cells by detecting oxidative damage in DNA, particularly oxidative damage to formamidopyrimidine residues, and specifically recognizing and cleaving Fapy-G, Fapy-A, and 8-oxoG (8-oxoguanine) residues in DNA.

[0003] Traditional methods for detecting formamide pyrimidine-DNA glycosylation enzymes mainly include gel electrophoresis, radiolabeling, high-performance liquid chromatography (HPLC), and mass spectrometry (MS). Among these, gel electrophoresis is commonly used for the detection of formamide pyrimidine-DNA glycosylation enzymes due to its relatively simple operation. Agarose gel electrophoresis is generally chosen for routine gel electrophoresis; however, agarose gel electrophoresis is generally used for the detection of large nucleic acids, and its resolution is relatively low when used for the detection of small nucleic acids. Summary of the Invention

[0004] This invention discloses a method for detecting the activity of formamide pyrimidine-DNA glycosylation enzyme, in order to solve the problem of low resolution in the prior art.

[0005] This invention provides a method for detecting the activity of formamide pyrimidine-DNA glycosylation enzyme, comprising:

[0006] A double-stranded probe is provided, the double-stranded probe comprising two complementary single-stranded probes containing 8-oxoG modification sites;

[0007] Mix Fpg enzyme, double-stranded probe, buffer solution and water thoroughly to form a mixed system;

[0008] The above mixture was subjected to PCR reaction. The Fpg enzyme cleaved the double-stranded probe containing the 8-oxoG modification site to obtain a longer and a shorter fragment sample.

[0009] Fragment samples were electrophoresed using non-denaturing polyacrylamide gel;

[0010] After electrophoresis, the non-denaturing polyacrylamide gel was stained by shaking, and the staining results were photographed to detect the activity of Fpg enzyme.

[0011] Optionally, the concentration of the non-denaturing polyacrylamide gel is selected from 20%, 15%, and 12%.

[0012] Optionally, the complementary base of the double-stranded probe has a length of 42 bp.

[0013] Optionally, the two single-stranded probe sequences of the double-stranded probe are as follows:

[0014] P: 5-CATCTCTTCTCCGAGCCGGTCGAAATAGTTGGT / oxoG / AAGAGATG-3;

[0015] PR: 5-CATCTCTT / oxoG / ACCAACTATTTCGACCGGCTCGGAGAAGAGATG-3;

[0016] Here, oxoG represents damaged 8-oxoguanine.

[0017] Optionally, the concentration of the non-denaturing polyacrylamide gel is 15%.

[0018] Optionally, the buffer solution is an NEB buffer solution.

[0019] Optionally, the PCR reaction conditions are 37℃ for 30 min and 65℃ for 5 min.

[0020] Optionally, the dye used in the staining process is SuperRed nucleic acid dye.

[0021] Compared with existing technologies, the advantages of this invention are: relatively simple and easy to operate, low reagent cost, and sensitive and easy-to-observe experimental results; no radioactive or carcinogenic pollution, and environmentally friendly. Attached Figure Description

[0022] Figure 1 This is a schematic diagram illustrating the principle of the activity detection method of the present invention;

[0023] Figure 2 This is a diagram showing the results obtained by the detection method of the present invention. Detailed Implementation

[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Example 1

[0026] Example 1 of this application provides a method for non-denaturing polyacrylamide gel electrophoresis separation, comprising the following steps:

[0027] (1) Preparation of acrylamide solution: Weigh 29g of acrylamide and 1g of N,N-methylenebisacrylamide. Add these substances to 60ml of deionized water and heat to 37℃ to promote dissolution. After dissolution, add deionized water to a total volume of 100ml. Filter the solution through a 0.22um filter membrane to obtain a 30% acrylamide stock solution, which should be stored in a brown bottle at 4℃.

[0028] (2) Preparation of AP: Weigh 1g of ammonium persulfate and add water to make up to 10ml. After it is completely dissolved, filter it with a 0.22um filter membrane to obtain 10% AP. After dispensing, store it at -20℃.

