A method for quantitatively detecting uracil in DNA by specific enzyme digestion combined with qPCR single base resolution

Abstract

The present invention discloses a method for quantitatively detecting uracil in DNA by combining specific enzyme cutting with qPCR single base resolution, and belongs to the field of biotechnology. The present invention is based on the fact that UdgX‑H109S can recognize and cut uracil (U) to produce apurinic site (AP), and apurinic endonuclease 1 (APE1) cuts the produced AP site to produce 1 nucleotide gap, resulting in the extension of Bst 3.0 DNA polymerase stopping. Real-time fluorescence quantitative PCR (qPCR) technology can amplify the difference of extension products, and then directly detect the content of uracil at a specific site. The method of the present invention has high sensitivity, good specificity, simple operation, high efficiency, and can not only be used for single base resolution determination of uracil in low-abundance biological samples, but also provides a new method for studying the function of uracil in organisms.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for quantitatively detecting uracil in DNA by combining specific enzyme cutting with qPCR single base resolution. Background Art

[0002] DNA mainly encodes genetic information through four bases: adenine (A), guanine (G), cytosine (C), and thymine (T). However, DNA is constantly damaged in the form of oxidation, depurination, alkylation, and deamination, which leads to genomic instability and affects the replication and transmission of genetic information. To avoid the loss of DNA genetic information, cells have evolved the base excision repair (BER) pathway to remove DNA damage. BER is a highly conserved pathway from bacteria to humans and is responsible for repairing the vast majority of endogenous DNA damage, including alkylation, oxidation, deamination and depurination, as well as single-strand breaks (SSBs). Therefore, BER can remove these lesions that are frequently generated in DNA and maintain the integrity of the genome and the stable transmission of genetic material.

[0003] Uracil and thymine have similar chemical structures, so both uracil and thymine in DNA form base pairing with adenine according to the Watson-Crick base complementary pairing principle. Therefore, it is impossible for most DNA polymerases to correctly distinguish and remove uracil from DNA, resulting in the appearance of uracil in DNA. In particular, the frequency of this situation increases when the synthesis of thymine is disturbed. There are three sources of uracil, one is the spontaneous hydrolysis and deamination of cytosine, the other is the deamination of cytosine by DNA cytosine deaminase of the AID / APOBEC family, and the other is the misincorporation of dUTP into DNA to form uracil. Uracil produced by cytosine deamination may cause mutations from C to U or G to A, while misincorporation of dUTP may cause mismatches between U and A. Uracil-DNA glycosylase (UNG), which is ubiquitous in cells, can effectively remove uracil from deoxyribose to generate an apyrimidine site (AP site), which will be further repaired by the BER pathway. Under normal circumstances, uracil is recognized and removed by UNG, but if too much uracil accumulates in DNA, it will cause DNA chain breaks, leading to apoptosis mediated by p53 or Poly (ADP-ribose) polymerase 1. This so-called "thymine-free cell death" has been used in a variety of chemotherapy drugs. In addition, uracil accumulation in DNA is also associated with clinical diseases including megaloblastic anemia, neural tube defects, and HIV immunity. Therefore, developing methods that can detect the location and specific levels of uracil has become a new research direction.

[0004] The most direct method for quantifying the total uracil content in DNA is LC-MS / MS. This method detects uracil at frequencies of ∼0.15 and ∼0.08 uracil / 10 in the human genome. 6 base, but this method cannot detect the specific location information of uracil. Therefore, according to the situation that UNG excises uracil and generates AP sites, many UNG-based uracil sequencing methods have been developed, such as Excision-seq, snAP-seq, and du-seq. However, although these methods will perform appropriate pretreatment to reduce some interference, pre-existing DNA chain breaks, AP sites, or other modified nucleotides containing aldehyde groups (including 5-formyldeoxyuridine (5fU) and 5-formyldeoxycytidine (5fC)) may produce false positives. Therefore, UdgX from Mycobacterium smegmatis in the UDG superfamily can specifically recognize and excise uracil, and at the same time, it can form an irreversible covalent bond with deoxyribose to reduce false positives. Based on the specific binding of UdgX to DNA containing uracil, DNA fragments containing uracil can be further pulled down for enrichment, thereby achieving single-base positioning analysis of uracil on the genome. However, the above-mentioned genome-wide uracil localization methods are highly dependent on sequencing depth and complex bioinformatics analysis, which to some extent limits the quantitative application to a few key uracil sites. Therefore, there is a need for site-specific uracil detection and quantification methods.

