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DNase I Footprinting to Identify Protein Binding Sites
脱氧核糖核酸酶I 足迹法识别蛋白结合位点   

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Abstract

DNase I footprinting is used to precisely localise the position that a DNA binding protein, e.g. a transcription factor, binds to a DNA fragment. A DNA fragment of a few hundred bp is labelled at one end and then incubated with the proteins suspected to bind. After a limited digestion with DNase I, the reaction is quenched, DNA is precipitated and analysed on a denaturing polyacrylamide gel. This protocol uses 32P-radioactively labeled DNA.

Materials and Reagents

  1. Oligonucleotides (usually 20-30 mer) to amplify a suitable fragment (100-400 bp) encompassing the region to be tested for protein binding ability
  2. Plasmid DNA carrying the cloned required region to use as template for the PCR amplification
  3. [γ-32P] ATP (3,000 Ci/mmole, 30 μCi = 3 μl/labeling ) (e.g. NEN, catalog number: BLU502A)
  4. Polynucleotide kinase (PNK) (e.g. Biolabs, catalog number: M0201 )
  5. Agarose
  6. Purified protein (or enriched crude bacterial extracts see below)
  7. DNase I (e.g. Sigma-Aldrich, catalog number: D5025 )
  8. Phenol
  9. Chloroform
  10. Herring sperm DNA (e.g. Sigma-Aldrich, catalog number: D6898 ; Roche, catalog number: 223 646 )
  11. BSA (e.g. Biolabs, catalog number: B9001 )
  12. Acrylamide
  13. Urea
  14. DTTP (e.g. Biolabs, catalog number: N0447 )
  15. Taq polymerase (5 units/μl) (e.g. Biolabs, catalog number: M0267 )
  16. DNA Marker (e.g. 100 bp ladder, Biolabs, catalog number: N3231 )
  17. Antarctic alkaline phosphatase (Biolabs, catalog number: M0296 )
  18. MspI (Biolabs, catalog number: N3032 )
  19. 40% Acrylamide stock (19:1 acrylamide: bis acrylamide) (e.g. Euromedex, catalog number: EU0076-C)
  20. TBE buffer
  21. Binding buffer (see Recipes)
  22. DNase I dilution buffer (see Recipes)
  23. DNase I stop buffer (see Recipes)
  24. DNase I stock (see Recipes)
  25. Loading formamide dyes (see Recipes)
  26. Denaturing Sequencing gel (6% acrylamide) (see Recipes)
  27. Hepes-Glutamate (see Recipes)

Equipment

  1. Suitable space for working with 32P radioactivity
  2. Image quantification apparatus (e.g. Typhoon GE Healthcare Life Sciences; X-ray film and developing materials)
  3. PCR machine
  4. Small horizontal agarose gel apparatus
  5. Transilluminator (preferably 365 nM)
  6. Apparatus for running a 30 cm sequencing gel (e.g. Model S2 Vertical sequencing apparatus, now sold by Biometra)
  7. Power supply capable of producing 2,000 volts and 60 watts)
  8. Geiger counter to monitor for radioactivity and any contamination.
  9. Heating block at 90 °C
  10. Gel drying apparatus

