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KMnO4 Footprinting
高锰酸钾足迹法   

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参见作者原研究论文

本实验方案简略版
Molecular Microbiology
Mar 2012

Abstract

The KMnO4 footprinting method offers a rapid and easy way to detect and localize single-stranded regions within a duplex DNA molecule, such as it occurs for instance within an actively transcribing RNA polymerase-DNA complex or during R-loop formation in DNA-RNA hybrid structures. The method is based on the selective oxidation of single-stranded thymines in DNA. The modified nucleotides react with strong bases by ring opening and subsequent phosphodiester cleavage. Because the modified nucleotides will not be recognized by DNA polymerase sites of modification can also be analyzed by primer extension with Klenow DNA polymerase, which stops elongation one residue before the modification. Hence, localization of the modified base positions can be performed on denaturing polyacrylamide gels either after piperidine catalyzed phosphodiester cleavage of 3'- or 5'-32P-end-labeled DNA or by primer extension with non-labeled DNA employing 32P-labeled oligonucleotide primers. Due to the fact that KMnO4 can penetrate through membranes the footprinting method can also be used for footprint analyses within living cells.

Keywords: Single-stranded DNA localization (单链DNA定位), Single-stranded thymine modification (单链碱基修饰), DNA footprinting (DNA足迹), RNA-DNA hybrid analysis (RNA-DNA杂交分析), Structural analysis of DNA (DNA的结构分析)

Materials and Reagents

  1. Radiolabeled DNA fragment of interest
  2. 14.3 M β-mercaptoethanol
  3. 500 mM EDTA
  4. Phenol
  5. Bromophenol blue (Sigma-Aldrich, catalog number: BO126 )
  6. Xylene cyanol (Sigma-Aldrich, catalog number: X4126 )
  7. Formamide deionized (Panreac Applichem, catalog number: A2156 )
  8. Cholorophorm
  9. Piperidine, purity grade: pro analysis (p.a.) (e.g. Sigma-Aldrich, catalog number: 411027 )
  10. Ethanol (purity grade: pro analysis) (p.a.)
  11. X-ray films
  12. Glycogen (1 μg/μl) (e.g. Roche, catalog number: 10901393001 )
  13. NaOAc (300 mM pH = 5.5)
    Optional for Procedure B (primer extension analysis)
  14. Non-radio labeled DNA fragment of interest
  15. 5'-32P-labeled desoxyoligonucleotide primer
  16. NaOH (10 mM)
  17. Tris
  18. MgSO4
  19. DTT
  20. Klenow fragment of DNA polymerase I (e.g. Biolabs, catalog number: MO210S )
  21. NH4OAc
  22. 370 mM KMnO4 stock solution (a 1:1 dilution with H2O is used for the reaction) (see Recipes)
  23. Phenol/Chloroform (see Recipes)
  24. Neutralization solution (see Recipes)
  25. dNTP mix (see Recipes)
  26. Stop mix (see Recipes)
  27. Electrophoresis loading buffer (see Recipes)

Equipment

  1. Table-top centrifuge
  2. Vortex shaker
  3. Incubator
  4. Polyacrylamide gel electrophoresis system
  5. Vacuum concentrator

Procedure

  1. Footprinting with 3'- or 5'-end-labeled DNA analyzed by piperidine-catalyzed strand scission.
    1. Add 4 μl of 160 mM KMnO4 solution to radiolabeled DNA fragments of interest (40 ng; 5,000 to 10,000 cpm) (in the presence or absence of a binding partner) in a total volume of 40 μl.
    2. Incubate samples for 2 min at 30 °C; mix gently.
    3. Stop reaction by the addition of 4.8 μl β-mercaptoethanol; mix again.
    4. Put samples on ice.
    5. Add 5.3 μl 500 mM EDTA.
    6. Extract samples three times with 100 μl phenol/chloroform by vigorous shaking on a vortex for 1 min each time.
    7. Centrifuge samples (5 min, 3,000 rpm) and take aqueous layer.
    8. Precipitate samples with 2.5 volumes absolute ethanol (-20 °C).
    9. Centrifuge tubes (10 min, 10,000 rpm, 4 °C).
    10. Dissolve wet ethanol pellets in 70 μl of 10% (v/v) piperidine.
    11. Incubate samples at 90 °C for 30 min.
    12. Lyophilize samples in a vacuum concentrator until dry.
    13. Add 30 μl distilled water and lyophilize again.
    14. Repeat step 9.
    15. Dissolve final pellets in 50 μl distilled water.
    16. Add 4 μl 300 mM NaOAc (pH 5.5).
    17. Add 1 μl glycogen (1 μg/μl) to aid precipitation.
    18. Precipitate samples with 2.5 volumes absolute ethanol (-20 °C).
    19. Wash pellets with 150 μl 70% ethanol.
    20. Dry samples briefly in a vacuum concentrator to get rid of excess ethanol and dissolve in 5 μl electrophoresis loading buffer.
    21. Separate on a 10% or 15 % denaturing polyacrylamide gel, respectively, depending on the size of the DNA fragment.
    22. Visualize radioactive bands by autoradiography (over-night X-ray exposure).