[0005] Uracil and thymine are difficult to distinguish, so developing methods to measure the amount of uracil in DNA is a very interesting challenge. A recent study showed that a mutant protein of UdgX in Mycobacterium smegmatis, UdgX-H109S, can cleave the N-glycosidic bond connecting uracil base and deoxyribose in single-stranded DNA and generate AP sites. 1-3 At the same time, UdgX-H109S has no recognition and cleavage activity for dihydroxyuracil, hydroxymethyluracil and hypoxanthine in DNA. 1 Based on the uracil-cleaving property of UdgX-H109S, the present invention establishes a method for quantitatively detecting uracil in DNA by combining specific enzyme cleavage with qPCR single-base resolution. Summary of the invention

[0006] The purpose of the present invention is to solve the problems existing in the prior art and to provide a method for quantitatively detecting uracil in DNA by combining specific enzyme cleavage with qPCR single-base resolution, that is, a site-specific uracil quantification method that achieves single-base resolution, high sensitivity, high specificity, and simple operation.

[0007] The principle of the present invention is shown in Figure 1The present invention proposes a method for quantitatively detecting uracil in DNA by UdgX-H109S-mediated extension termination combined with qPCR single-base resolution. In this method, UdgX-H109S can specifically recognize and cut uracil to produce AP sites, and apurinic / apyrimidinic nuclease 1 (APE1) can break AP sites, resulting in a nucleotide gap at the uracil site; however, UdgX-H109S cannot recognize cytosine and thymine in DNA, so no nucleotide gap is generated. Based on this, during the extension process, Bst 3.0 DNA polymerase terminates the extension at the nucleotide gap of DNA modified with uracil, resulting in less extension products; however, it extends normally in DNA without uracil, and the extension products are not reduced. Later, the difference between uracil and cytosine or thymine is amplified by real-time quantitative PCR (qPCR), and the content of uracil in DNA can be quantitatively evaluated. Based on the uracil-cleaving property of UdgX-H109S, the uracil content in DNA can be detected with single-base resolution.

[0008] The principle of qPCR single-base resolution quantitative analysis of uracil: U% uracil modification content at a specific site - (total DNA - amount of DNA not containing U) / total DNA * 100%. When processing actual samples, one group used Bst 3.0 DNA polymerase to extend DNA samples that were not treated with UdgX-H109S but were treated with APE1, and qPCR amplification was used to obtain the total amount of DNA at a specific site; one group used Bst 3.0 DNA polymerase to extend DNA samples that were treated with UdgX-H109S and APE1, and qPCR amplification obtained the amount of DNA that did not contain U at a specific site. Based on this, during the experiment, the samples were equally divided into two parts, one without treatment with UdgX-H109S and the other with UdgX-H109S. After both were treated with APE1, they were extended using Bst 3.0 DNA polymerase, and the extension products were amplified by qPCR to obtain the corresponding C T Value, according to C T The quantitative equation plotted with the values ​​can achieve single-base resolution quantification of uracil.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] A method for quantitatively detecting uracil in DNA by combining specific enzyme digestion with qPCR single base resolution comprises the following steps:

[0011] (1) Synthesize a DNA standard chain containing the target site. Dilute the DNA chain to a series of different concentrations, extend it using DNA polymerase and perform qPCR, then plot the DNA concentration and the corresponding C TThe linear equation of the value.

[0012] (2) The DNA sample to be tested is equally divided into two parts, one part is not treated with UdgX-H109S (adding the corresponding protein buffer, without adding protein), and the other part is treated with UdgX-H109S. Then the two samples are treated with APE1 enzyme at the same time. The amino acid sequence of UdgX-H109S is shown in SEQ ID NO.1.

[0013] (3) The same DNA probe is added to the two DNA samples. One part of the probe is completely complementary to the downstream of the target DNA site, and the other part is unpaired with the DNA standard chain and is used for the subsequent qPCR reaction. Then, DNA polymerase is used for extension reaction.