Procedure


I.   Preparation of the labeled DNA fragment

  1. Label the 5' end of one of the oligonucleotides to be used to make the fragment to footprint. Choose the oligo so that the suspected binding site is not too far from the labeled end. Normally the footprint should be performed on both strands of the DNA, i.e. using two DNA fragments labeled at either end. Use a 0.5 ml tube suitable for PCR.
    3 μl Oligo 1, 10 pmoles/μl 
    2 μl 10x PNK buffer (supplied by manufacturer of PNK)
    3 μl [γ- 32P] ATP (3,000 Ci/mmole)
    11 μl H2O
    1 μl (10 units) PNK
    Incubate 30 min 37 °C
    N.B. Take suitable precautions for use of radioactivity. Perform in approved location.
  2. Precipitate the labeled oligo. Add
    80 μl 0.1 M Sodium acetate (natural pH about 9.0)
    1 μl 10 mM Sodium phosphate buffer, pH 7.4
    250 μl ethanol (96%)
    Incubate in dry ice for 30 min (or at -80 °C for > 1 h).
  3. Centrifuge 15 min 4 °C 12,000 x g.
  4. Carefully remove the supernatant.
    N.B. Very radioactive, Discard accordingly.
  5. Rinse the (tiny) pellet with 100 μl 96% ethanol (or 70% ethanol at -20 °C). Centrifuge 5 min 4 °C 12,000 x g.
  6. Carefully remove the supernatant. Dry in vacuo 5 min.
  7. Resuspend the labeled Oligo 1 in 35 μl H2O. Vortex well and give a quick centrifugation to place all labeled oligo in bottom of tube.
  8. Add 5 μl Thermopol buffer (Biolabs or other suitable Taq polymerase buffer).
    5 μl deoxyNTP mix containing 2.5 mM each dATP, dCTP, dGTP, DTTP.
    4 μl Oligo 2 10 pmoles/μl (Oligo 2 corresponds to the other end of fragment to be amplified).
    1 μl template DNA (e.g. plasmid DNA carrying the cloned region to be amplified, about 50 ng depending on size of plasmid. We usually use 1 μl 1/10 dilution of standard mini plasmid DNA preparation).
    Mix well, quick centrifugation and add 0.5 μl (2.5 units) Taq polymerase (5 units/μl) and immediately start PCR.
  9. PCR cycling
    1. Denature 94 °C for 2 min
    2. 94 °C for 30 sec
    3. 55 °C* for 30 sec
    4. 72 °C for 30 sec*
    5. Repeat b-d 25 times
    6. Final extension 5 min 72 °C
    * The temperature of annealing depends upon the oligonucleotides used and the extension time at 72 °C on the length of the fragment amplified. Footprints on fragments greater than 500 bp are not recommended and so 30 sec is usually good for most fragments.
  10. Test 1-2 μl on small agarose gel (containing ethidium bromide or other suitable DNA detection reagent) for amplification of a fragment of the correct size and check you have a fragment of the correct size in good yield (i.e. most or all of the oligos have been used up) 30 pmoles of oligos can give maximally 2 μg of a fragment of 100 bp and 10 μg of a fragment 500 bp. Proceed to purification. In theory you can use the PCR as it is or after passage through a spin column. However any minor, shorter length contaminants will produce artifactual bands in the footprint and the presence of unincorporated radioactivity will not allow you to estimate the amount of radioactive DNA in the footprinting reactions. So we always proceed to purify the labeled DNA from an agarose gel. In exceptional cases e.g. to eliminate a close running contaminant band from a short (100-200 bp) fragment, the fragments can be purified on a native acrylamide gel (see step I-14 below).
  11. Run the PCR mixture, mixed with 10 μl of loading dyes, on a small 1% agarose gel in 50 mM TBE buffer. Usually the whole 50 μl PCR can be loaded in 3 wells.
  12. Visualize the gel on a long wavelength (365 nm) transilluminator (to minimize damage to the DNA by short wavelength) and cut out the agarose containing the radioactive fragment. Discard rest of gel as radioactive waste.
  13. Extract the DNA from agarose using a gel purification kit (e.g. Machery-Nagel Nucleo-spin Gel and PCR clean-up). Elute in 50 μl elution buffer.
  14. To purify the DNA from an acrylamide gel: Run the PCR mix on a native acrylamide gel (5-8% depending upon size in 50 mM TBE at room temperature i.e. do not allow the gel to heat up. Depending upon the size of the apparatus used 100-200 volts should be adequate). Locate the radioactive DNA by short exposure of the wet gel wrapped in Saran wrap to a phosphorimager screen (or X-ray film). The piece of acrylamide containing the radioactive band is cut out and the radioactive DNA eluted by shaking overnight at 37 °C in 1 ml of 0.5 M ammonium acetate, 0.1% SDS, 1 mM EDTA. Separate the aqueous phase from the acrylamide gel piece by centrifugation and transfer to another tube. Extract with 0.5 ml phenol/CHCl3 and precipitate the DNA with 2.5 volumes ethanol in dry ice for at least 30 min. Centrifuge 10 min 4 °C, remove all the supernatant, dry in vacuo 2-3 min) Resuspend in 50 μl elution buffer (as for DNA eluted from agarose.).
  15. Run 1 μl on a new small agarose gel and estimate the quantity by comparison to the staining intensity of marker DNAs (e.g. 100 bp ladder).
  16. Count 1 μl by Kerenkov radiation in a scintillation counter or estimate using a Geiger counter. Expect to have 50,000-150,000 cpm/μl with about 5-50 ng DNA/μl (yield in range 10-50% of the moles of starting oligonucleotide). You can calculate the molar concentration of the DNA fragment from the length of the fragment in bp and using 1 bp corresponds to a molecular mass of 660.


