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Analysis of Telomeric G-overhangs by in-Gel Hybridization
凝胶杂交分析端粒的G悬突   

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参见作者原研究论文

本实验方案简略版
PLOS Genetics
Oct 2014

Abstract

Telomeric DNA in majority of eukaryotes consists of an array of TG-rich tandem repeats. The TG-rich DNA strand is oriented with its 3’ end towards chromosome termini and is usually longer than its complementary CA-rich strand, thus forming 3’ single stranded overhang (G-overhang). G-overhangs arise from incomplete replication of chromosome termini by the lagging strand mechanism and post-replicative nucleolytic processing. The G-overhang is important for telomere protection as it serves as a binding platform for specific proteins and is required for t-loop formation. Hence, structure of telomeric G-overhang is an important indicator of telomere maintenance and functionality. Here we describe a method for analysis of G-overhangs in a model plant Arabidopsis thaliana by in-gel hybridization technique. This method allows relative quantification of the amount of single stranded telomeric DNA. Short telomeric probes are radioactively labeled and hybridized to DNA under non-denaturing conditions to specifically detect ssDNA. Total telomeric DNA can be measured using denaturing conditions in the same gel and this procedure usually follows the non-denaturing in-gel hybridization. Terminal nature of the ssDNA is verified by exonuclease treatment. This technique was originally developed in yeast and now is used as a major tool for G-overhang analysis in a variety of organisms ranging from human to plants.

Keywords: Telomere (端粒), G-overhang (G overhang), In-gel hybridization (在凝胶杂交), Arabidopsis (拟南芥), Telomere analysis (端粒长度的分析)

Materials and Reagents

  1. Plastic wrap (common food wrap or FisherbrandTM Clear Plastic Wrap) (Thermo Fisher Scientific, catalog number: 22-305654 )
  2. Whatman® papers (Sigma-Aldrich, catalog number: 3030917 )
  3. GeneJET Plant Genomic DNA Purification Kit (Life Technologies, catalog number: K0792 )
    Note: Currently, it is “Thermo Fisher Scientific, catalog number: K0792”.
  4. T4 DNA polymerase (NEB, catalog number: M0203S )
  5. NEB buffer #2 (NEB, catalog number: B7002S )
  6. dNTPs (Life Technologies, catalog number: R0181 )
    Note: Currently, it is “Thermo Fisher Scientific, catalog number: R0181”.
  7. Isopropanol
  8. Absolute ethanol (EtOH)
  9. HindIII (Thermo Fisher Scientific, catalog number: ER0501 )
  10. 3 M NaOAc (pH 5.2)
  11. DNA Gel Loading Dye (6x) (Life Technologies, catalog number: R0611 )
    Note: Currently, it is “Thermo Fisher Scientific, catalog number: R0611”.
  12. PeqGold Universal agarose (VWR, catalog number: 732-2789 )
  13. Ethidium Bromide (EtBr) solution (1%) (Applichem, catalog number: A1152 )
  14. T4 polynucleotide kinase (PNK) (10 U/µl) (Thermo Fisher Scientific, catalog number: EK0031 )
  15. Custom oligonucleotide probe, (TA3C3)4 or (TA3C3)3 (10 pmol/µl) (Sigma-Aldrich)
  16. γ32P-ATP (> 6,000 Ci/mmol) (HARTMANN ANALYTIC GmbH, catalog number: SRP-501 )
  17. QIAquick Nucleotide Removal Kit (QIAGEN, catalog number: 28304 )
  18. Tris
  19. Acetic acid
  20. EDTA (pH 8.0)
  21. NaCl
  22. Sodium citrate (pH 7.0)
  23. SDS
  24. Na-phosphate buffer (pH 7.2)
  25. BSA
  26. Tris-Cl (pH 8)
  27. 1x TAE (see Recipes)
  28. 20x SSC (see Recipes)
  29. Hybridisation buffer (see Recipes)
  30. Wash solution-1 (see Recipes)
  31. Wash solution-2 (see Recipes)
  32. Denaturation solution (see Recipes)
  33. Neutralization solution (see Recipes)

