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DNA Fragmentation Analysis
DNA片段化分析

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Abstract

DNA fragmentation with length corresponding to multiple integer of approximately 180 base pairs is a distinct feature of apoptosis in animals and programmed cell death in plants. This feature can simply be detected by DNA gel electrophoresis followed by ethidium bromide staining, although in some cases it is difficult to distinguish the DNA laddering. We herein describe a protocol to detect a programmed cell death-associated DNA laddering of plant tissues. After agarose-gel electrophoresis of genomic DNA, Southern hybridization using DIG-labeled genomic DNA probe is performed, that improves detection of DNA laddering.

Keywords: Programmed cell death(程序性细胞死亡), DNA laddering(DNA梯), Southern hybridization(Southern杂交)

Materials and Reagents

  1. Fresh plant tissues
  2. Nucleon PhytoPure DNA extraction kits (General Electric Company, catalog number: RPN8510 )
  3. GeneMate LE Agarose (BioExpress, catalog number: E-3120 )
  4. Biodyne Plus Nylon Membrane (Pall, catalog number: 60406 )
  5. Wizard DNA Clean-Up System (Promega, catalog number: A7280 )
  6. DIG-High Prime DNA Labeling and Detection Starter Kit II (Roche Diagnostics, catalog number: 11585614910 )
  7. X-ray film
  8. Saline sodium citrate (SSC)
  9. Depurination solution (see Recipes)
  10. Denaturation solution (see Recipes)
  11. Neutralization solution (see Recipes)
  12. 1x TAE buffer (see Recipes)

Equipment

  1. Plastic container for gel and membrane treatment
  2. Centrifuge (Eppendorf, model: 5415R )
  3. Spectrophotometer (Shimadzu, model: BioSpec-nano )
  4. Shaker (BIO CRAFT, model: BC-700 )
  5. Vacuum transfer apparatus (BIO CRAFT, model: BS-31 )
  6. Hybridization oven and UV crosslinker (UVP, model: HybriLinker HL-2000 )
  7. Vacuum concentrator (SavantTM, model: DNA SpeedVac DNA110 )
  8. Hybridization incubator (TAITEC, model: HB-80 )