[0014] (4) Design qPCR primers. The 5' end primer is completely consistent with the 5' end sequence of the DNA standard chain, and the 3' end primer is completely consistent with the downstream sequence on the DNA probe that is not complementary to the standard chain. Then perform qPCR on the extension product and calculate the amount of DNA according to the linear equation. For DNA samples that have not been treated with UdgX-H109S, qPCR detects the total amount of DNA; for DNA samples that have been treated with UdgX-H109S, qPCR detects the amount of DNA that does not contain U. The uracil modification content at a specific site is calculated as follows:

[0015] The uracil modification content of a specific site (U%) = (total amount of DNA - amount of DNA without U) / total amount of DNA * 100%.

[0016] In the above method, the DNA polymerase is preferably Bst 3.0 DNA polymerase.

[0017] In step (1), the sequence of the DNA standard strand is preferably a DNA sequence containing the target site and 20-30 nt upstream and downstream thereof.

[0018] In step (2), the conditions for treating the DNA to be detected with UdgX-H109S are preferably as follows: UdgX-H109S is reacted with the DNA to be detected in UdgX-H109S buffer (50 mM Tris-HCl pH 8.0, 1 mM Na2EDTA, 1 mM DTT, 25 μg / mL BSA) at 37°C for 0.3-12 h.

[0019] In step (2), the conditions for the treatment with APE1 enzyme are preferably as follows: adding APE1 enzyme and APE1 buffer to the reaction system and reacting at 37° C. for 1-16 hours.

[0020] In step (2), the DNA to be detected is treated with UdgX-H109S and APE1, and the specific steps are as follows:

[0021] 1) Prepare the following UdgX-H109S reaction system (10 μL): 50 mM Tris-HCl pH 8.0, 1 mM Na2EDTA, 1 mM DTT, 25 μg / mL BSA, 8 pmol UdgX-H109S, 1-10 μg DNA to be tested, add water to make up to 10 μL; incubate the reaction system at 37°C for 20 min.

[0022] 2) APE1 enzyme treatment: 1) After the reaction, add 1 μL 10U / μL APE1 enzyme and 1 μL 10×APE1 buffer, and place the reaction system at 37°C for 60 min.

[0023] A kit for quantitatively detecting uracil in DNA, comprising UdgX-H109S protein, APE1 enzyme, and DNA polymerase. The DNA polymerase is preferably Bst 3.0 DNA polymerase

[0024] Furthermore, the kit also contains a reaction buffer of the corresponding protein or enzyme.

[0025] The present invention realizes the detection of the content of uracil in DNA with single base resolution based on the cleavage uracil property of UdgX-H109S. Based on this, the present invention further provides the application of UdgX-H109S in quantitatively detecting uracil in DNA, and the application of UdgX-H109S in preparing a kit for quantitatively detecting uracil in DNA.

[0026] The method of the present invention has high sensitivity, good specificity, simple operation and high efficiency. It can not only be used for single-base resolution determination of uracil in biological samples with low abundance, but also provides a new method for studying the function of uracil in organisms. The advantages and beneficial effects of the present invention are as follows:

[0027] 1) The method of the present invention is simple and easy to understand, the process is concise and easy to operate, the cost is low, the instrument requirements are low, and the complete experimental process can be completed within one day, which is convenient for promotion and use.

[0028] 2) The present invention can precisely locate uracil and accurately quantify the uracil content at a specific site.

[0029] 3) The present invention can quickly evaluate the uracil modification content at different sites on the genome without complex optimization and quantification.

[0030] 4) The present invention does not require the use of complex, time-consuming and expensive second-generation and third-generation sequencing technologies, and qPCR amplification can accurately quantify uracil in the genome with single-base resolution.

[0031] 5) The present invention can be used to analyze the content changes of important uracil sites in clinical samples, providing a powerful tool for further studying the role of uracil in different diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is a schematic diagram of the specific enzyme cleavage combined with qPCR single-base resolution quantitative detection of uracil in DNA in the present invention.

[0033] Figure 2 The denaturing PAGE images show that UdgX-H109S in the present invention does not cleave dsT:A and dsC:G (A), and cleaves the corresponding dsU:A and dsU:G (B, C).

[0034] Figure 3 The results of quantitative analysis of uracil at the specific site Chr1:1378925 in HEK293T cells in the present invention. (A) qPCR fluorescence amplification curves generated using different concentrations of dsC:G double strands. (B) C T The linear relationship between the value and the dsC:G double-strand content. (C) Real-time fluorescence amplification curve of qPCR and C T The bar graph shows the results of detecting uracil levels at position Chr1:1378925.