[0029] (3) Preparation of TBE buffer: Weigh 108g of tris(hydroxymethyl)aminomethane (Tris), 55g of boric acid, and 7.44g of disodium ethylenediaminetetraacetate (EDTA*2Na). Add these substances to 60ml of deionized water and heat to 37℃ to promote dissolution. After dissolution, add deionized water to a total volume of 1L to obtain a 10x TBE buffer. Store at room temperature.

[0030] (4) Preparation of native-PAGE (non-denaturing polyacrylamide) gel:

[0031] Table 1. Native-PAGE gel preparation system

[0032]

[0033] Following the above reaction system, add the appropriate reagents and gently pipette until well mixed. After standing for a few seconds to allow air bubbles to dissipate, pour the mixture into a gel plate. After pouring, insert a comb and wait approximately 30-60 minutes for the gel to solidify.

[0034] After the non-denaturing polyacrylamide gel has solidified, remove the gel plate, wipe it clean, and store it at -4°C.

[0035] In this embodiment, a 20% native-PAGE gel can separate DNA fragments smaller than 12 bp, a 15% native-PAGE gel has an effective separation range of 25-150 bp, and a 12% native-PAGE gel can separate DNA fragments larger than 60 bp.

[0036] Example 2

[0037] Example 2 of this application provides a method for detecting the activity of formamide pyrimidine-DNA glycosylation enzyme, comprising the following steps:

[0038] (1) Preparation of NEB buffer: Weigh 1.21g Tris, 0.95g magnesium chloride (MgCl2), and 0.154g dithiothreitol (DTT) respectively, add them to 90ml of ultrapure water, and after complete dissolution, adjust the pH of the reagents to 7.0 with dilute hydrochloric acid. Then, bring the solution to a final volume of 100ml. Filter the solution through a 0.22um filter membrane to obtain 10*NEB buffer, and store it at 4℃.

[0039] (2) Preparation of double-stranded probes: Two complementary single-stranded probes containing 8-oxoG sites were designed to enable Fpg enzyme to recognize and cleave the two single strands. The two single-stranded probes were P and PR.

[0040] P: 5-CATCTCTTCTCCGAGCCGGTCGAAATAGTTGGT / oxoG / AAGAGATG-3;

[0041] PR: 5-CATCTCTT / oxoG / ACCAACTATTTCGACCGGCTCGGAGAAGAGATG-3;

[0042] The two probes, P and PR, were mixed and denatured at 92℃ for 5 min, annealed at 63℃ for 30 s, and stored at 4℃ to form double-stranded probes of P and PR. The two synthesized probes were diluted to a final concentration of 10 μM.

[0043] (3) PCR reaction:

[0044] Table 2 PCR reaction system

[0045]

[0046] The Fpg enzyme was purchased from NEB. The NEB buffer solution used was designed for use with this Fpg enzyme. It is understood that when applying the detection method provided in this application to the detection of other brands or self-made Fpg enzymes, the appropriate buffer solution should be replaced as needed.

[0047] Following the above reaction system, add the appropriate reagents and gently pipette until well mixed. After mixing, gently centrifuge the solution to the bottom of the PCR tube, then place the sample in the PCR instrument and set the conditions as follows: 37℃ for 30 min, 65℃ for 5 min.

[0048] Add 1 μl of 10* loading buffer (sample loading buffer, purchased from TAKARA) to each of the reacted samples and mix well.

[0049] (4) Electrophoresis

[0050] Place a 15% non-denaturing polyacrylamide gel in the electrophoresis tank, clamp it, and pour 1xTBE buffer over it to submerge the groove.

[0051] After aspirating the prepared sample with a pipette, slowly inject it into the groove on the gel. Once completed, turn on the power and run the sample at 300V for approximately 120 minutes.

[0052] (5) Staining

[0053] Dilute the SuperRed 10,000x stock solution approximately 3300 times with ultrapure water into 0.1M NaCl to prepare a 3x staining solution. After electrophoresis, carefully place the gel into a container, slowly add sufficient 3x staining solution to submerge the gel, and incubate at room temperature with shaking for approximately 30 minutes.

[0054] The stained gels were observed and photographed using a UV imager. SuperRed and EB have the same spectrum and are both excited near 300 nm UV light.