II.  The footprinting reaction

  1. All protein and DNA dilutions are made in 1x binding buffer. Protein dilutions are made at 4 °C to minimize any instability of the protein in dilute solutions. Binding reactions can be made at RT or at 30 °C or 37 °C depending upon the experiment. E.g. Most prokaryotic transcription factors bind DNA at RT. E.coli RNA polymerase will only form open complexes at 37 °C.
  2. The binding buffer we usually use is Hepes-Glutamate. The concentration of K glutamate can be increased or decreased according to the experiment; generally higher salt favors specificity but decreases affinity. Mg++ salts (1-10 mM) can be added according to the experiment, e.g. Mg++ is required for RNA polymerase binding. The BSA is added to stabilize dilute proteins and prevent non-specific absorption to the microfuge tube. The reaction mix in general consists of 20 μl DNA solution to which we add 20 μl of the diluted protein (if several components need to be tested at the same time the volume for each component can be reduced accordingly to give a final volume of 40 μl).
  3. To test a range of protein concentrations for binding to the labeled DNA. Make a suitable volume of binding buffer and keep on ice. Prepare a series of dilution tubes for the protein to test over a range of concentrations. This range depends entirely on the protein under study as binding constants can vary from nM to nearly mM. E.g. for 10 serial dilution of 1/2 concentration each step, Prepare 10 tubes with 25 μl binding buffer on ice.
  4. Prepare a suitable volume of labeled DNA e.g. For 12 reactions prepare 240 μl binding buffer and add labeled DNA fragment to have about 20,000 -100,000 cpm/reaction. Mix well. Ideally this should give a final concentration of the DNA of about 1 nM in the 40 μl footprinting reaction, if binding constants of protein to DNA are in the nM range.
  5. Dispense 20 μl of the DNA mix into 12 1.5 ml microtubes at RT.
  6. Make the protein dilutions on ice and immediately mix the diluted protein with the DNA at RT. E.g. Dilute the protein to 1 μM in 50 μl binding buffer.
    1. Add 20 μl to one tube of DNA (to give final concentration of 500 nM). Mix by pipetting. Leave at RT until finished all mixes.
    2. Take 25 μl of the 1 μM dilution and mix with 25 μl binding buffer in next dilution tube.
    3. Add 20 μl of this dilution to the next DNA tube (final concentration 250 nM).
    4. Take 25 μl of the 0.5 μM dilution and mix with 25 μl binding buffer in next dilution tube.
    5. Continue for the whole series of 10 dilutions.
  7. Add 20 μl 1x binding buffer to two DNA samples for the (essential) controls without protein.
  8. Incubate for 10 min at RT (or at chosen temperature for chosen time).
  9. Meanwhile prepare a suitable dilution of DNase I in DNase I dilution buffer. It is crucial to find the correct DNase I concentration to get limited DNase attack so that not all the DNA is degraded. As there should in theory be no more than 1 break/DNA strand, the majority (80-90%) of the DNA should be full length.
    It is usually necessary to make a pilot experiment with each new stock of DNase I to test a series of dilutions of DNase I with a DNA fragment (in the absence of binding proteins) to find the concentration which gives an adequate ladder but leaves full length DNA at the top of the gel. The amount of DNase I used should be lower for longer DNA fragments. The amount given below is a guideline only.
  10. DNase I stock of 5 mg/ml in DNase I storage buffer (store in aliquots at -20 °C).
    Dilute about 10,000 fold (e.g. 2 μl to 200 μl, in DNase I dilution buffer, then take 2 μl of the first dilution to 200 μl DNase I dilution buffer) to give 0.5 μg/ml.
  11. To make the footprint reactions:
    T= 0 add 4 μl dilute DNase I to the footprint mix 1. Mix gently by pipetting.
    T= 15 sec Add 4 μl dilute DNase I to the footprint mix 2. Mix gently by pipetting.
    T= 30 sec Add 4 μl dilute DNase I to the footprint mix 3. Mix gently by pipetting.
    T= 45 sec Add 4 μl dilute DNase I to the footprint mix 4. Mix gently by pipetting.
    T= 60 sec Add 100 μl phenol pH 8.0 to the footprint mix 1. Vortex to stop reaction
    T= 75 sec Add 100 μl phenol pH 8.0 to the footprint mix 2. Vortex to stop reaction.
    T= 90 sec Add 100 μl phenol pH 8.0 to the footprint mix 3. Vortex to stop reaction.
    T= 105 sec Add 100 μl phenol pH 8.0 to the footprint mix 4. Vortex to stop reaction.
    Repeat until all the tubes have been treated.
    Precise timings are crucial to get precise and comparable DNase I digestions in each lane (some researchers prefer to use longer times and more dilute DNase I).
  12. Add 200 μl DNase I STOP solution to each reaction. Vortex well.
  13. Centrifuge 10 min RT.
  14. Transfer the aqueous upper phase (careful not to take any phenol) to a clean 1.5 ml microfuge tube.
  15. Add 600 μl 96% ethanol. Mix well and incubate in dry ice for 1 h (or overnight at -80 °C).
  16. Centrifuge 15 min, 4 °C, 12,000 x g.
  17. Remove the supernatant carefully with a pipette and 1 ml pipette tip. Depending upon the number of cpm used per reaction and the sensitivity of the available Geiger counter, verify that some radioactivity is in the (invisible) pellet with a Geiger counter. If the herring sperm DNA, used in the DNase I STOP to aid precipitation, has not been sufficiently sonicated or too much has been used, the DNA pellet might not adhere to the microfuge tube and can be lost with the ethanol.
  18. Centrifuge again, 5 min 4 °C. Remove the rest of the liquid with a 20 μl pipette tip. Verify that the radioactivity is still in the tube.
  19. Dry in vacuo 5 min.
  20. Resuspend in 5 μl H2O and add 6 μl gel loading formamide dyes (deionized formamide with Bromophenol blue and Xylene cyanol). Vortex well. Quick centrifuge to put all liquid in bottom of tube.
  21. Heat to 90 °C for 2 min. Quench in ice and immediately load (5 μl) onto a denaturing (7 M urea) acrylamide sequencing gel, acrylamide (19:1 acrylamide: bis-acrylamide), which has been prerun for 30 min to 1 h to get hot. Voltage and wattage depends upon the apparatus used. We use 60 watts for the Model S2. Use with 5-10% final concentration acrylamide depending on the size of the fragment under investigation.
  22. To locate the site of protein binding it is necessary to calibrate gels with molecular weight markers. E.g. DNA marker pBR322 digested with MspI, treated with alkaline phosphatase (e.g. Antarctic alkaline phosphatase) and labeled with [γ-32P] ATP and PNK or a sequencing ladder prepared using the same Oligo 1, which was used for the radioactive labeling of the fragment.
  23. Electrophorese at 60 watts for a 30 x 30 plates and 1 mM thick gels using the Model S2 apparatus. Adjust volts and power for other sized gels. Time of migration depends upon the size of the fragment and expected position of protein binding sites (generally 80 min to 3 h).
  24. After migration, allow to cool slightly, open plates, transfer the gel to Whatman 3 mm paper. Cover with Saran wrap and dry on a gel drying apparatus.
  25. Put the dried gel, still covered with Saran to expose in a Phosphorimager cassette overnight (alternatively expose to X-ray film with an intensifying screen).
  26. The pattern of DNase I cleavages with and without protein indicates the site of protein binding (Figure 1). Deformations such as bent or looped DNA are indicated by hypersensitive cleavages compared to the control (free DNA), since bending of the DNA has facilitated attack by DNase I in the wider minor groove formed on the outside of the loop or bend (Figure 2).


