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In vitro DNA Protection Assay Using Oxidative Stress

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FEMS Microbiology Letters
Nov 2014



A wide range of stresses such as oxidative stress, acid, alkaline, UV, and metal can damage DNA. Here, we describe a protocol to measure the DNA nicking damage by Fenton reaction-mediated oxidative stress. Fenton reaction (Fe2+ + H2O2 → Fe3+ + OH- + ∙OH) produces the highly deleterious hydroxyl radicals that damage the cellular components such as DNA, lipid and proteins.

Keywords: DNA damage (DNA损伤), DNA protection (DNA的保护), Oxidative stress (氧化应激), Fenton reaction (芬顿反应)

Material and Reagents

  1. 300 ng/µl plasmid DNA
    We used 7.2 kbp pMK 3 plasmid purified from E. coli JM109 by QIA filterWe used 7.2 kbp pMK3 plasmid purified from E. coli JM109 by QIA filterTM (Plasmid Midi kit). The high quality plasmid abundant in supercoiled form is required to monitor the reduction of the supercoiled form.
  2. Proteins to be tested on their DNA protection ability
    a. BSA (Wako Pure Chemical Industries, catalog number: 01317843 )
    b. Lysozyme (Wako Pure Chemical Industries, catalog number: 12202673 )
  3. Agarose
  4. Ethidium bromide (EtBr) (Invitrogen, catalog number: 15585011 )
    Caution: Ethidium bromide is toxic and strong mutagen. Use appropriate gloves, safety goggles and lab coat
  5. Ferrous ammonium sulfate (1.5 mM) (Wako Pure Chemical Industries, catalog number: 01412172 )
    Note: The solution must be prepared just prior to the experiment.
  6. 200 mM hydrogen peroxide (Wako Pure Chemical Industries, catalog number: 08104215 )
  7. NaCl (Wako Pure Chemical Industries, catalog number: 19101665 )
  8. Sodium dodecyl sulfate (SDS) (Wako Pure Chemical Industries, catalog number: 08104215)
  9. Tris (Nacalai tesque, catalog number: 3543421 )
  10. Acetic acid (Nacalai tesque, catalog number: 0021243 )
  11. EDTA (Nacalai tesque, catalog number: 15111 )
  12. CIA (chloroform:isoamyl alcohol = 24:1) (Nacalai tesque, catalog number: 0840255 )
    Caution: Chloroform is suspect mutagen and harmful if inhaled. Avoid breathing vapor and prolonged contact with skin, using fume food and safety gloves. Follow the safety rule in your institute.
  13. 300 ng/µl plasmid DNA (pMK3) (see Recipes)
  14. 1 µg/µl bovine serum albumin (BSA) (see Recipes)
  15. 1 µg/µl lysozyme (see Recipes)
  16. Protein/binding buffer (see Recipes)
  17. 1.5 mM ferrous ammonium sulfate (see Recipes)
  18. 200 mM hydrogen peroxide (see Recipes)
  19. 10% sodium dodecyl sulfate (SDS) (see Recipes)
  20. 1x TAE buffer (see Recipes)
  21. 50x TAE buffer (see Recipes)


  1. Eppendorf tubes (1.5 ml)
  2. Eppendorf tubes (2.0 ml)
  3. Tips
  4. Thermostat bath
  5. Centrifuge machine
  6. Electrophoresis apparatus
  7. UV Trans illuminator and recording system (Nippon Genetics, model: e.g. FAS III system )