[0035] Figure 4 These are the results of quantitative analysis of uracil at specific sites Chr1:1378925 (A) and Chr4:50566961 (B) in HEK293T cells in the present invention. DETAILED DESCRIPTION

[0036] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0037] Example 1: Optimization of conditions for UdgX-H109S cleavage of double-stranded DNA containing uracil modifications

[0038] Prepare annealing double-stranded DNA reaction system (10 μL): 1 μL 100 mM Tris-HCl (pH 7.0), 0.5 μL 1M LiCl, 4 μL 10 μM ssU single-stranded DNA (5'-ATTCTCCTGCCTCAGCCTCC U GAGTAGCTGG GACTACAGGC-3'), 4.5 μL 10 μM A / G complementary single-stranded DNA (5'-GCCTGTAGTCCCAGCTACTC A GGAGGCTGAGGCAGGAGAAT-3' / 5'-GCCTGTAGTCCCAGCTACTC GThe reaction system was placed at 95°C for 5 min, and then cooled to 25°C at 1°C / 1s to form double-stranded dsU:A and dsU:G.

[0039] Optimization of the optimal reaction conditions for UdgX-H109S to cleave 2 pmol of dsU:A / dsU:G substrate:

[0040] The reaction system is 10 μL: 50 mM Tris-HCl pH 8.0, 1 mM Na2EDTA, 1 mM DTT, 25 μg / mL BSA, UdgX-H109S 2 , 2 pmol of DNA to be tested, add water to make the total volume 10 μL; the reaction system was placed at 37°C for reaction.

[0041] 1) Optimization of UdgX-H109S protein dosage: Under the above reaction conditions, 0, 0.125, 0.25, 0.5, 1, 2, 4, and 8 pmol of UdgX-H109S were added to 2 pmol of dsU:A and dsU:G reaction substrate systems, respectively, and the reaction was carried out at 37°C for 30 min. Then, UdgX-H109S was inactivated by heating at 85°C for 10 min, and then 0.2 U of APE1 was added and the reaction was carried out at 37°C for 60 min. The results showed that 8 pmol of UdgX-H109S could completely remove 2 pmol of dsU:A / dsU:G substrates.

[0042] 2) Optimize the reaction time: add 8pmol UdgX-H109S to 2pmol dsU:A and dsU:G reaction substrate systems, react at 37°C for 0, 1, 2, 5, 10, 20, 30, 40, 50, 60min, then heat at 85°C for 10min to inactivate UdgX-H109S, then add 0.2U APE1, react at 37°C for 60min. The results show that 2pmol dsU:A / dsU:G substrates are completely removed at 20min. Therefore, the final reaction was carried out under the conditions of 8pmol UdgX-H109S and 37°C for 20min.

[0043] Example 2: Elongation termination method mediated by UdgX-H109S

[0044] 1) UdgX-H109S cleaves uracil: 2 pmol dsC:G (5'-ATTCTCCTGCCTCAGCCTCC) annealed to double strands C GAGTAGCTGGGACTACAGGC-3',3'-GCCTGTAGTCCCAGCTACTC GGGAGGCTGAGGCAGGAGAAT-5') and dsU:G(5'-ATTCTCCTGCCTCAGCCTCC U GAGTAGCTGGG ACTACAGGC-3',3'-GCCTGTAGTCCCAGCTACTC G 50 mM Tris-HCl pH 8.0, 1 mM Na2EDTA, 1 mM DTT, 25 μg / mL BSA, and 8 pmol UdgX-H109S were added to the strand of GGAGGCTGAGGCAGGAGAAT-5') and reacted at 37°C for 20 min.

[0045] 2) APE1 cleavage of AP site: add 1 μL 10U / μL APE1 enzyme and 1 μL 10×APE1 buffer to the reaction product of 1), place the reaction system at 37°C for 60 min, then place the reaction system at 65°C for 20 min to terminate the reaction.