    Figure 1. Principle of DNase I footprinting



    Figure 2. Example of DNase I footprinting

  27. Using the Image Quant program, it is possible to quantify the amount of radioactivity in specific bands or regions, corresponding to protein protected sites and non-protected regions, and hence to derive binding constants for the proteins.

Recipes

  1. Binding buffer
    25 mM Hepes (pH 8.0)
    100 mM K glutamate (pH 8.0)
    0.5 mg/ml BSA
  2. DNase I dilution buffer
    10 mM Tris (pH 8.0)
    10 mM MgCl2
    10 mM CaCl2
    125 mM KCl
    0.1 mM DTT
  3. DNase I stop buffer
    0.5 M Na acetate pH 5.0
    10 μg/ml DNA (e.g. sonicated herring sperm DNA. Suspend DNA in 10 mg ml-1 H2O and allow to hydrate overnight at 4 °C. Sonicate until the solution loses all viscosity.)
    2.5 mM EDTA
  4. DNase I stock e.g. 5 mg/ml in
    50% glycerol
    100 mM NaCl
    10 mM Tris (pH 8.0)
    10 mM MgCl2
  5. Loading formamide dyes
    1 ml deionized formamide
    10 μl 5% solution xylene cyanol and bromophenol blue.
  6. Denaturing Sequencing gel (6% acrylamide)
    6% acrylamide (19:1 acrylamide: bis acrylamide) (dilute from 40% stock)
    7 M urea
    1x TBE
    For 80 ml of acrylamide/urea/TBE mixture add 0.4 ml 10% ammonium persulphate and 40 μl TEMED to polymerise the acrylamide, mix gently and carefully pour between the gel plates (0.4 mM thick spacers) avoiding bubbles. Insert a comb to make the wells and leave to polymerise horizontally.
  7. Hepes-Glutamate
    25 mM Hepes (pH 8.0)
    100 mM K glutamatew
    0.5 mg/ml BSA

Acknowledgments

The use of DNAse I to identify protein bindinging sites on DNA was first described by Galas and Schmitz (1978). Since then it has been exploited and adapted in very many laboratories. Our protocol is based on that in use in the laboratory of Annie Kolb (Colland et al., 2000; Marschall et al., 1998), who introduced the use of potassium glutamate in the binding buffer, which mimics in vivo conditions and increases the affinity of most proteins for their DNA targets. We are extremely grateful to Annie Kolb for her continuous advice and interest. Work in our lab has been funded by the Centre National de Recherche Scientifique (CNRS) to UPR9073 (now renamed FRE3630), by Université Paris 7, Denis Diderot; Agence National de Recherche [(ANR-09-Blanc 0399 (GRONAG)] and by the "Initiative d'Excellence" program from the French state [ANR-11-LBX-0011-01 (DYNAMO)].

References

  1. Brenowitz, M., Senear, D. F., Shea, M. A. and Ackers, G. K. (1986). Quantitative DNase footprint titration: a method for studying protein-DNA interactions. Methods Enzymol 130: 132-181.
  2. Brechemier-Baey, D., Dominguez-Ramirez, L. and Plumbridge, J. (2012). The linker sequence, joining the DNA-binding domain of the homologous transcription factors, Mlc and NagC, to the rest of the protein, determines the specificity of their DNA target recognition in Escherichia coli. Mol Microbiol 85(5): 1007-1019.
  3. Colland, F., Barth, M., Hengge-Aronis, R. and Kolb, A. (2000). Sigma factor selectivity of Escherichia coli RNA polymerase: role for CRP, IHF and lrp transcription factors. EMBO J 19(12): 3028-3037.
  4. El Qaidi, S. and Plumbridge, J. (2008). Switching control of expression of ptsG from the Mlc regulon to the NagC regulon. J Bacteriol 190(13): 4677-4686. 
  5. Galas, D. J. and Schmitz, A. (1978). DNase footprinting: a simple method for the detection of protein-DNA binding specificity. Nucleic Acids Res 5(9): 3157-3170.
  6. Marschall, C., Labrousse, V., Kreimer, M., Weichart, D., Kolb, A. and Hengge-Aronis, R. (1998). Molecular analysis of the regulation of csiD, a carbon starvation-inducible gene in Escherichia coli that is exclusively dependent on sigma s and requires activation by cAMP-CRP. J Mol Biol 276(2): 339-353.
  7. Plumbridge, J. and Kolb, A. (1991). CAP and Nag repressor binding to the regulatory regions of the nagE-B and manX genes of Escherichia coli. J Mol Biol 217(4): 661-679. 