Equipment

  1. EppendorfTM ThermoMixerTM C
  2. Centrifuge (Eppendorf AG)
  3. Horizontal Midi Gel Electrophoresis system (PerfectBlue Gel System Midi S, Peqlab)
  4. Gel DocTM XR+ System (Bio-Rad Laboratories)
  5. Gel dryer (Bio-Rad Laboratories, model: 583 )
  6. Imaging plate (Fujifilm, catalog number: BAS-IP MS 2025 )
    Note: Currently, it is “GE Healthcare, catalog number: BAS-IP MS 2025”.
  7. Vacuum pump (Gardner Denver Thomas GmbH, ILMVAC, catalog number: 412711 )
  8. Water bath with shaking
  9. Pharos FX Plus Molecular Imager (Bio-Rad Laboratories)
  10. PC with an image analysis software (e.g., Image Lab from Bio-Rad Laboratories)

Software

  1. Image Lab 5.1 (Bio-Rad Laboratories) (freely available at http://www.bio-rad.com/en-us/product/image-lab-software)

Procedure

  1. Non-denaturing in-gel hybridization
    1. Genomic DNA was prepared using standard procedures. DNA was prepared as method described in Kazda et al. (2012) or using GeneJET Plant Genomic DNA Purification Kit. When 2x CTAB DNA extraction procedure (Borevitz et al., 2003) was used, incubation was done at 55 °C (for 1 h) instead of 65 °C to reduce a risk of DNA melting.
    2. Negative control was generated by removing terminal G-overhangs with T4 DNA polymerase that has a strong 3’ exonuclease activity on ssDNA. Genomic DNA (2 µg) was treated with 30U of T4 DNA polymerase in presence of 0.1 mM dNTPs in total reaction volume of 200 µl of 1x NEB buffer #2. Reaction was incubated for 30 min at 37 °C. DNA was precipitated with 200 µl isopropanol, centrifuged 30 min at max speed at RT and washed with 70% EtOH. Resuspension of dry DNA was carried out at 37 °C in 175 µl dH2O. Samples were processed immediately.
    3. Samples and their T4 DNA polymerase pretreated negative controls (2 µg each) were digested with 50 U of HindIII in total reaction volume of 200 µl of 1x Buffer R. Reaction was incubated overnight at 37 °C. DNA was precipitated with 20 µl of 3 M NaAc (pH 5.2) and 200 µl of isopropanol, centrifuged 30 min at max speed at RT and washed with 70% EtOH. Resuspension of dry DNA was carried out at 37 °C in 20-25 µl dH2O. Samples were stored at -20 °C or processed immediately and 4-5 µl of DNA Gel Loading Dye (6x) was added.
    4. Digested DNA was separated using agarose gel electrophoresis. Gel was prepared in 1x TAE at a final concentration of 0.9% agarose. Samples were loaded and separated at 25 V for 15 h. DNA was stained by ethidium bromide. Gel was soaked in 200 ml of 2x SSC with 20 µl of 1% ethidium bromide at RT for 30 min while shaking.
    5. DNA was visualized by UV and picture was acquired using Gel DocTM XR+ System (Figure 1). Attention was paid that lanes were not over saturated (Figure 2, for accurate signal quantification).


      Figure 1. In-gel hybridization analysis of wild type and ku80 mutant A. thaliana. The digested DNA of two biological replicates of wild type (wt) and mutant (ku80) was separated on an agarose gel and hybridized with a radioactively labeled (TAAACCC) 3 probe first under native and then under denaturing conditions (left panel). The same procedure was used for T4 treated samples (right panel).


      Figure 2. Volume analysis using Image Lab 5.1 software. A. “Rectangle”; Using “Rectangle” function in “Volume Tools”, we selected relevant lane area and background. Background subtraction was set to Global and “Adj. Vol.” values were exported for further analysis; B. Lane profile; Attention was paid that lanes were not showing the signal saturation. Saturation of the signal can be seen as red color in lane 12. In the lane profile we can see flattening of the signal peak where we cannot fully observe the total amount of the signal.