[0055] (6) Results Analysis

[0056] Figure 1 This is a schematic diagram illustrating the principle of the activity detection method of the present invention; Figure 2 This is a graph showing the results obtained by the detection method of the present invention. According to... Figure 2 As shown, the marker, serving as a standard for the molecular weight of DNA double strands, has a base pair of 100 bp at its lowest point. In the unenzyme-added sample, due to the lack of Fpg enzyme cleavage, only one DNA band of 42 bp size for the P and PR double strands is displayed. In the enzyme-added sample, Fpg enzyme specifically recognizes and cleaves the 8-OXOG sites in the P and PR double strands, separating the DNA double strands into DNA fragments of 34 bp and 8 bp molecular weights, respectively. The 15% PAGE gel effectively separates the two DNA fragments that differ by 26 bp; therefore, two DNA bands of different sizes are clearly observed.

[0057] The Fpg enzyme activity detection method provided in this application involves the Fpg enzyme specifically recognizing and cleaving 8-oxoG on the DNA strand under the reaction system and conditions, thereby breaking the DNA into two strands. Relying on the addition of non-denaturing polyacrylamide gel and superRed dye, the activity of the Fpg enzyme can be clearly and accurately determined. This method can simply and accurately detect two activities of the Fpg enzyme: (1) N-terminal glycosylation enzyme activity, which specifically recognizes and cleaves damaged purine bases on double-stranded DNA, generating an apurinyl cleavage (AP) site; (2) AP lyase activity, which cleaves the 3' or 5' end of the AP site, generating a phosphate gap, thereby breaking the strand and forming two short strands. This method is simple and reliable, and can improve the accuracy and safety of Fpg enzyme activity detection methods.

[0058] This method can be used not only for the detection of Fpg enzyme activity, but also for the detection of a variety of specific nucleases, providing a new approach to enzyme function detection.

[0059] The above are merely embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention are included within the scope of the claims of the present invention pending approval.

Claims

1. A method for detecting the activity of formamide pyrimidine-DNA glycosylation enzyme, characterized in that, include: A double-stranded probe is provided, the double-stranded probe comprising two complementary single-stranded probes containing 8-oxoG modification sites; Mix Fpg enzyme, double-stranded probe, buffer solution and water thoroughly to form a mixed system; The above mixture was subjected to PCR reaction. The Fpg enzyme cleaved the double-stranded probe containing the 8-oxoG modification site to obtain a longer and a shorter fragment sample. Fragment samples were electrophoresed using non-denaturing polyacrylamide gel; After electrophoresis, the non-denaturing polyacrylamide gel was stained by shaking, and the staining results were photographed to detect the activity of Fpg enzyme.

2. The method for detecting the activity of formamide pyrimidine-DNA glycosylation enzyme according to claim 1, characterized in that, The concentration of the non-denaturing polyacrylamide gel is selected from 20%, 15%, and 12%.

3. The method for detecting the activity of formamide pyrimidine-DNA glycosylation enzyme according to claim 1, characterized in that, The complementary base of the double-stranded probe is 42 bp in length.

4. The method for detecting the activity of formamide pyrimidine-DNA glycosylation enzyme according to claim 3, characterized in that, The two single-stranded probe sequences of the double-stranded probe are as follows: P: 5-CATCTCTTCTCCGAGCCGGTCGAAATAGTTGGT / oxoG / AAGAGATG-3; PR: 5-CATCTCTT / oxoG / ACCAACTATTTCGACCGGCTCGGAGAAGAGATG-3; Here, oxoG represents damaged 8-oxoguanine.

5. The method for detecting the activity of formamide pyrimidine-DNA glycosylation enzyme according to claim 3, characterized in that, The concentration of the non-denaturing polyacrylamide gel is 15%.

6. The method for detecting the activity of formamide pyrimidine-DNA glycosylation enzyme according to claim 1, characterized in that, The buffer solution is NEB buffer.

7. The method for detecting the activity of formamide pyrimidine-DNA glycosylation enzyme according to claim 1, characterized in that, The PCR reaction conditions were 37℃ for 30 min and 65℃ for 5 min.

8. The method for detecting the activity of formamide pyrimidine-DNA glycosylation enzyme according to claim 1, characterized in that, The dye used in the staining process is SuperRed nucleic acid dye.

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