Procedure

  1. Grind plant tissues in liquid nitrogen and extract genomic DNA using PhytoPure according to user manual.
    Notes:
    1. Isopropanol precipitation is done at -20 °C for at least 30 min.
    2. Other plant genomic DNA isolation procedures are acceptable.
  2. Quantity and quality of the isolated genomic DNAs are checked by spectrophotometer and/or agarose gel electrophoresis.
    Note: We used DNAs with OD260/280 > 1.8.
  3. 1-5 μg of genomic DNAs are loaded to a 2% agarose gel.
    Note: 100-bp ladder marker is also loaded to estimate the molecular weight of fragmented DNA.
    Note: Smaller amount (< 1 μg) of DNA may be possible to detect, although we have never tried it.
  4. Electrophoresis at 50 volt for 2 h in 1x TAE buffer.
    Note: We used an agarose gel of 110 x 100 x 6 mm.
  5. Ethidium bromide staining and exposure to ultraviolet light.
    Note: Check the positions of the bands of molecular marker in the gel.
  6. Place the agarose gel into a plastic container in depurination solution (250 ml) and gently agitate on a shaker for 10 min (or until bromophenol blue turned yellow).
    Note: The depurination solution partially hydrolyzes large DNA fragments to help sufficient transfer of DNA fragments.
  7. Rinse the gel by dH2O for three times.
  8. Treat the gel in denaturation solution (250 ml) for 15 min twice.
    Note: The denaturation solution denatures double-stranded DNA for efficient hybridization.
  9. Rinse the gel by dH2O for three times.
  10. Treat the gel in neutralization solution (250 ml) for 15 min twice.
    Note: The neutralization solution neutralizes pH of the gel.
  11. Place the gel on a vacuum transfer apparatus with Biodyne Plus membrane presoaked in 20x SSC buffer and perform transfer for 2 h according to the manufacturer’s instructions.
    Note: Filter blotting using 20x SSC is optional.
  12. Fix the DNA to the membrane by UV crosslinking and wash the membrane with sterilized dH2O.
    Notes:
    1. The membrane is wrapped in Saran Wrap prior to exposure to UV.
    2. UV energy dosage is 120 mJ/cm2.
  13. Dry the membrane.
    Note: This dried membrane can be stored in wrap at 4 °C.
  14. Preparation of DIG-labeled probe.
    1. Digest 2 μg of genomic DNA by 50 units of restriction enzyme at 37 °C for 12 h.
      Note: 4-base cutters (e.g. HaeIII, MspI) are suitable.
    2. Purify the DNA using Wizard DNA Clean-Up System.
      Note: Phenol-chloroform extraction and ethanol precipitation is optional.
    3. Dry the purified DNA solution using vacuum concentrator.
    4. Resolve the DNA with 16 μl of sterilized dH2O.
    5. Boil the DNA sample for 10 min and chill on ice to maintain the denatured single-strand DNA.
    6. Add 4 μl of DIG-High Prime (5x conc.) and mix.
    7. Incubate for 20 h at 37 °C.
    8. Stop the reaction by heating to 65 °C for 10 min.
    9. The reaction mixture can be stored at -20 °C.
  15. Place the membrane in a hybridization bottle and perform pre-hybridization for at least one hour and hybridization over-night using DIG Easy Hyb at 44 °C in a hybridization oven.
    Note: In the case of the probe above mentioned, 2-3 μl of probe is added to the 5 ml hybridization buffer.
  16. Wash the membrane into a plastic container with 1x SSC and 0.2% SDS at 68 °C in a hybridization incubator for 15 min twice.
    Note: Shake the container as fast as possible, making sure that the membrane keeps moving, and the solution should be changed every time.
  17. Wash the membrane into a plastic container with 0.1x SSC and 0.5% SDS at 68 °C in a hybridization incubator for 20 min twice.
  18. Perform detection according to the DIG-High Prime DNA Labeling and Detection Starter Kit II user manual.
  19. Expose chemiluminescent signal to X-ray film or to an imager. DNA laddering is visualized (Figure 1).


    Figure 1. Detection of DNA laddering. Seedlings of Arabidopsis wild-type (WT) and ire1a/b mutant were treated with tunicamycin (5 µg/ml) for 48 h. (A) Ethidium bromide staining of genomic DNAs extracted form aerial parts of seedlings. M, 100-bp ladder marker. (B) Southern blot of the agarose gel in (A) hybridized with DIG-labeled HaeIII/MspI-digested Arabidopsis genomic DNA probe.

Recipes

  1. Depurination solution (500 ml)
    Mix 25 ml of 5 N HCl (0.25 N) with 475 ml of dH2O
    Stored at room temperature
  2. Denaturation solution (500 ml)
    10 g of NaOH (0.5 N)
    43.83 g of NaCl (1.5 M)
    Add dH2O to 500 ml
    Stored at room temperature
  3. Neutralization solution (500 ml)
    30.3 g of Tris (0.5 M)
    87.66 g of NaCl (3 N)
    Add dH2O to 450 ml
    Adjust pH to 7.5 by conc. HCl
    Add dH2O to 500 ml
    Stored at room temperature
  4. 1x TAE buffer
    40 mM Tris-acetate
    1 mM EDTA

Acknowledgments

This protocol was adapted from Mishiba et al. (2013).

References

  1. Mishiba, K., Nagashima, Y., Suzuki, E., Hayashi, N., Ogata, Y., Shimada, Y. and Koizumi, N. (2013). Defects in IRE1 enhance cell death and fail to degrade mRNAs encoding secretory pathway proteins in the Arabidopsis unfolded protein response. Proc Natl Acad Sci U S A 110(14): 5713-5718.
  2. Wyllie, A. H. (1980). Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284(5756): 555-556.