    6. Agarose gel was dried using a gel drier. Gel was sandwiched between two Whatman papers and dried in a gel drier under vacuum at RT for 10 min per side. Gel was processed immediately.
    7. For G-overhang detection, 10 pmol of (TA3C3) 4 or (TA3C3) 3 oligonucleotide probe resuspended in dH2O was labeled in presence of 5 µl of γ-32P-ATP (> 6,000 Ci/mmol) using 10 U T4 PNK in total reaction volume of 20 µl of 1x PNK A Buffer. Reaction was incubated at 37 °C for 30 min and purified with QIAquick nucleotide removal kit according to manufacturer´s instructions.
    8. Dried gel was prehybridized in 50 ml hybridization buffer (in a plastic container) in 50 °C water bath with shaking (35 rpm) for 1-2 h. A heavy item was used to fix the container on the bottom of the water bath, so it does not float. Entire volume of purified probe was added and hybridized overnight at 50 °C in the water bath with shaking (35 rpm).
    9. Gel was removed from the hybridization solution and rinsed with wash solution-1 briefly, followed by two additional washes with wash solution-1 at RT, 35 rpm shaking for one hour. Later two washes with wash solution-2 at 38 °C, 35 rpm shaking for one hour was given.
    10. Gel was dried at RT between Whatman papers in gel drier under vacuum. Attention was paid not to over dry the gel (maximum 1 min drying time in total).
    11. Gel was wrapped in plastic wrap and exposed to imaging plate without a delay for up to 3 days at 4 °C to avoid contamination. Freezing and thawing causes gel to become brittle and was avoided.
    12. Signal was detected on Pharos FX (Figure 1).

  2. Denaturing in-gel hybridization
    This procedure usually follows the non-denaturing in-gel hybridization to detect total telomeric DNA in analyzed samples.
    1. Gel was unwrapped and DNA was denatured by soaking and rocking gel lightly in denaturing solution (100-200 ml) for 30 min at RT (35 rpm).
    2. Denaturing solution was removed and gel was rinsed with dH2O prior to adding neutralization solution (100-200 ml) for 15 min at RT.
    3. Equilibration was done in 100-200 ml of 5x SSC for 15 min at RT and gel was prehybridized in 50 ml hybridization solution for 1 h as indicated previously.
    4. Hybridization was done overnight with the same probe and conditions as was used for the non-denaturing in-gel hybridization.
    5. Washing and exposure were done as for the non-denaturing in-gel hybridization. Shorter exposure (1 d) is usually sufficient for detecting strong signal (Figure 1).

  3. Signal calculation by “Image Lab 5.1” software
    The G-overhang signal from samples is normalized to DNA loading determined by ethidium bromide staining, and calculated by subtracting corresponding negative control pretreated with T4 DNA polymerase.
    1. EtBr gel (loading control)
      1. “Volume tools” “Rectangle” was used for quantification.
        1. Selected area. Always the same rectangle was used for all lanes (first rectangle was copied and pasted). The area was selected in a way that the whole lane is covered as best as possible. However, the area around the lanes was not included in the rectangle (Figure 2A).
        2. Selected background. The “Global” subtraction method was used and the rectangle was also used for this purpose (Figure 2A).
      2. “Analysis table” was used.
        1. Values were copied to clip board and transferred to excel. Attention was paid to decimal point. Adjusted values “Adj. Vol.” were used (values corrected for background by the software Figure 2A).
        2. Ratio was calculated (highest value “wt_high” was set to 1, other value “mut” - was corrected, Figure 3A).
          X= EtBr (“Adj. Vol.”) / EtBr (highest “Adj. Vol.”)
          e.g. wt_high=5,000 and mut=3,000        -> Xmut= 3,000/5,000= 0.6
                                                                               -> Xwt= 5,000/5,000= 1.0
    2. Native gel (G-overhang signal)
      1. Volume analysis was done as previously.
      2. Adjusted values were corrected for loading (T4 untreated and T4 treated samples, Figure 3A).
        Y= native (“Adj. Vol.”) / X
        e.g. mut= 4,500 and Xmut= 0.6          -> Ymut= 4,500/0.6= 7,500
               wt = 5,000 and Xwt=1.0               -> Ywt= 5,000/ 1.0= 5,000
      3. Values were corrected for T4 treatment. Values were subtracted from T4 treated sample.
        Z= Y(T4-) – Y(T4+)
        e.g. Ymut= 7,500 and Ymut_T4= 1,200     -> Zmut= 6,300
               Ywt= 5,000 and Ywt_T4= 1,000          -> Zwt= 4,000
    3. Ratio calculation (set highest wild type control sample “highest wt” to 1, Figure 3A).
      A= Z / Z(highest wt)
      e.g. Zwt= 4,000 and Zmut= 6,300      -> Amut= 6,300/4,000= 1,575
                                                                      -> Awt= 1.0
    4. Graphical representation
      Relative values from the point 3 are used to present the result of the analysis in a graph (Figure 3B).