  2. Footprinting with non-labeled DNA analyzed by primer extension
    1. Add 4 μl of 160 mM KMnO4 solution to 40 μl non-labeled DNA fragments (~100 ng) in presence or absence of a binding partner.
    2. Follow steps 2 to 8 of procedure A.
    3. Dissolve pellet in 35 μl distilled water.
    4. Add 1 μl of an appropriate 15 to 20mer 5'-32P-labeled desoxyoligonucleotide primer complementary to the downstream sequence of interest (~ 5 x 105 cpm). For convenient resolution the primer should not bind farther than 50 to 80 nucleotides downstream to the region of interest.
    5. Add 4 μl 10 mM NaOH.
    6. Incubate for 2 min at 80 °C.
    7. Put samples on ice for 5 min.
    8. Add 4.5 μl of neutralization solution and mix samples.
    9. Incubate for 3 min at 68 °C for hybridization.
    10. Put samples on ice and add 5 μl of all four dNTPs (5 mM each), 1 unit Klenow fragment of DNA polymerase I and mix gently.
    11. Extension reaction is carried out for 10 min at 50 °C.
    12. Put samples on ice and terminate the reaction by the addition of 17 μl stop mix.
    13. Samples are precipitated with 2.5 volumes ethanol (-20 °C).
    14. Wash pellets with 150 μl 70% ethanol.
    15. Dry samples briefly in a vacuum concentrator to get rid of excess ethanol and dissolve in 5 μl electrophoresis loading buffer.
    16. Separate on a 10% or 15% denaturing polyacrylamide gel, respectively, depending on the size of the cDNA fragments expected.
    17. Visualize radioactive bands by autoradiography (over-night X-ray exposure).

Recipes

  1. KMnO4 stock solution (370 mM)
    KMnO4 (MW = 158.04) has a solubility limit of ~60 g/L (corresponding to 0.37 M).
    A 200 ml stock solution is prepared by adding 12 g KMnO4 to 220 ml distilled water. The solution is boiled until it reaches a final volume of 200 ml. This stock solution can be kept in a dark bottle for several months.
  2. Phenol/Chloroform
    A 1:1 mixture (v/v) of phenol and chloroform, purity grade: pro analysis (p.a.) is used for extraction. The phenol has been saturated with 1 M Tris-HCl (pH 7.9) before and 0.1% (final concentration) 8-Hydroxychinolin should be added to the phenol to avoid oxidation.
  3. Neutralization solution
    0.5 M Tris-HCl (pH 7.2)
    0.1 M MgSO4
    2 mM DTT (optional for procedure B)
  4. dNTP mix
    5 mM each, dATP, dCTP, dGTP, dTTP (optional for procedure B)
  5. Stop mix
    4 M NH4OAc
    20 mM EDTA (optional for procedure B)
  6. Electrophoresis loading buffer
    0.1% (w/v) bromophenol blue
    0.1% xylene cyanol
    95% formamide deionized
    25 mM EDTA (pH 8.0)

Acknowledgments

We like to thank people from the laboratory for helpful discussions. Work from this laboratory was funded by the Deutsche Forschungsgemeinschaft (DFG) SPP 1258 [Wa455/13-2] and [PU 435/1-1].

References

  1. Hayatsu, H. and Ukita, T. (1967). The selective degradation of pyrimidines in nucleic acids by permanganate oxidation. Biochem Biophys Res Commun 29(4): 556-561.
  2. Stratmann, T., Pul, U., Wurm, R., Wagner, R. and Schnetz, K. (2012). RcsB-BglJ activates the Escherichia coli leuO gene, encoding an H-NS antagonist and pleiotropic regulator of virulence determinants. Mol Microbiol 83(6): 1109-1123.
  3. Sasse-Dwight, S. and Gralla, J. D. (1989). KMnO4 as a probe for lac promoter DNA melting and mechanism in vivo. J Biol Chem 264(14): 8074-8081.