简介

具有对应于大约180个碱基对的多个整数的长度的DNA片段是动物中凋亡和植物中程序性细胞死亡的独特特征。 这个特征可以简单地通过DNA凝胶电泳随后溴化乙锭染色检测,尽管在一些情况下难以区分DNA梯状。 我们在本文中描述了用于检测植物组织的程序性细胞死亡相关DNA梯状的方案。 在基因组DNA的琼脂糖凝胶电泳后,进行使用DIG标记的基因组DNA探针的Southern杂交,其改善DNA梯状的检测。

关键字:程序性细胞死亡, DNA梯, Southern杂交

材料和试剂

  1. 新鲜植物组织
  2. Nucleon PhytoPure DNA提取试剂盒(通用电气公司,目录号:RPN8510)
  3. GeneMate LE琼脂糖(BioExpress,目录号:E-3120)
  4. Biodyne Plus Nylon Membrane(Pall,目录号:60406)
  5. Wizard DNA Clean-Up System(Promega,目录号:A7280)
  6. DIG-High Prime DNA标记和检测起始试剂盒II(Roche Diagnostics,目录号:11585614910)
  7. X光片
  8. 盐水柠檬酸钠(SSC)
  9. 净化溶液(见配方)
  10. 变性溶液(参见配方)
  11. 中和解决方案(参见配方)
  12. 1x TAE缓冲区(请参阅配方)

设备

  1. 用于凝胶和膜处理的塑料容器
  2. 离心机(Eppendorf,型号:5415R)
  3. 分光光度计(Shimadzu,型号:BioSpec-nano)
  4. Shaker(BIO CRAFT,型号:BC-700)
  5. 真空转印装置(BIO CRAFT,型号:BS-31)
  6. 杂交烘箱和UV交联剂(UVP,型号:HybriLinker HL-2000)
  7. 真空浓缩器(Savant TM ,型号:DNA SpeedVac DNA110)
  8. 杂交孵育器(TAITEC,型号:HB-80)

程序

  1. 使用PhytoPure根据用户手册在液氮中研磨植物组织并提取基因组DNA 注意:
    1. 异丙醇在-20℃下沉淀至少30分钟。
    2. 其他植物基因组DNA分离程序是可以接受的。
  2. 通过分光光度计和/或琼脂糖凝胶电泳检查分离的基因组DNA的数量和质量 注意:我们使用具有OD <260> 280> 1.8。
  3. 将1-5μg基因组DNA加载到2%琼脂糖凝胶上 注意:还装载100-bp梯形标记以估计片段化DNA的分子量。
    注意:虽然我们从未尝试过,但是可以检测到较小量(<1μg)的DNA。
  4. 在50伏电泳,在1x TAE缓冲液中电泳2小时 注意:我们使用110 x 100 x 6 mm的琼脂糖凝胶。
  5. 溴化乙锭染色和暴露于紫外光。
    注意:检查凝胶中分子标记带的位置。
  6. 将琼脂糖凝胶放入脱盐溶液(250 ml)中的塑料容器中,在摇床上轻轻搅拌10分钟(或直到溴酚蓝变黄)。 注意:脱嘌呤溶液部分水解大DNA片段以帮助充分转移DNA片段。
  7. 通过dH 2 O冲洗凝胶三次。
  8. 在变性溶液(250ml)中处理凝胶15分钟两次 注意:变性溶液使双链DNA变性以进行有效杂交。
  9. 通过dH 2 O冲洗凝胶三次。
  10. 在中和溶液(250ml)中处理凝胶15分钟两次 注意:中和溶液会中和凝胶的pH值。
  11. 将凝胶放置在真空转移装置上,用Biodyne Plus膜预浸在20x SSC缓冲液中,根据制造商的说明进行转移2小时。
    注意:使用20x SSC的滤纸是可选的。
  12. 通过UV交联将DNA固定到膜上,并用灭菌的dH 2 O洗涤膜。
    注意:
    1. 在暴露于UV之前,将膜包裹在Saran Wrap中。
    2. UV能量剂量为120mJ/cm 2
  13. 干燥膜。
    注意:这种干膜可以在4℃下保存。
  14. DIG标记的探针的制备。
    1. 用50单位的限制酶在37℃消化2μg基因组DNA 12小时 注意:4基刀具(例如HaeIII,MspI)是适合的。
    2. 使用Wizard DNA Clean-Up System纯化DNA。
      注意:酚 - 氯仿萃取和乙醇沉淀是可选的。
    3. 使用真空浓缩器干燥纯化的DNA溶液
    4. 用16μl无菌dH 2 O溶液分解DNA
    5. 煮沸DNA样品10分钟,冰上冷却保持变性的单链DNA
    6. 加入4μlDIG-High Prime(5x浓度)并混合
    7. 在37℃下孵育20小时。
    8. 通过加热至65℃10分钟停止反应
    9. 反应混合物可储存在-20℃下
  15. 将膜置于杂交瓶中,进行至少1小时的预杂交,并在杂交炉中在44℃下使用DIG Easy Hyb杂交过夜。
    注意:在上述探针的情况下,将2-3μl探针加入到5ml杂交缓冲液中。
  16. 将膜用1×SSC和0.2%SDS在68℃下在杂交孵育箱中洗涤两次15分钟,以塑料容器。
    注意:尽可能快地摇动容器,确保膜保持移动,并且每次都应更换溶液。
  17. 用杂交孵育器在68℃下用0.1×SSC和0.5%SDS将膜洗涤到塑料容器中20分钟两次。
  18. 根据DIG-High Prime DNA标记和检测启动器Kit II用户手册进行检测。
  19. 将化学发光信号暴露于X射线胶片或成像器。 DNA梯状可视化(图1)