      Figure 3. Graphical representation of the In-gel hybridization analysis. A. Signal calculation; Signal from the EtBr gel was used to create a loading ratio (X). Signal from the native gel hybridization was first corrected for DNA loading (Y) and signal from T4 treated samples was subtracted from not treated samples (Z). A fold change in G-overhang was calculated to the wt sample with the highest value of Z (A); B. Relative quantification of the G-overhang signal; The relative G-overhang signal (calculated ratio) is represented as average fold changes in G-overhang signal relative to wild type with highest signal.

Recipes

  1. 1x TAE
    40 mM Tris
    20 mM acetic acid
    1 mM EDTA (pH 8)
  2. 20x SSC
    3.0 M NaCl
    0.3 M sodium citrate (pH 7.0)
    Dilute accordingly
  3. Hybridization buffer
    7% SDS
    0.25 M Na-phosphate buffer (pH 7.2)
    0.1 g/L BSA
  4. Wash solution-1
    0.25 x SSC
    0.01% SDS
  5. Wash solution-2
    0.25 x SSC
  6. Denaturation solution
    150 mM NaCl
    0.5 M NaOH
  7. Neutralization solution
    150 mM NaCl
    0.5 M Tris-Cl (pH 8)

Acknowledgments

This protocol was used to analyze G-overhang structure in Arabidopsis (Kazda et al., 2012; Derboven et al., 2014) and was adapted from the previously published studies Dionne et al. (1996), Wei et al. (2002) and Heacock et al. (2007). This work was supported by the EMBO installation grant (1304130933) and the program SoMoPro II (3SGA5833) co-financed by EU and the South Moravian Region. This publication reflects only the author's views and the Union is not liable for any use that may be made of the information contained therein.

References

  1. Borevitz, J. O., Liang, D., Plouffe, D., Chang, H. S., Zhu, T., Weigel, D., Berry, C. C., Winzeler, E. and Chory, J. (2003). Large-scale identification of single-feature polymorphisms in complex genomes. Genome Res 13(3): 513-523.
  2. Derboven, E., Ekker, H., Kusenda, B., Bulankova, P. and Riha, K. (2014). Role of STN1 and DNA polymerase alpha in telomere stability and genome-wide replication in Arabidopsis. PLoS Genet 10(10): e1004682.
  3. Dionne, I. and Wellinger, R. J. (1996). Cell cycle-regulated generation of single-stranded G-rich DNA in the absence of telomerase. Proc Natl Acad Sci U S A 93(24): 13902-13907.
  4. Heacock, M. L., Idol, R. A., Friesner, J. D., Britt, A. B. and Shippen, D. E. (2007). Telomere dynamics and fusion of critically shortened telomeres in plants lacking DNA ligase IV. Nucleic Acids Res 35(19): 6490-6500.
  5. Kazda, A., Zellinger, B., Rossler, M., Derboven, E., Kusenda, B. and Riha, K. (2012). Chromosome end protection by blunt-ended telomeres. Genes Dev 26(15): 1703-1713.
  6. Wei, C., Skopp, R., Takata, M., Takeda, S. and Price, C. M. (2002). Effects of double-strand break repair proteins on vertebrate telomere structure. Nucleic Acids Res 30(13): 2862-2870.