简介

DNase I足迹用于精确定位DNA结合蛋白例如转录因子结合DNA片段的位置。 在一端标记几百bp的DNA片段,然后与怀疑结合的蛋白质一起孵育。 在用DNA酶I有限消化后,淬灭反应,沉淀DNA并在变性聚丙烯酰胺凝胶上分析。 该方案使用32-放射性标记的DNA。

材料和试剂

  1. 用于扩增合适片段(100-400bp)的寡核苷酸(通常为20-30mer),包括待测蛋白质结合能力的区域
  2. 携带克隆的所需区域的质粒DNA用作PCR扩增的模板
  3. [γ-sup 32 P] ATP(3000Ci/mmole,30μCi=3μl/标记)(例如,NEN,目录号:BLU502A)
  4. 多核苷酸激酶(PNK)(例如Biolabs,目录号:M0201)
  5. 琼脂糖
  6. 纯化蛋白(或富集的粗细菌提取物见下文)
  7. DNase I(例如Sigma-Aldrich,目录号:D5025)
  8. 苯酚
  9. 氯仿
  10. 鲱鱼精子DNA(例如Sigma-Aldrich,目录号:D6898; Roche,目录号:223 646)
  11. BSA(例如Biolabs,目录号:B9001)
  12. 丙烯酰胺
  13. 尿素
  14. DTTP(例如Biolabs,目录号:N0447)
  15. Taq聚合酶(5单位/μl)(例如Biolabs,目录号:M0267)
  16. DNA标记(例如 100bp阶梯,Biolabs,目录号:N3231)
  17. 南极碱性磷酸酶(Biolabs,目录号:M0296)
  18. MspI(Biolabs,目录号:N3032)
  19. 40%丙烯酰胺原料(19:1丙烯酰胺:双丙烯酰胺)(例如Euromedex,目录号:EU0076-C)
  20. TBE缓冲区
  21. 绑定缓冲区(参见配方)
  22. DNase I稀释缓冲液(参见配方)
  23. DNase I停止缓冲区(参见配方)
  24. DNase I股票(见配方)
  25. 装载甲酰胺染料(参见配方)
  26. 变性测序凝胶(6%丙烯酰胺)(参见配方)
  27. Hepes-Glutamate(参见食谱)

设备

  1. 适合使用 32 P放射性的空间
  2. 图像量化设备(例如 Typhoon GE Healthcare Life Sciences; X光片和显影材料)
  3. PCR机
  4. 小平面琼脂糖凝胶装置
  5. 透照器(优选365nM)
  6. 用于运行30cm测序凝胶的装置(例如现在由Biometra销售的S2型垂直测序装置)
  7. 电源能够产生2000伏和60瓦)
  8. 盖革计数器监测放射性和任何污染
  9. 加热块在90℃下
  10. 凝胶干燥装置