  1. Image J (NIH) (http://imagej.nih.gov/ij/)


  1. Pre-incubate plasmid DNA (300 ng pMK3) with or without protein sample: MrgA, MrgA*, BSA, or Lysozyme (10 and 20 µg) in 30 µl of protein-binding buffer: For 1 h at 37 °C.
  2. Add 8 µl of 1.5 mM fresh ferrous ammonium sulfate solution, gently mix the samples by pipetting, and then incubate for 5 min at room temperature (25 °C).
    Note: Ferroxidation of ferrous iron by oxygen does not proceed significantly before the addition of hydrogen peroxide at this time scale. This 5-min incubation in our experiment is to allow MrgA to sequestrate ferrous iron in its core.
  3. Add 2 µl of 200 mM hydrogen peroxide (final conc. 10 mM) and gently mix the samples by pipetting, and then incubate for 5 min at room temperature (25 °C).
  4. Add 10 µl of 10% SDS and 50 µl of CIA, and vortex. This is to denature and remove the proteins from DNA, and to recover DNA in aqueous phase in the next step.
  5. Recover supernatant by centrifugation at ≥13,000 x g for 5 min at 4 °C.
  6. Separate 20 µl of the supernatant on 1% agarose gel in TAE buffer (without EtBr) and electrophorese at 100 V for 40 min at room temperature. Following the electrophoresis, soak the gel in TAE buffer containing EtBr to visualize DNA.
  7. Measure the signal intensities of supercoiled DNA using NIH image J software. In detail:
    1. Take agarose gel images by UV-trans illuminator and CCD camera (FASIII system). Scan the photo copy by conventional scanner (if you can get high quality digital image from CCD camera, it can be directly used for the measurement. We scanned the photocopy because the resolution of the digital image is low in case of FASIII system). Measure the signal intensities of supercoiled DNA were using NIH image J software. Measure each signal intensity by subtracting the background.
    2. Calculate relative signal intensities using the signal intensity of the supercoiled plasmid DNA without oxidative stress.
    3.  Calculate the mean and standard deviation of the relative intensities from multiple independent experiments. Evaluate the statistical significance by Student’s t-test.

    Figure 1 shows the result of recombinant MrgA and MrgA*. MrgA and MrgA* were prepared as described previously (Ushijima et al., 2014), and their purities were higher than 95% in SDS-PAGE stained with Coomassie brilliant blue. MrgA has both ferroxidase activity and DNA binding activity, while MrgA* is impaired in its ferroxidase activity. As a negative control, either 1 µg/µl bovine serum albumin (BSA) or 1 µg/µl lysozyme can be used (see Recipes).

    Figure 1. Representative data. A. Relative amount of supercoiled plasmid DNA. Mean and SD values of no protein (n=9), MrgA (n=4), MrgA* (n=3), BSA (n=6), and lysozyme (n=4) are shown. All proteins are 10 µg. *: Significant difference compared to oxidative stress exposure (lane 2) in t-test, p < 0.05. ns: Not significant, p >0.25. B. Representative gel image of DNA protection assay. Lanes 1: Plasmid DNA without oxidative stress. Lanes 2: Plasmid DNA treated with oxidative stress (Fe2+/H2O2). Lanes 3~6: Plasmid DNA was pre-incubated with 10 µg of MrgA (lane 3), MrgA* (lane 4), BSA (lane 5) or lysozyme (lane 6) prior to the Fe2+ addition. SC: Supercoil, N: Nicked. DNA protection by MrgA was dependent on the intact ferroxidase activity, and DNA binding by MrgA* didn’t contribute to DNA protection (Ushijima et al., 2014).


  1. We confirmed that at least Tris or HEPES based buffer can be used for the DNA nicking damage by Fenton reaction-mediated oxidative stress.
  2. 1.5 mM ferrous ammonium sulfate should be freshly prepared and the dissolved solution should not exceed pH 7.0 (pH of MilliQ water is 5.50-6.86). The solution with pH values over 7.0 oxidizes ferrous iron to ferric iron immediately (Morgan and Lahav, 2007).
  3. Note that signal intensities can vary depending on experiments: some of the signal intensities in Figure 1B representative image are different from the mean intensities in Figure A. At least three independent experiments and the statistical evaluation are recommended. To compare distinct gel images, put reference samples that can be used to normalize the signal intensities, e.g. marker DNAs with known quantities. We employed one-side Student’s t-test.
  4. EDTA can be used to stop the Fenton reaction. However, when we tried to add EDTA before purification, it did not increase the DNA signal intensity, but rather generated smear signal.