简介

在大多数真核生物中的端粒DNA由富含TG的串联重复阵列组成。富含TG的DNA链以其3'末端朝向染色体末端定向,并且通常比其互补的富含CA的链更长,从而形成3'单链突出端(G突出端)。 G突出由染色体末端的不完全复制通过滞后链机制和复制后核酸水解加工产生。 G突出端对于端粒保护是重要的,因为其用作特异性蛋白质的结合平台并且是t环形成所需的。因此,端粒G突出端的结构是端粒维持和功能的重要指标。在这里我们描述了通过凝胶内杂交技术在模拟植物拟南芥中分析G突出端的方法。该方法允许单链端粒DNA的量的相对定量。短端粒探针放射性标记并在非变性条件下与DNA杂交以特异性检测ssDNA。可以使用在相同凝胶中的变性条件测量总端粒DNA,并且该程序通常在非变性凝胶内杂交之后。 ssDNA的末端性质通过核酸外切酶处理来验证。这种技术最初是在酵母中开发的,现在被用作从人类到植物的多种生物体中的G突出端分析的主要工具。

关键字:端粒, G overhang, 在凝胶杂交, 拟南芥, 端粒长度的分析

材料和试剂

  1. 塑料包装(普通食品包装或Fisherbrand TM 透明塑料包装)(Thermo Fisher Scientific,目录号:22-305654)
  2. Whatman ?纸(Sigma-Aldrich,目录号:3030917)
  3. GeneJET植物基因组DNA纯化试剂盒(Life Technologies,目录号:K0792)
    注意:目前,它是"Thermo Fisher Scientific,目录号:K0792"。
  4. T4 DNA聚合酶(NEB,目录号:M0203S)
  5. NEB缓冲液#2(NEB,目录号:B7002S)
  6. dNTP(Life Technologies,目录号:R0181)
    注意:目前,它是"Thermo Fisher Scientific,目录号:R0181"。
  7. 异丙醇
  8. 无水乙醇(EtOH)
  9. HindIII(Thermo Fisher Scientific,目录号:ER0501)
  10. 3 M NaOAc(pH 5.2)
  11. DNA凝胶负载染料(6x)(Life Technologies,目录号:R0611)
    注意:目前,它是"Thermo Fisher Scientific,目录号:R0611"。
  12. PeqGold通用琼脂糖(VWR,目录号:732-2789)
  13. 溴化乙啶(EtBr)溶液(1%)(Applichem,目录号:A1152)
  14. T4多核苷酸激酶(PNK)(10U /μl)(Thermo Fisher Scientific,目录号:EK00??31)
  15. 定制的寡核苷酸探针(TA3C3)4或(TA3C3)3(10pmol/μl)(Sigma-Aldrich)
  16. γH 32 P-ATP(> 6,000Ci/mmol)(HARTMANN ANALYTIC GmbH,目录号:SRP-501)
  17. QIAquick核苷酸去除试剂盒(QIAGEN,目录号:28304)
  18. Tris
  19. 乙酸
  20. EDTA(pH 8.0)
  21. NaCl
  22. 柠檬酸钠(pH 7.0)
  23. SDS
  24. 磷酸钠缓冲液(pH 7.2)
  25. BSA
  26. Tris-Cl(pH 8)
  27. 1x TAE(请参阅配方)
  28. 20x SSC(请参阅配方)
  29. 杂交缓冲液(参见配方)
  30. 洗液-1(见配方)
  31. 洗液-2(见配方)
  32. 变性溶液(参见配方)
  33. 中和解决方案(参见配方)