程序


I.   标记的DNA片段的制备

  1. 标记用于使片段占据的寡核苷酸之一的5'端。 选择寡核苷酸,使可疑的结合位点不远离标记末端。 通常的占位面积应该是 在两条链的DNA上进行,即使用两端标记的两个DNA片段。 使用0.5 ml适合PCR的管。
    3μlOligo 1,10 pmoles /μl
    2μl10x PNK缓冲液(由PNK制造商提供)
    3μl[γ-32 P] ATP(3,000Ci/mmole) 11μlH 2 O 2/b 1μl(10 units)PNK
    在37℃下孵育30分钟
    N.B. 采取适当的放射性使用预防措施。 在批准的位置执行。
  2. 沉淀标记的寡核苷酸。 添加
    80μl0.1M乙酸钠(天然pH约9.0) 1μl10mM磷酸钠缓冲液,pH7.4 250μl乙醇(96%)
    在干冰中孵育30分钟(或在-80℃下进行> 1小时)
  3. 离心15分钟4°C 12,000 x g
  4. 小心地清除上清液。
    N.B.非常放射性,因此舍弃。
  5. 用100μl96%乙醇(或-20%的70%乙醇)冲洗(微小)沉淀。离心5分钟4℃12,000×g 。
  6. 小心取出上清液。真空干燥5分钟。
  7. 将标记的Oligo 1重悬在35μlH 2 O中。涡旋并快速离心以将所有标记的寡核苷酸置于管底部。
  8. 加入5μlThermopol缓冲液(Biolabs或其他合适的Taq聚合酶缓冲液) 5μl含有2.5mM dATP,dCTP,dGTP,DTTP的脱氧NTP混合物 4μlOligo 2 10 pmoles /μl(Oligo 2对应于待扩增片段的另一端)。
    1μl模板DNA(例如,携带待扩增的克隆区域的质粒DNA,约50ng,取决于质粒的大小)通常使用1μl1/10稀释的标准微型质粒DNA制备物) br /> 混匀,快速离心,加入0.5μl(2.5 units)Taq聚合酶(5单位/μl),立即开始PCR。
  9. PCR循环
    1. 94℃变性2分钟
    2. 94℃30秒
    3. 55℃* 30秒
    4. 72℃,30秒*
    5. 重复b-d 25次
    6. 最终延伸5分钟72℃
    *退火温度取决于所用的寡核苷酸和72℃下扩增片段长度的延伸时间。不建议在大于500 bp的碎片上使用足迹,因此30秒通常适用于大多数碎片
  10. 在小琼脂糖凝胶(含有溴化乙锭或其他合适的DNA检测试剂)上测试1-2μl用于扩增正确大小的片段,并检查您是否具有良好产量的正确大小的片段(即大多数或所有寡核苷酸已被用尽),30 pmol寡核苷酸可以产生最多2μg100bp的片段和10μg500bp的片段。继续纯化。理论上,你可以使用PCR,或者在通过离心柱后。然而,任何较小的,较短长度的污染物将在印迹中产生人为的条带,并且存在未并入的放射性将不允许估计足迹反应中的放射性DNA的量。因此,我们总是从琼脂糖凝胶中纯化标记的DNA。在例外情况下,例如为了从短(100-200bp)片段消除接近运行的污染带,可以在天然丙烯酰胺凝胶上纯化片段(参见下面的步骤I-14)。
  11. 运行PCR混合物,与10微升加载染料,在50%TBE缓冲液的小1%琼脂糖凝胶上。通常整个50μlPCR可以加载到3个孔中。
  12. 在长波长(365 nm)透照仪(以最小化短波长对DNA的损害)可视化凝胶,并切出含有放射性片段的琼脂糖。丢弃其余的凝胶放射性 浪费
  13. 使用凝胶纯化试剂盒(例如Machery-Nagel Nucleo-spin Gel和PCR清洗)从琼脂糖中提取DNA。在50μl洗脱缓冲液中洗脱
  14. 为了从丙烯酰胺凝胶纯化DNA:在天然丙烯酰胺凝胶上进行PCR混合物(5-8%,取决于在室温下50mM TBE中的大小,即不允许凝胶升温。根据所使用的设备的尺寸,100-200伏特应当是足够的)。通过短暂暴露包裹在Saran包装中的湿凝胶到phosphorimager筛(或X射线胶片)来定位放射性DNA。切下含有放射性条带的丙烯酰胺片段,通过在37℃下在1ml的0.5M乙酸铵,0.1%SDS,1mM EDTA中振荡过夜来洗脱放射性DNA。通过离心将水相与丙烯酰胺凝胶块分离并转移到另一个管中。用0.5ml苯酚/CHCl 3提取,并用2.5体积的乙醇在干冰中沉淀DNA至少30分钟。离心10分钟4℃,除去所有上清液,真空干燥2-3分钟)重悬于50μl洗脱缓冲液(从琼脂糖洗脱的DNA)。
  15. 在新的小琼脂糖凝胶上运行1μl,并通过与标记DNA的染色强度(例如100bp梯度)相比较来估计数量。
  16. 在闪烁计数器中通过Kerenkov辐射计数1μl或使用盖革计数器估计。预期具有约50,000-150,000cpm /μl和约5-50ng DNA /μl(产率范围为起始寡核苷酸的摩尔数的10-50%)。您可以根据片段长度以bp计算DNA片段的摩尔浓度,使用1bp对应于660的分子量。