  1. 300 ng/µl plasmid DNA (pMK3)
    Extract pMK3 purified from E. coli JM109 by Plasmid Midi Kit
    Adjust concentration with MilliQ water
  2. 1 µg/µl bovine serum albumin (BSA)
    10 mg BSA
    Adjust total volume to 10 ml with protein/binding buffer
  3. 1 µg/µl lysozyme
    10 mg lysozyme
    Adjust total volume to 10 ml with protein/binding buffer
  4. Protein/binding buffer
    20 mM Tris-HCl (pH 8.0)
    200 mM NaCl
  5. 1.5 mM ferrous ammonium sulfate
    1.17 mg Ferrous ammonium sulfate
    2 ml MilliQ water
  6. 200 mM hydrogen peroxide
    10.2 µl Hydrogen peroxide (30% w/v)
    439.8 µl MilliQ water
  7. 10% sodium dodecyl sulfate (SDS)
    10 g sodium dodecyl sulfate
    Adjust total volume to 100 ml with MilliQ water
  8. 1x TAE buffer
    20 ml 50x TAE
    980 ml MilliQ water
  9. 50x TAE buffer
    242 g Tris
    57.1 ml acetic acid
    100 ml 0.5 M EDTA
    Adjust total volume to 1 L with MilliQ water


This protocol was modified from in vitro DNA damage assay described previously (Martinez and Kolter, 1997).


  1. Martinez, A. and Kolter, R. (1997). Protection of DNA during oxidative stress by the nonspecific DNA-binding protein Dps. J Bacteriol 179(16): 5188-5194.
  2. Morgan, B. and Lahav, O. (2007). The effect of pH on the kinetics of spontaneous Fe(II) oxidation by O2 in aqueous solution--basic principles and a simple heuristic description. Chemosphere 68(11): 2080-2084.
  3. Ushijima, Y., Ohniwa, R. L., Maruyama, A., Saito, S., Tanaka, Y. and Morikawa, K. (2014). Nucleoid compaction by MrgA(Asp56Ala/Glu60Ala) does not contribute to staphylococcal cell survival against oxidative stress and phagocytic killing by macrophages. FEMS Microbiol Lett 360(2): 144-151.


诸如氧化应激,酸,碱,UV和金属的各种应力可以损害DNA。 在这里,我们描述了一个协议,以测量DNA切口损伤芬顿反应介导的氧化应激。 Fenton反应(Fe 2+ + H 2 O 2 O 2→Fe 3+ +)+ OH - +∙OH)产生损害细胞组分如DNA,脂质和蛋白质的高度有害的羟基自由基。