设备

  1. Eppendorf TM ThermoMixer TM C
  2. 离心机(Eppendorf AG)
  3. 水平Midi凝胶电泳系统(PerfectBlue Gel System Midi S,Peqlab)
  4. Gel Doc TM XR +系统(Bio-Rad Laboratories)
  5. 凝胶干燥器(Bio-Rad Laboratories,型号:583)
  6. 成像板(Fujifilm,目录号:BAS-IP MS 2025)
    注意:目前,它是"GE Healthcare,目录号:BAS-IP MS 2025"。
  7. 真空泵(Gardner Denver Thomas GmbH,ILMVAC,目录号:412711)
  8. 水浴摇动
  9. Pharos FX Plus Molecular Imager(Bio-Rad Laboratories)
  10. PC的图像分析软件(例如来自Bio-Rad Laboratories的Image Lab)

软件

  1. Image Lab 5.1(Bio-Rad Laboratories)(免费提供于 http: //www.bio-rad.com/zh-TW/product/image-lab-software

程序

  1. 非变性凝胶内杂交
    1. 使用标准程序制备基因组DNA。制备DNA 方法描述于Kazda等人(2012)或使用GeneJET Plant Genomic DNA纯化试剂盒。当使用2x CTAB DNA提取程序(Borevitz等人, ?et al。,2003),在55℃(1小时)代替 65°C以降低DNA熔解的风险。
    2. 阴性对照 通过用T4 DNA聚合酶去除末端G突出而产生 对ssDNA具有强的3'外切核酸酶活性。基因组DNA(2μg) 在0.1mM dNTP存在下用30U T4 DNA聚合酶处理 总反应体积为200μl的1x NEB缓冲液#2。反应 在37℃温育30分钟。用200μl沉淀DNA 异丙醇,在室温下以最大速度离心30分钟,并用70% EtOH。干燥DNA的重悬在37℃在175μldH 2 O中进行。 立即处理样品。
    3. 样品及其T4 DNA 聚合酶预处理的阴性对照(各2μg) 50U HindIII,在总反应体积为200μl的1x Buffer R中。 将反应在37℃温育过夜。用20沉淀DNA μl的3M NaAc(pH 5.2)和200μl异丙醇,在37℃离心30分钟 ?最大速度,并用70%EtOH洗涤。干DNA的重悬 在37℃在20-25μldH 2 O中进行。将样品在-20℃或储存 立即处理并加入4-5μl的DNA Gel Loading Dye(6x)。
    4. 使用琼脂糖凝胶电泳分离消化的DNA。凝胶 在1x TAE中制备至终浓度为0.9%的琼脂糖。样品 并在25V下分离15小时。 DNA通过乙锭染色 溴化物。将凝胶浸泡在200ml含有20μl1%乙酸的2x SSC中 溴化物在RT搅拌30分钟
    5. 通过UV显现DNA 并使用Gel Doc TM X/TM系统(图1)获得图片。注意 ?车道不过饱和(图2,为准确 信号量化)。


      图1.凝胶中杂交分析 野生型和 ku80 突变体。 thaliana 。两个的消化DNA 分离野生型(wt)和突变体(ku80)的生物复制物 在琼脂糖凝胶上并与放射性标记(TAAACCC)3杂交 ?探针首先在天然下,然后在变性条件下(左 面板)。相同的程序用于T4处理的样品(右图) 面板)。