简介

KMnO 4足迹法提供了检测和定位双链DNA分子内的单链区域的快速和简单的方法,例如其在活性转录的RNA聚合酶-DNA复合物内或在R期间发生在DNA-RNA杂交结构中形成。该方法基于DNA中单链胸腺嘧啶的选择性氧化。修饰的核苷酸通过开环和随后的磷酸二酯切割与强碱反应。因为修饰的核苷酸不能被DNA聚合酶识别,还可以通过用Klenow DNA聚合酶引物延伸来分析修饰的位点,其在修饰前停止延伸一个残基。因此,修饰的碱基位置的定位可以在变性聚丙烯酰胺凝胶上在哌啶催化磷酸二酯切割3'-或5'-端32 P-端标记的DNA之后进行,或者通过用非 - 使用32 P标记的寡核苷酸引物。由于KMnO 4可以穿透膜的事实,足迹法也可以用于活细胞内的足迹分析。

关键字:单链DNA定位, 单链碱基修饰, DNA足迹, RNA-DNA杂交分析, DNA的结构分析

材料和试剂

  1. 放射性标记的目标DNA片段
  2. 14.3 Mβ-巯基乙醇
  3. 500mM EDTA
  4. 苯酚
  5. 溴酚蓝(Sigma-Aldrich,目录号:BO126)
  6. 二甲苯Cyanol(Sigma-Aldrich,目录号:X4126)
  7. 甲酰胺去离子(Panreac Applichem,目录号:A2156)
  8. Cholorophorm
  9. 哌啶,纯度等级:pro分析(p.a。)(例如,Sigma-Aldrich,目录号:411027)
  10. 乙醇(纯度等级:分析)(p.a.)
  11. X光片
  12. 糖原(1μg/μl)(例如,Roche,目录号:10901393001)
  13. NaOAc(300mM pH = 5.5) 可选用于程序B(引物延伸分析)
  14. 非无线标记的目标DNA片段
  15. 5' - <32> P标记的脱氧寡核苷酸引物
  16. NaOH(10mM)
  17. Tris
  18. MgSO 4 4 /
  19. DTT
  20. DNA聚合酶I的Klenow片段(例如Biolabs,目录号:MO210S)
  21. NH 4 OAc
  22. 使用370mM KMnO 4储备溶液(用H 2 O进行1:1的稀释用于反应)(参见配方)
  23. 苯酚/氯仿(见配方)
  24. 中和解决方案(参见配方)
  25. dNTP mix(参见配方)
  26. 停止混合(参见配方)
  27. 电泳加载缓冲液(参见配方)

设备

  1. 台式离心机
  2. 涡流振动器
  3. 孵化器
  4. 聚丙烯酰胺凝胶电泳系统
  5. 真空浓缩器

程序

  1. 通过哌啶催化的链断裂分析具有3'-或5'-末端标记的DNA的足迹。
    1. 将4μl的160mM KMnO 4溶液加入放射性标记的目的DNA片段(40ng; 5,000至10,000cpm)(在存在或不存在结合配偶体的情况下),总体积为40μl。
    2. 在30°C孵育样品2分钟; 轻轻混匀。
    3. 通过加入4.8μlβ-巯基乙醇停止反应; 再次混合
    4. 将样品放在冰上。
    5. 加入5.3μl500mM EDTA
    6. 用100μl苯酚/氯仿提取样品三次,每次用涡旋振荡振荡1分钟
    7. 离心样品(5分钟,3,000rpm),并取水层
    8. 用2.5倍体积的无水乙醇(-20℃)沉淀样品
    9. 离心管(10分钟,10,000rpm,4℃)
    10. 将湿乙醇沉淀溶于70μl10%(v/v)哌啶中
    11. 在90℃孵育样品30分钟。
    12. 在真空浓缩器中冻干样品直至干燥
    13. 加入30μl蒸馏水,并再次冻干
    14. 重复步骤9.
    15. 将最终的颗粒溶解在50μl蒸馏水中
    16. 加入4μl300mM NaOAc(pH5.5)
    17. 加入1μl糖原(1μg/μl)以帮助沉淀
    18. 用2.5倍体积的无水乙醇(-20℃)沉淀样品
    19. 用150μl70%乙醇洗涤沉淀。
    20. 在真空浓缩器中短暂干燥样品以除去过量的乙醇并溶解在5μl电泳上样缓冲液中
    21. 分别在10%或15%变性聚丙烯酰胺凝胶上,取决于DNA片段的大小
    22. 通过放射自显影(过夜X射线曝光)显现放射性带