关键字:DNA损伤, DNA的保护, 氧化应激, 芬顿反应


  1. 300 ng /μl质粒DNA 我们使用从E中纯化的7.2kbp pMK 3质粒。 通过QIA过滤器的大肠杆菌JM109使用通过QIA过滤器TM (Plasmid Midi试剂盒)从大肠杆菌JM109纯化的7.2kbp pMK3质粒。 需要超级螺旋形式丰富的高质量质粒来监测超螺旋形式的还原。
  2. 要测试其DNA保护能力的蛋白质
    一个。 BSA(Wako Pure Chemical Industries,目录号:01317843)
    b。 溶菌酶(Wako Pure Chemical Industries,目录号:12202673)
  3. 琼脂糖
  4. 溴化乙锭(EtBr)(Invitrogen,目录号:15585011) 注意:溴化乙锭是有毒和强诱变剂。 使用合适的手套,护目镜和实验室外套。
  5. 硫酸亚铁铵(1.5mM)(Wako Pure Chemical Industries,目录号:01412172)
  6. 200mM过氧化氢(Wako Pure Chemical Industries,目录号:08104215)
  7. NaCl(Wako Pure Chemical Industries,目录号:19101665)
  8. 十二烷基硫酸钠(SDS)(Wako Pure Chemical Industries,目录号:08104215)
  9. Tris(Nacalai tesque,目录号:3543421)
  10. 乙酸(Nacalai tesque,目录号:0021243)
  11. EDTA(Nacalai tesque,目录号:15111)
  12. CIA(氯仿:异戊醇= 24:1)(Nacalai tesque,目录号:0840255)
    注意:氯仿是可疑的诱变剂,吸入会有害。 避免吸入蒸气和长期接触皮肤,使用烟气食品和安全手套。 按照您所在学院的安全规则。
  13. 300 ng /μl质粒DNA(pMK3)(参见配方)
  14. 1μg/μl牛血清白蛋白(BSA)(见Recipes)
  15. 1μg/μl溶菌酶(见配方)
  16. 蛋白质/结合缓冲液(见配方)
  17. 1.5 mM硫酸亚铁铵(见配方)
  18. 200 mM过氧化氢(见配方)
  19. 10%十二烷基硫酸钠(SDS)(参见配方)
  20. 1x TAE缓冲区(请参阅配方)
  21. 50x TAE缓冲区(请参阅配方)


  1. Eppendorf管(1.5ml)
  2. Eppendorf管(2.0ml)
  3. 提示
  4. 恒温槽
  5. 离心机
  6. 电泳仪
  7. UV Trans照明器和记录系统(Nippon Genetics,型号:例如FAS III系统)


  1. 图片J(NIH)( http://imagej.nih.gov/ij/


  1. 在30μl蛋白质结合缓冲液中预孵育具有或不具有蛋白质样品:MrgA,MrgA *,BSA或溶菌酶(10和20μg)的质粒DNA(300ng pMK3):在37℃下1小时。 />
  2. 加入8μl1.5 mM新鲜的硫酸亚铁铵溶液,轻轻混合样品,通过移液,然后在室温(25°C)孵育5分钟。
  3. 加入2μl200mM过氧化氢(最终浓度为10mM),并通过移液轻轻混合样品,然后在室温(25℃)下孵育5分钟。
  4. 加入10μl的10%SDS和50μl的CIA,并涡旋。这是为了变性并从DNA中除去蛋白质,并在下一步骤中在水相中回收DNA
  5. 通过在≥13,000×g下离心5分钟,在4℃下回收上清液
  6. 在TAE缓冲液(无EtBr)中的1%琼脂糖凝胶上分离20μl上清液,并在室温下在100V电泳40分钟。电泳后,将凝胶浸泡在含有EtBr的TAE缓冲液中以显现DNA
  7. 使用NIH图像J软件测量超螺旋DNA的信号强度。 详细:
    1. 通过紫外透射照明器和CCD相机采取琼脂糖凝胶图像 (FASIII系统)。 通过常规扫描仪扫描照片副本(如果可以   从CCD相机获得高质量的数字图像,可直接使用   用于测量。 我们扫描了影印件,因为分辨率   在FASIII系统的情况下数字图像低)。 测量信号 超螺旋DNA的强度使用NIH图像J软件。 测量 每个信号强度减去背景。
    2. 使用没有氧化应激的超螺旋质粒DNA的信号强度计算相对信号强度
    3.  计算相对强度的平均值和标准偏差 来自多个独立实验。 评估统计 通过学生t检验的显着性
    图1显示重组MrgA和MrgA *的结果。如先前所述制备MrgA和MrgA *(Ushijima等人,2014),并且在用考马斯亮蓝染色的SDS-PAGE中它们的纯度高于95%。 MrgA具有铁氧化酶活性和DNA结合活性,而MrgA *的铁氧化酶活性受损。作为阴性对照,可以使用1μg/μl牛血清白蛋白(BSA)或1μg/μl溶菌酶(参见配方)。