[0046] 3) Bst 3.0 DNA polymerase extension: After the above reaction, add 10 pmol DNA probe (5'-ATATGCTGTCTAGTGACTAGTCTCAGGCATGGTCGCAGGCGCCTGTAGTCCCAGCTAC-3') to the dsC:G and dsU:G samples respectively. React at 95°C for 10 min to open the double-stranded DNA, and then react at 55°C for 20 min to anneal the DNA template and probe. Then add 200 nmol Mg 2+ , 1× Isothermal Amplification Buffer II (containing 2 mM Mg 2+ ), 0.013U Bst3.0 DNA polymerase, 35nmol dNTP mixture, add water to a final volume of 25μL. React at 65℃ for 30min, denature at 85℃ for 5min, and place the sample at 4℃.

[0047] 4) qPCR amplification extension primer: qPCR system is 10 μL qPCR Green MasterMix (No Rox), 8 pmol F-922 (5'-AACATTCAGATTCTCCTGCCTCA-3'), 8 pmol R-922 (5'-ACCAATATGCTGTCTAGTCTCAGGC-3'), 1 μL 3) extension product, add water to a total volume of 20 μL. The qPCR program was performed on the CFX Connect real-time system (Bio-Rad Laboratories, Hercules, USA). The specific qPCR program was: 95°C, 10 min, (95°C, 5 s; 61°C, 30 s; 72°C, 45 s) × 45 cycles.

[0048] The cleavage efficiency of UdgX-H109S was analyzed by agarose gel electrophoresis. Figure 2 A,dsU:A(5'-ATTCTCCTGCCTCAGCCTCC U GAGTAGCTGGGACTACAGGC-3',3'-GCCTGTAGTCCCAGCTACTC A GGAGGCTGAGGCAGGAGAAT-5') and dsU:G(5'-ATTCTCCTGCCTCAGCCTCC U GAGTAGCTGGGACTACAGGC-3',3'-GCCTGTAGTCCCAGCTACTC G There were no bands in the lanes with UdgX-H109S added, i.e., the second and fourth lanes; and obvious bands in the lanes without UdgX-H109S added, i.e., the first and third lanes. The results showed that UdgX-H109S could effectively cleave dsU:A and dsU:G.

[0049] The effect of UdgX-H109S-mediated elongation termination was analyzed by agarose gel electrophoresis. Figure 2 There was almost no product in lanes B and 2C, dsU:G and dsU:A, indicating that UdgX-H109S could effectively prevent the extension of uracil sites, while dsC:G (5'-ATTCTCCTGCCTCAGCCTCC C GAGTAGCTGGGACTACAGGC-3',3'-GCCTGTA GTCCCAGCTACTC G GGAGGCTGAGGCAGGAGAAT-5') and dsT:A(5'-ATTCTCCTGCCTCAGCCTCC TGAGTAGCTGGGACTACAGGC-3',3'-GCCTGTAGTCCCAGCTACTC A There are obvious bands in the lanes of GGAGGCT GAGGCAGGAGAAT-5'), which indicate that the extension termination method mediated by UdgX-H109S can effectively distinguish uracil from cytosine or thymine.

[0050] Example 3: Quantification of uracil modification levels at Chr1:1369922 in HEK293T cells

[0051] The specific steps are as follows:

[0052] 1) First, a series of different concentrations of dsC:G double-stranded (5'-ATTCTCCTGCCTCAGCCTCC C GAGTAGCTGGGACTACAGGC-3',3'-GCCTGTAGTCCCAGCTACTC G GGAGGCTGAGGCAGG AGAAT-5') was used for Bst 3.0 DNA polymerase-mediated extension reaction and qPCR, and a linear equation for subsequent quantitative calculation was drawn.

[0053] 2) Human embryonic kidney 293T cell line (HEK293T) was cultured in DMEM medium containing 10% (v / v) fetal bovine serum, 100 U / mL penicillin and 100 g / mL streptomycin at 37°C and 5% CO2. The cultured cells were collected and DNA was extracted. 400 ng of DNA was equally divided into two parts, one was treated with UdgX-H109S and the other was not treated with UdgX-H109S. The two samples were subjected to qPCR reaction to obtain the corresponding C T value.

[0054] The DNA probe and qPCR primer sequences used to detect Chr1:1369922 locus are as follows:

[0055] DNA probe oligo 922: 5'-CAATATGCTGTCTAGTGACTAGTCTCAGGCATGGTCGCAGGCGCCTGTAGTCCCAGCTAC-3';

[0056] qPCR primers: F-922: 5'-AACATTCAGATTCTCCTGCCTCA-3',

[0057] R-922: 5'-ACCAATATGCTGTCTAGTCTCAGGC-3'.