II。 足迹反应

  1. 所有蛋白和DNA稀释液在1×结合缓冲液中制备。在4℃下制备蛋白质稀释液以使蛋白质在稀释溶液中的任何不稳定性最小化。结合反应可以在RT或在30℃或37℃进行,这取决于实验。 例如大多数原核转录因子在RT结合DNA。大肠杆菌 RNA聚合酶仅在37℃下形成开放复合物。
  2. 我们通常使用的结合缓冲液是Hepes-谷氨酸。谷氨酸钾的浓度可以根据实验增加或减少;通常较高的盐有利于特异性但降低亲和力。可以根据实验添加Mg ++盐(1-10mM),例如,RNA聚合酶结合需要Mg ++。加入BSA以稳定稀释的蛋白质并防止微量离心管的非特异性吸收。反应混合物通常由20μlDNA溶液组成,向其中加入20μl稀释的蛋白质(如果需要同时测试多种组分,则每种组分的体积可相应减少,以使最终体积为40μl )。
  3. 测试一系列蛋白质浓度与标记的DNA的结合。制备适当体积的结合缓冲液,并保持在冰上。准备一系列稀释管的蛋白质,以测试一系列浓度。该范围完全取决于所研究的蛋白质,因为结合常数可以从nM变化到几乎mM。 例如对于每步1/2浓度的10次连续稀释,在冰上用25μl结合缓冲液制备10管。
  4. 制备合适体积的标记的DNA例如。对于12个反应,制备240μl结合缓冲液,并加入标记的DNA片段以具有约20,000-100,000cpm /反应。混合好。理想的情况是,如果蛋白质与DNA的结合常数在nM范围内,则在40μl覆盖反应中应该使DNA的最终浓度为约1nM。
  5. 在RT下将20μlDNA混合物分配到12个1.5ml微量管中
  6. 使蛋白质稀释在冰上,立即混合稀释的蛋白质与DNA在室温。 例如在50μl结合缓冲液中稀释蛋白质至1μM。
    1. 加入20微升到一个DNA管(最终浓度为500 nM)。通过吸移混合。离开RT,直到完成所有混合。
    2. 取25μl的1μM稀释液,并与25μl结合缓冲液在下一个稀释管混合。
    3. 加入20μl此稀释液到下一个DNA试管(终浓度250 nM)
    4. 取25μl的0.5μM稀释液,并与25μl结合缓冲液在下一个稀释管混合。
    5. 继续整个10个稀释系列。
  7. 加入20μl1x结合缓冲液的两个DNA样品的(必需)控件没有蛋白质。
  8. 在室温下孵育10分钟(或在选定的温度下选择时间)。
  9. 同时在DNase I稀释缓冲液中制备合适的DNase I稀释液。至关重要的是找到正确的DNA酶I浓度,以获得有限的DNA酶攻击,从而不是所有的DNA都被降解。因为在理论上应该不超过1条断裂/DNA链,所以大部分(80-90%)的DNA应该是全长的。
    通常需要用每种新的DNase I储备液进行试验性实验,以用DNA片段(在没有结合蛋白质的情况下)测试一系列DNase I稀释液,以发现产生足够梯度但留下全长的浓度DNA在凝胶的顶部。对于更长的DNA片段,使用的DNase I的量应该更低。以下给出的数额仅供参考
  10. DNase I储备液,在DNase I储存缓冲液中为5mg/ml(以等分试样在-20℃下储存)。
    在DNase I稀释缓冲液中稀释约10,000倍(例如,2μl至200μl,然后取2μl的第一稀释液至200μlDNA酶I稀释缓冲液),得到0.5μg/ml。
  11. 要进行足迹反应:
    T = 0加入4μl稀释的DNA酶I到足迹混合1.通过移液轻轻混合。
    T = 15秒将4μl稀释的DNase I加入足迹混合物2.用移液器轻轻混匀 T = 30秒将4μl稀释的DNase I加入足迹混合物3.用移液器轻轻混匀 T = 45秒将4μl稀释的DNase I加入足迹混合物4.通过移液轻轻混匀 T = 60秒将100μl苯酚pH 8.0加入足迹混合物1.涡旋以停止反应
    T = 75秒将100μl苯酚pH 8.0加入足迹混合物2.涡旋以停止反应 T = 90秒将100μl苯酚pH 8.0加入足迹混合物3.涡旋以停止反应 T = 105秒将100μl苯酚pH 8.0加入足迹混合物4.涡旋以停止反应 重复,直到所有的管被处理 精确的时间对于在每个泳道中获得精确和可比较的DNA酶I消化至关重要(一些研究者喜欢使用更长的时间和更稀释的DNA酶I)。
  12. 每个反应加入200μlDNase I STOP溶液。 旋涡。
  13. 离心10分钟RT
  14. 将上层水相(小心不要取任何苯酚)转移到干净的1.5 ml微量离心管中
  15. 加入600μl96%乙醇。 充分混合并在干冰中孵育1小时(或在-80℃下过夜)
  16. 离心15分钟,4℃,12,000×g。
  17. 用移液管和1 ml移液管吸头小心地除去上清液。根据每次反应使用的cpm数量和可用的盖革计数器的灵敏度,使用盖革计数器验证一些放射性在(不可见)沉淀中。如果用于DNase I STOP以帮助沉淀的鲱鱼精DNA没有被充分超声处理或使用太多,则DNA沉淀可能不附着于微量离心管并且可能与乙醇一起丢失。
  18. 再次离心,5℃4℃。用20μl移液器吸头除去其余的液体。验证放射性仍在管中。
  19. 真空干燥5分钟。
  20. 重悬在5μlH 2 O中,加入6μl凝胶负载甲酰胺染料(去离子甲酰胺,溴酚蓝和二甲苯蓝)。涡旋井。快速离心机将所有液体置于试管底部
  21. 加热至90℃2分钟。在冰中猝灭并立即加载(5μl)到变性(7M尿素)丙烯酰胺测序凝胶,丙烯酰胺(19:1丙烯酰胺:双丙烯酰胺)上,其已经预热30分钟至1小时以变热。电压和功率取决于所使用的装置。我们对S2型使用60瓦。使用5-10%最终浓度的丙烯酰胺,取决于所研究的片段的大小
  22. 为了定位蛋白质结合位点,有必要用分子量标记物校准凝胶。例如,用MspI消化的DNA标记物pBR322,用碱性磷酸酶处理(例如 南极 碱性磷酸酶),并用[γ-32 P] ATP和PNK标记或使用用于片段的放射性标记的相同Oligo 1制备的测序梯。
  23. 对于30×30板和1mM厚凝胶,使用S2型装置以60瓦特进行电泳。调整电压和功率为其他大小的凝胶。迁移时间取决于片段的大小和蛋白质结合位点的预期位置(通常为80分钟至3小时)。
  24. 迁移后,允许稍微冷却,打开板,将凝胶转移到Whatman 3 mm纸。用Saran包裹并在凝胶干燥装置上干燥
  25. 放置干燥的凝胶,仍然用Saran覆盖,在Phosphorimager暗盒中暴露过夜(或者用增强屏幕暴露于X-射线胶片)。
  26. 具有和不具有蛋白质的DNA酶I切割的模式指示蛋白质结合的位点(图1)。与对照(游离DNA)相比,由于DNA的弯曲促进DNA酶I在环或弯曲外侧形成的更宽的小沟中的攻击,变形如弯曲或环状DNA由过敏性裂解指示(图2) 。