      图2.使用Image Lab 5.1软件进行的卷分析。 A. "长方形";使用"卷工具"中的"矩形"功能,我们选择 相关车道区域和背景。背景减法设置为 全球和" Vol。"值输出用于进一步分析;乙。 车道轮廓;注意,车道没有显示信号 饱和。信号的饱和度可以看作是在车道中的红色 在泳道谱中,我们可以看到信号峰的平坦化 我们不能完全观察信号的总量
    6. 使用凝胶干燥器干燥琼脂糖凝胶。将凝胶夹在两个之间 Whatman纸,并在真空下在RT下在凝胶干燥器中干燥10分钟 每侧。立即处理凝胶。
    7. 对于G悬 检测,10pmol(TA3C3)4或(TA3C3)3寡核苷酸探针 在5μl的γ-32 P-ATP(>10μM)的存在下标记重悬于dH 2 O中的标记。 6,000 Ci/mmol),使用10U T4 PNK,在总反应体积为20μl的1x中 ?PNK A缓冲区。将反应在37℃下温育30分钟并纯化 用QIAquick核苷酸去除试剂盒根据制造商 说明。
    8. 将干凝胶在50ml杂交中预杂交 ?缓冲液(在塑料容器中)在50℃水浴中摇动(35℃) rpm)1-2小时。使用较重的物品将容器固定在底部 ?的水浴,所以它不浮。纯净的整个体积 探针,并在50℃的水浴中与50℃杂交过夜 ?摇动(35rpm)
    9. 从杂交中除去凝胶 溶液,并用洗涤溶液-1短暂冲洗,然后用两次冲洗 在室温下用洗涤溶液-1另外洗涤,35rpm振荡一次 小时。然后在38℃下用洗涤溶液-2洗涤两次,35rpm振荡 ?一小时。
    10. 凝胶在Whatman纸之间在室温干燥 在真空下的凝胶干燥器中。注意不要过度干燥凝胶 (总共最长1分钟干燥时间)
    11. 将凝胶包裹在塑料中 ?包裹并暴露于成像板,无延迟,最多3天,在4 ?°C以避免污染。冷冻和解冻导致凝胶变得 脆性并避免。
    12. 在Pharos FX上检测到信号(图1)。

  2. 变性凝胶内杂交
    该程序通常遵循非变性凝胶内杂交以检测分析样品中的总的端粒DNA
    1. 解开凝胶,通过浸泡和摇动凝胶使DNA变性 在变性溶液(100-200ml)中轻微在室温(35rpm)下30分钟
    2. 除去变性溶液,并用dH 2 O预漂洗凝胶 在室温下加入中和溶液(100-200ml)15分钟
    3. 在100-200ml的5×SSC中在RT和凝胶中进行平衡15分钟 如所示在50ml杂交溶液中预杂交1小时 之前。
    4. 用与非变性凝胶杂交相同的探针和条件杂交过夜
    5. 按照非变性凝胶进行洗涤和暴露 杂交。较短的暴露(1d)通常就足够了 检测强信号(图1)。

  3. "Image Lab 5.1"软件的信号计算
    将来自样品的G突出端信号标准化为从溴化乙锭染色确定的DNA负载,并通过减去用T4 DNA聚合酶预处理的相应阴性对照进行计算。
    1. EtBr凝胶(上样对照)
      1. "体积工具""矩形"用于量化。
        1. 选择区域。总是使用相同的矩形用于所有车道 (第一个矩形被复制和粘贴)。该地区是以某种方式选择的 整个车道尽可能地被覆盖。但是,该地区 在车道周围不包括矩形(图2A)。
        2. 选定的背景。使用"全局"减法方法,矩形也用于此目的(图2A)。
      2. 使用"分析表"
        1. 值被复制到剪贴板并转移到Excel。注意 ?支付到小数点。调整值"Adj。 Vol。" (由软件图2A校正背景的值)
        2. 计算比率(最高值"wt_high"设置为1,其他值"mut" - 被校正,图3A)。
          X = EtBr("Adj.Vol。")/EtBr(最高"Adj.Vol。")
          例如 wt_high = 5,000和mut = 3,000       - > Xmut = 3,000/5,000 = 0.6
                                                                           - > Xwt = 5,000/5,000 = 1.0
    2. 天然凝胶(G突出信号)
      1. 体积分析如前所述。
      2. 针对负载校正调整值(T4未处理和T4处理的样品,图3A) Y = native("Adj。Vol。")/X
        例如 mut = 4,500和Xmut = 0.6         - > Ymut = 4,500/0.6 = 7,500
              wt = 5,000和Xwt = 1.0                - > Ywt = 5,000/1.0 = 5,000
      3. 对T4处理校正值。从T4处理的样品中减去值 Z = Y(T4-)-Y(T4 +)
        例如。 Ymut = 7,500和Ymut_T4 = 1,200    - > Zmut = 6,300
              Ywt = 5,000和Ywt_T4 = 1,000         - > Zwt = 4,000
    3. 比率计算(将最高野生型对照样品"最高重量"设定为1,图3A) A = Z/Z(最高wt)
      例如 Zwt = 4,000和Zmut = 6,300     - > Amut = 6,300/4,000 = 1,575
                                                                  - > Awt = 1.0
    4. 图形表示
      来自点3的相对值用于在图表中呈现分析结果(图3B)。