[0058] According to the linear equation obtained in the first step and C obtained in the second step T The values ​​were used to calculate the level of uracil modification.

[0059] The results are as follows Figure 3 As shown, the linear equation ( Figure 3 B) is C T =-3.1937lgC dsC:G +2.7166, R 2 =0.9967, the amplification efficiency is 105%, so the linear equation meets the quantitative requirements. T The total amount of DNA at Chr1:1369922 can be calculated from the value; according to the qPCR amplification of the C T The value can be used to calculate the amount of DNA at Chr1:1369922 that does not contain U. The result is as follows Figure 3 Real-time fluorescence amplification curve of qPCR in C and C T The uracil content at Chr1:1369922 was calculated to be 18% using the formula U% = (total amount of DNA at Chr1:1369922 - amount of DNA at Chr1:1369922 without U) / total amount of DNA at Chr1:1369922 * 100%.

[0060] Example 4: Rapidly determine whether other sites on the genome contain uracil modifications

[0061] The method of the present invention was used to detect another uracil site Chr4:50566961 in HEK293T cells reported in the literature. 4 and another control site Chr1: 1378925 that does not contain uracil modification. The experimental methods and operations are consistent with the above, but the DNA probes and qPCR primers used in the experiment need to be replaced with probes and primers of the corresponding sites.

[0062] The DNA probe and qPCR primer sequences used to detect the Chr1:1378925 locus are as follows:

[0063] DNA probe oligo 925: 5'-GTCAACTCATCCGATCTCTCCCACCGTCGTACTGAAGCAGGTGCTCCCATTTAGGC-3';

[0064] qPCR primers: F-925: 5'-AGCCCAGCTGTTGGCCATTC-3',

[0065] R-925: 5'-CTCATCCGATCTCTCCCACCG-3'.

[0066] The DNA probe and qPCR primer sequences used to detect Chr4:50566961 locus are as follows:

[0067] DNA probe oligo 961: 5'-CTGCTGACGACCTGACGACTACACAACACAAAGAAGTTTCTGAGAATGCTTCTTT-3',

[0068] qPCR primers: F-961: 5′-GTGAAAAAGGAAATATCTTCTCCTAAAAAC-3′;

[0069] R-961: 5'-ATGCTGACGACCTGACGACTC-3'.

[0070] Results Figure 4 , for the detection of Chr4:50566961, sample C of the UdgX-H109S treatment group was used T The value was significantly increased compared with the sample of the group not treated with UdgX-H109S, which showed that Chr4:50566961 contained uracil modification. T C values ​​in DNA samples treated with and without UdgX-H109S T The value did not change, which indicated that there was no uracil modification at Chr1:1378925. C T (未添加UdgX-H109S处理)–C T (添加UdgX-H109S处理) The uracil modification level at Chr4:50566961 was calculated to be 46%. Therefore, the method of the present invention can quickly and easily analyze the uracil modification level at any position of the genome.

[0071] 1. Sang, PB; Srinath, T.; Patil, AG; Woo, EJ; Varshney, U., A uniqueuracil-DNAbinding protein of the uracil DNA glycosylase superfamily. NucleicAcids Res 2015, 43(17), 8452-63.

[0072] 2.Ahn,W.C.;Aroli,S.;Kim,J.H.;Moon,J.H.;Lee,G.S.;Lee,M.H.;Sang,P.B.;Oh,B.H.;Varshney,U.;Woo,E.J.,Covalent binding of uracil DNA glycosylase UdgXto abasic DNA upon uracil excision.Nat Chem Biol 2019,15(6),607-614.

[0073] 3.Jia,Q.;Zeng,H.;Tu,J.;Sun,L.;Cao,W.;Xie,W.,Structural insights intoan MsmUdgX mutant capable of both crosslinking and uracil excisioncapability.DNA Repair(Amst)2021,97,103008.