    图1. DNase I足迹原则


    图2. DNase I足迹示例

  27. 使用Image Quant程序,可以量化的量 特定条带或区域中的放射性,对应于蛋白质 保护位点和非保护区,因此得到结合 蛋白质的常数

食谱

  1. 绑定缓冲区
    25mM Hepes(pH8.0) 100mM K谷氨酸(pH8.0) 0.5 mg/ml BSA
  2. DNase I稀释缓冲液
    10mM Tris(pH8.0) 10mM MgCl 2/
    10mM CaCl 2
    125 mM KCl
    0.1 mM DTT
  3. DNase I停止缓冲区
    0.5M乙酸钠pH5.0 / 10μg/ml DNA(例如超声处理的鲱鱼精子DNA)悬浮在10mg ml -1 H-H 2 O中的DNA,并允许水合过夜 超声处理,直至溶液失去所有粘度。)
    2.5mM EDTA
  4. 例如在
    中5mg/ml的DNA酶I储备液 50%甘油 100 mM NaCl
    10mM Tris(pH8.0) 10mM MgCl 2/
  5. 装载甲酰胺染料
    1ml去离子甲酰胺 10μl5%溶液二甲苯蓝和溴酚蓝
  6. 变性测序凝胶(6%丙烯酰胺)
    6%丙烯酰胺(19:1丙烯酰胺:双丙烯酰胺)(用40%原料稀释) 7 M尿素
    1x TBE
    对于80ml丙烯酰胺/尿素/TBE混合物,加入0.4ml 10%过硫酸铵和40μlTEMED以聚合丙烯酰胺,轻轻混合并小心地倾倒在凝胶板(0.4mM厚的间隔物)之间,避免气泡。插入梳子,使井和水平聚合。
  7. 肝 - 谷氨酸
    25mM Hepes(pH8.0) 100mM K谷氨酸 0.5 mg/ml BSA

致谢

Galas和Schmitz(1978)首次描述了使用DNAse I鉴定DNA上的蛋白质结合位点。从那时起,它已经在许多实验室中被利用和改编。我们的方案是基于在Annie Kolb的实验室中使用的(Colland等人,2000; Marschall等人,1998),他们介绍了使用钾谷氨酸盐,其模拟体内条件并增加大多数蛋白质对其DNA靶标的亲和力。我们非常感谢Annie Kolb不断的建议和兴趣。我们实验室的工作由国家科学研究中心(CNRS)资助,UPR9073(现改名为FRE3630),巴黎7大学,丹尼斯狄德罗大学;和来自法国的"卓越倡议"计划[ANR-11-LBX-0011-01(DYNAMO)]。

参考文献

  1. Brenowitz,M.,Senear,D.F.,Shea,M.A。和Ackers,G.K。(1986)。 定量DNase足迹滴定:一种研究蛋白质-DNA相互作用的方法。 Methods Enzymol 130:132-181
  2. Brechemier-Baey,D.,Dominguez-Ramirez,L.and Plumbridge,J。(2012)。 将同源转录因子(Mlc和NagC)的DNA结合结构域连接的接头序列其余的蛋白质决定了它们在大肠杆菌中的DNA靶标识别的特异性。


    Mol Microbiol 85(5):1007-1019。 >
  3. Colland,F.,Barth,M.,Hengge-Aronis,R。和Kolb,A。(2000)。 大肠杆菌RNA聚合酶的西格玛因子选择性:CRP,IHF和lrp转录因子的作用。 a> EMBO J 19(12):3028-3037
  4. El Qaidi,S.and Plumbridge,J。(2008)。 将ptsG的表达从Mlc调节子切换到NagC调节子。 J Bacteriol 190(13):4677-4686。
  5. Galas,D.J。和Schmitz,A。(1978)。 DNA酶足迹:检测蛋白质-DNA结合特异性的简单方法。 em> Nucleic Acids Res 5(9):3157-3170
  6. Marschall,C.,Labrousse,V.,Kreimer,M.,Weichart,D.,Kolb,A.and Hengge-Aronis,R。(1998)。 csiD的调节的分子分析,csiD是一种碳饥饿诱导型基因,在大肠杆菌中是完全依赖的 并且需要通过cAMP-CRP激活。 J Mol Biol 276(2):339-353。
  7. Plumbridge,J。和Kolb,A。(1991)。 CAP和Nag阻遏物与 nagE-B 的调节区域结合, 和 manX 基因 。 J Mol Biol 217(4):661-679。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Gaugué, I., Bréchemier-Baey, D. and Plumbridge, J. (2013). DNase I Footprinting to Identify Protein Binding Sites. Bio-protocol 3(14): e824. DOI: 10.21769/BioProtoc.824.
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