    图1.代表性数据。A.超螺旋质粒DNA的相对量。显示了无蛋白质(n = 9),MrgA(n = 4),MrgA *(n = 3),BSA(n = 6)和溶菌酶(n = 4)的平均值和SD值。所有蛋白质均为10μg。 *:在t检验中与氧化应激暴露(泳道2)相比显着差异,p < 0.05。 ns:不显着,p> 0.25。 B.DNA保护测定的代表性凝胶图像。泳道1:没有氧化应激的质粒DNA。泳道2:用氧化应激(Fe 2+ +/H 2 O 2 O 2)处理的质粒DNA。泳道3〜6:在Fe 2+反应之前,将质粒DNA与10μgMrgA(泳道3),MrgA *(泳道4),BSA(泳道5)或溶菌酶(泳道6) sup>添加。 SC:超线圈,N:镀镍。 MrgA的DNA保护依赖于完整的铁氧化酶活性,而MrgA *的DNA结合对DNA保护没有贡献(Ushijima等人,2014)。


  1. 我们证实,至少Tris或HEPES基缓冲液可用于通过Fenton反应介导的氧化应激的DNA切口损伤。
  2. 应该新制备1.5mM硫酸亚铁铵,溶解的溶液不应超过pH 7.0(MilliQ水的pH为5.50-6.86)。 pH值大于7.0的溶液立即氧化二价铁到三价铁(Morgan和Lahav,2007)。
  3. 注意,信号强度可以根据实验而变化:图1B中代表性图像中的一些信号强度不同于图A中的平均强度。推荐至少三次独立实验和统计评价。为了比较不同的凝胶图像,使用可用于标准化信号强度的参考样品,例如具有已知量的标记DNA。我们采用单边学生t检验。
  4. EDTA可用于停止Fenton反应。然而,当我们在纯化之前试图加入EDTA时,它不增加DNA信号强度,而是产生涂片信号。


  1. 300ng /μl质粒DNA(pMK3)
    通过Plasmid Midi Kit提取从大肠杆菌JM109纯化的pMK3 用MilliQ水调节浓度
  2. 1μg/μl牛血清白蛋白(BSA)
    10 mg BSA
    将总体积调整至10 ml
  3. 1μg/μl溶菌酶 10mg溶菌酶 用蛋白质/结合缓冲液
    将总体积调整至10 ml
  4. 蛋白/结合缓冲液
    20mM Tris-HCl(pH8.0) 200 mM NaCl
  5. 1.5mM硫酸亚铁铵
    2ml MilliQ水
  6. 200mM过氧化氢
  7. 10%十二烷基硫酸钠(SDS)
    10g十二烷基硫酸钠 用MilliQ水将总体积调整到100 ml
  8. 1x TAE缓冲区
    20 ml 50x TAE
    980ml MilliQ水
  9. 50x TAE缓冲区
    57.1ml乙酸 100ml 0.5M EDTA
    用MilliQ水将总体积调整为1 L /


该方案从之前描述的体外 DNA损伤测定(Martinez和Kolter,1997)修改。


  1. Martinez,A。和Kolter,R。(1997)。 在非特异性DNA结合蛋白Dps的氧化应激过程中保护DNA。 J Bacteriol 179(16):5188-5194。
  2. Morgan,B。和Lahav,O。(2007)。 pH对由O 2引起的自发Fe(II)氧化的动力学的影响/sub>在水溶液中 - 基本原理和简单的启发式描述。 68(11):2080-2084。
  3. Ushijima,Y.,Ohniwa,R.L.,Maruyama,A.,Saito,S.,Tanaka,Y.and Morikawa,K。 MrgA(Asp56Ala/Glu60Ala)的核仁压实对葡萄球菌细胞对氧化应激和吞噬细胞的存活无贡献 FEMS Microbiol Lett 360(2):144-151。
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引用:Ushijima, Y., Ohniwa, R. L. and Morikawa, K. (2015). In vitro DNA Protection Assay Using Oxidative Stress. Bio-protocol 5(14): e1538. DOI: 10.21769/BioProtoc.1538.