[0074] 4.Wang,Y.;Zhang,X.;Han,S.;Yang,W.;Chen,Z.;Wu,F.;Liu,J.;Weng,X.;Zhou,X.,Base-Resolution Analysis of Deoxyuridine at the Genome Scale Based on theArtificial Incorporation Modified Nucleobase.ACS Cent Sci 2021,7(6),973-979。

Claims

1. A method for quantitatively detecting uracil in double-stranded DNA by specific enzyme digestion combined with qPCR single base resolution, characterized in that: The following steps are included: (1) Synthesize a double-stranded DNA standard chain containing the target site, dilute it into a series of standards with different concentrations, extend it using DNA polymerase and perform qPCR to draw a C T The linear equation of the value and DNA concentration; (2) The double-stranded DNA sample to be tested is equally divided into two parts, one part is not treated with UdgX-H109S, and the other part is treated with UdgX-H109S; the two samples are then treated with APE1 enzyme; wherein the amino acid sequence of UdgX-H109S is shown in SEQ ID NO.1; (3) DNA probes are added to the two DNA samples respectively, and the two DNA samples are extended with DNA polymerase; one part of the DNA probe used is completely complementary to the downstream of the target DNA site, and the other part is used for subsequent qPCR amplification; (4) The obtained extension products are subjected to qPCR, and the uracil content in the DNA is calculated according to the linear equation; for DNA samples not treated with UdgX-H109S, the total amount of DNA is detected by qPCR; for DNA samples treated with UdgX-H109S, the amount of DNA without U is detected by qPCR; the uracil modification level at a specific site in the DNA to be detected is calculated using the following formula: Uracil modification level U% = (total amount of DNA - amount of DNA without U) / total amount of DNA * 100%.

2. The method for quantitatively detecting uracil in double-stranded DNA by specific enzyme digestion combined with qPCR single-base resolution according to claim 1, characterized in that: The DNA polymerase is Bst 3.0 DNA polymerase.

3. The method for quantitatively detecting uracil in double-stranded DNA by specific enzyme digestion combined with qPCR single-base resolution according to claim 1, characterized in that: In step (1), the sequence of the double-stranded DNA standard strand is a DNA sequence containing the target site and 20-30 nt upstream and downstream thereof.

4. The method for quantitatively detecting uracil in double-stranded DNA by specific enzyme digestion combined with qPCR single-base resolution according to claim 1, characterized in that: In step (2), the conditions for treating the double-stranded DNA to be detected with UdgX-H109S are as follows: UdgX-H109S is reacted with the double-stranded DNA to be detected in UdgX-H109S buffer at 37° C. for 0.3-12 h.

5. The method for quantitatively detecting uracil in double-stranded DNA by specific enzyme digestion combined with qPCR single-base resolution according to claim 1, characterized in that: In step (2), the conditions for using APE1 enzyme treatment are: adding APE1 enzyme and APE1 buffer to the reaction system and reacting at 37°C for 1-16 h.

6. The method for quantitatively detecting uracil in double-stranded DNA by specific enzyme digestion combined with qPCR single-base resolution according to claim 1, characterized in that: In step (2), the steps of treating the double-stranded DNA to be detected with UdgX-H109S and APE1 are as follows: 1) Prepare the following UdgX-H109S reaction system: 50 mM Tris-HCl pH 8.0, 1 mM Na2EDTA, 1 mM DTT, 25 μg / mL BSA, 8 pmol UdgX-H109S, 1-10 μg double-stranded DNA to be detected, add water to make up to 10 μL; incubate the reaction system at 37°C for 20 min; 2) APE1 enzyme treatment: 1) After the reaction, add 1 μL 10 U / μL APE1 enzyme and 1 μL 10 × APE1 buffer, and place the reaction system at 37°C for 60 min.

7. A kit for quantitatively detecting uracil in double-stranded DNA, characterized in that: It comprises UdgX-H109S protein, APE1 enzyme and DNA polymerase; the amino acid sequence of UdgX-H109S is shown in SEQ ID NO.

1.

8. The kit according to claim 7, characterized in that: Also contains the reaction buffer for the corresponding protein or enzyme.

9. Application of UdgX-H109S in quantitative detection of uracil in double-stranded DNA, characterized in that: The amino acid sequence of UdgX-H109S is shown in SEQ ID NO.

1.

10. Use of UdgX-H109S in preparing a kit for quantitatively detecting uracil in double-stranded DNA, characterized in that: The amino acid sequence of UdgX-H109S is shown in SEQ ID NO.1.

Images