  2. 通过引物延伸分析用非标记DNA的足迹
    1. 在存在或不存在结合配偶体的情况下,将4μl160mM KMnO 4溶液加入到40μl非标记的DNA片段(〜100ng)中。
    2. 按照程序A的步骤2至8.
    3. 将沉淀溶于35μl蒸馏水中。
    4. 加入1μl与目的下游序列互补的适当的15至20个寡核苷酸5' - 寡核苷酸32-标记的脱氧寡核苷酸引物(〜5×10 5,/sup> cpm)。 为了方便分辨,引物应该不超过感兴趣区域下游的50至80个核苷酸结合
    5. 加入4μl10mM NaOH
    6. 在80℃下孵育2分钟。
    7. 将样品在冰上5分钟。
    8. 加入4.5μl中和溶液并混合样品
    9. 在68℃孵育3分钟进行杂交
    10. 将样品置于冰上,加入5μl所有四种dNTP(各5mM),1单位DNA聚合酶I的Klenow片段,并轻轻混合。
    11. 延伸反应在50℃下进行10分钟
    12. 将样品置于冰上,通过加入17μl终止混合物终止反应
    13. 样品用2.5体积的乙醇(-20℃)沉淀
    14. 用150μl70%乙醇洗涤沉淀。
    15. 在真空浓缩器中短暂干燥样品以除去过量的乙醇并溶解在5μl电泳上样缓冲液中
    16. 分别在10%或15%变性聚丙烯酰胺凝胶上,取决于预期的cDNA片段的大小。
    17. 通过放射自显影(过夜X射线曝光)显现放射性带

食谱

  1. wmnO 4储备溶液(370mM) KMnO 4(MW = 158.04)的溶解度极限为〜60g/L(对应于0.37M)。
    通过将12g KMnO 4加入220ml蒸馏水中制备200ml储备溶液。 将溶液煮沸直到其达到200ml的最终体积。 该储备溶液可以在黑暗的瓶子中保存几个月。
  2. 苯酚/氯仿
    使用苯酚和氯仿的1:1混合物(v/v),纯度等级:前分析(p.a.)用于提取。 苯酚用1M Tris-HCl(pH7.9)饱和,然后向苯酚中加入0.1%(终浓度)8-羟基喹啉以避免氧化。
  3. 中和溶液
    0.5M Tris-HCl(pH7.2) 0.1M MgSO 4 2 mM DTT(步骤B可选)
  4. dNTP mix
    5mM,dATP,dCTP,dGTP,dTTP(对于方法B可选)
  5. 停止混合
    4 M NH 4 OAc
    20 mM EDTA(对于步骤B可选)
  6. 电泳加载缓冲液
    0.1%(w/v)溴酚蓝
    0.1%二甲苯蓝 95%甲酰胺去离子
    25mM EDTA(pH8.0)

致谢

我们喜欢感谢实验室的人们进行有益的讨论。 该实验室的工作由Deutsche Forschungsgemeinschaft(DFG)SPP 1258 [Wa455/13-2]和[PU 435/1-1]资助。

参考文献

  1. Hayatsu,H.and Ukita,T。(1967)。 高锰酸盐氧化对核酸中嘧啶的选择性降解。生物化学 Res Commun 29(4):556-561。
  2. Stratmann,T.,Pul,U.,Wurm,R.,Wagner,R.and Schnetz,K。(2012)。 RcsB-BglJ激活大肠杆菌 leuO基因,编码一个H- NS拮抗剂和毒力决定子的多效调节剂。 Mol Microbiol 83(6):1109-1123。
  3. Sasse-Dwight,S.and Gralla,J.D。(1989)。 KMnO 作为lac启动子DNA的探针熔解和机制 > in vivo 。 J Biol Chem 264(14):8074-8081。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Pul, Ü., Wurm, R. and Wagner, R. (2012). KMnO4 Footprinting. Bio-protocol 2(21): e280. DOI: 10.21769/BioProtoc.280.
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Kenji Murakami
Stanford Univ.
Thank you very much for your very good protocol.

What is the concentration of primers at the step d in your protocol 2?

What is the purpose to add 10 mM NaOH at the step e in your protocol 2?
4/12/2014 1:02:06 PM Reply