      图3.In-gel杂交的图形表示 A.信号计算;使用来自EtBr凝胶的信号 创建加载比(X)。来自天然凝胶杂交的信号 ?首先校正DNA负载(Y)和来自T4处理的样品的信号 从未处理的样品(Z)中扣除。 G突出端的倍数变化 ?计算为具有Z(A)的最高值的wt样品;乙。 G-突出端信号的相对定量;相对 G突出端信号(计算比率)表示为平均折叠 相对于具有最高信号的野生型的G突出端信号的变化。

食谱

  1. 1x TAE
    40 mM Tris
    20mM乙酸
    1mM EDTA(pH8)
  2. 20x SSC
    3.0 M NaCl
    0.3 M柠檬酸钠(pH 7.0) 相应稀释
  3. 杂交缓冲区
    7%SDS
    0.25M磷酸钠缓冲液(pH7.2) 0.1 g/L BSA
  4. 洗液-1
    0.25×SSC
    0.01%SDS
  5. 洗液-2
    0.25×SSC
  6. 变性溶液
    150mM NaCl 0.5 M NaOH
  7. 中和溶液
    150mM NaCl 0.5 M Tris-Cl(pH 8)

致谢

该方案用于分析拟南芥中的G突出端结构(Kazda等人,2012; Derboven等人,2014),并且改编自先前公开的研究Dionne et al。 (1996),Wei et al。(2002)和Heacock et al。(2007)。这项工作是由欧洲联盟和南摩拉维亚州共同资助的EMBO安装补助金(1304130933)和程序SoMoPro II(3SGA5833)支持。本出版物仅反映作者的观点,联盟不对其中所含信息的任何使用负责。

参考文献

  1. Borevitz,J.O.,Liang,D.,Plouffe,D.,Chang,H.S.,Zhu,T.,Weigel,D.,Berry,C.C.,Winzeler,E.and Chory,J。(2003)。 大规模鉴定复杂基因组中的单一特征多态性 基因组Res 13(3):513-523。
  2. Derboven,E.,Ekker,H.,Kusenda,B.,Bulankova,P.和Riha,K。(2014)。 STN1和DNA聚合酶α在端粒稳定性和全基因组复制中的作用拟南芥。 PLoS Genet 10(10):e1004682。
  3. Dionne,I。和Wellinger,R.J。(1996)。 在不存在端粒酶的情况下,细胞周期调节产生单链富含G的DNA。 Proc Natl Acad Sci USA 93(24):13902-13907。
  4. Heacock,M.L.,Idol,R.A.,Friesner,J.D.,Britt,A.B.and Shippen,D.E。(2007)。 端粒动力学和缺乏DNA连接酶IV的植物中严重缩短的端粒的融合 Nucleic Acids Res 35(19):6490-6500。
  5. Kazda,A.,Zellinger,B.,Rossler,M.,Derboven,E.,Kusenda,B.and Riha,K。 由端粒端粒染色体末端保护。 基因Dev 26(15):1703-1713。
  6. Wei,C.,Skopp,R.,Takata,M.,Takeda,S.and Price,C.M。(2002)。 双链断裂修复蛋白对脊椎动物端粒结构的影响核Acids Res 30(13):2862-2870。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Valuchova, S., Derboven, E. and Riha, K. (2016). Analysis of Telomeric G-overhangs by in-Gel Hybridization. Bio-protocol 6(7): e1775. DOI: 10.21769/BioProtoc.1775.
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