    图1.检测DNA梯状。将拟南芥野生型(WT)和ire1a/b 突变体的幼苗用衣霉素/ml)48小时。 (A)从幼苗的地上部分提取的基因组DNA的溴化乙啶染色。 M,100 bp梯形标记。 (B)与DIG-标记的Hae III/MspI-消化的拟南芥基因组DNA探针杂交的(A)中的琼脂糖凝胶的Southern印迹。< br /

食谱

  1. 净化溶液(500ml)
    将25ml 5N HCl(0.25N)与475ml dH 2 O混合 在室温下贮存
  2. 变性溶液(500ml) 10g NaOH(0.5N)
    43.83g NaCl(1.5M) 将dH <2> O添加到500ml
    在室温下贮存
  3. 中和溶液(500ml) 30.3g Tris(0.5M)
    87.66g NaCl(3N)
    将dH 2加到450ml
    中 通过浓缩将pH调节至7.5。 HCl
    将dH <2> O添加到500ml
    在室温下贮存
  4. 1x TAE缓冲区
    40mM Tris-乙酸盐 1mM EDTA

致谢

该协议改编自Mishiba等人(2013)。

参考文献

  1. Mishiba,K.,Nagashima,Y.,Suzuki,E.,Hayashi,N.,Ogata,Y.,Shimada,Y.and Koizumi,N。(2013)。 IRE1中的缺陷增强细胞死亡,并且不能降解拟南芥中编码分泌途径蛋白的mRNA unfolded protein response。 Proc Natl Acad Sci USA 110(14):5713-5718。
  2. Wyllie,A.H。(1980)。 糖皮质激素诱导的胸腺细胞凋亡与内源性内切核酸酶激活有关。 284(5756):555-556。
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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Mishiba, K., Nagashima, Y., Hayashi, N. and Koizumi, N. (2014). DNA Fragmentation Analysis. Bio-protocol 4(15): e1203. DOI: 10.21769/BioProtoc.1203.
  2. Mishiba, K., Nagashima, Y., Suzuki, E.,Hayashi, N., Ogata, Y., Shimada, Y. and Koizumi, N. (2013). Defectsin IRE1 enhance cell death and fail to degrade mRNAs encoding secretory pathwayproteins in the Arabidopsis unfolded protein response. Proc Natl Acad Sci U S A 110(14): 5713-5718.
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