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Terminal Restriction Fragments (TRF) Method to Analyze Telomere Lengths
末端限制性片段(TRF)技术测定端粒长度   

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Abstract

Chromosome ends - telomeres - are a focus of intensive research due to their importance for the maintenance of chromosome stability. Their shortening due to incomplete replication functions as a molecular clock counting the number of cell divisions, and ultimately results in cell-cycle arrest and cellular senescence. Determination of telomere lengths is an essential approach in telomere biology for research and diagnostic applications. Terminal Restriction Fragments (TRF) analysis is the oldest approach to analyze telomere lengths and remains the “gold standard” even in current studies. This technique relies on the fact that repeated minisatellite telomeric units do not contain target sites for restriction enzymes. Consequently, telomeres remain in relatively long fragments (TRF), whereas the genomic DNA is digested into short pieces. Fragments of telomeric DNA are then visualized by hybridization with radioactively labeled telomeric probe. As TRF include besides telomeres also a short region of telomere-associated DNA up to the first restriction site, results are slightly shifted towards higher TRFs values. Therefore, the use of frequent cutters or their mixtures is recommended to minimize this difference. Moreover, by using TRF analysis it is possible to distinguish genuine (terminal) telomeres from interstitial telomeric repeats (ITR) (Richards and Ausubel, 1988). In this approach, BAL31 digestion is first applied on high molecular weight DNA. The enzyme progressively degrades linear DNA from its ends. The degraded DNA is then digested with one or more restriction enzymes and fragments are separated by gel electrophoresis. After blotting, membranes are probed with either a terminal marker sequence or telomeric sequence. Genuine TRF can be distinguished from ITR due to their progressive shortening with increasing BAL31 digestion time, while ITR are BAL31-resistant. The TRF BAL31 digestion pattern at the time zero indicates the approximate telomere lengths (Fajkus et al., 2005).

Keywords: Telomere(端粒), Analysis(分析), Evaluation(评价), Terminal restriction fragments(末端限制性片段), Southern hybridisation(Southern杂交)

Materials and Reagents

  1. DNA isolated from plant or animal tissues (Note 1)
  2. Plug molds (Bio-Rad Laboratories, catalog number: 170-3713 )
  3. Restriction enzymes-Frequent-cutters restriction enzymes e.g. MseI (New England Biolabs, catalog number: R0525L ), AluI (New England Biolabs, catalog number: R0137S ), BstNI (New England Biolabs, catalog number: R0168S ), HaeIII (New England Biolabs, catalog number: R0108S ) , HinfI (New England Biolabs, catalog number: R0155L ), RsaI (New England Biolabs, catalog number: R0167S )
    Note: Reaction buffers are supplied by the manufacturer.
  4. BAL31 nuclease (New England Biolabs, catalog number: M0213L )
  5. 50 mM Ethylene glycol bis(2-aminoethylether)-N, N, N', N'-tetra acetic acid (EGTA) (pH 8.0) (SERVA Electrophoresis GmbH, catalog number: 11 290.02 )
  6. 0.5 M EDTA (pH 8.0) (Duchefa Biochemie, catalog number: E0511 )
  7. Agarose for electrophoresis (SERVA Electrophoresis GmbH, catalog number: 11404.05 )
  8. Agarose for pulsed field gel electrophoresis (PFGE) (Bio-Rad Laboratories, catalog number: 162-0138 )
  9. Low melt agarose (Bio-Rad Laboratories, catalog number: 161-3112 )
  10. Ethidium bromide aqueous solution 1% w/v (10 mg/ml) (SERVA Electrophoresis GmbH, catalog number: 21 251.01 )
  11. DNA loading buffer (6x concentrated) (Thermo Fisher Scientific, catalog number: R0611 )
  12. DNA marker for conventional agarose electrophoresis (e.g. GeneRuler 1 kb DNA Ladder) (Thermo Fisher Scientific, catalog number: SM0311 )
  13. DNA marker for PFGE [e.g. Mid Range or Low Range PFG Marker (New England Biolabs, catalog number: N3551S or N0350S )]
  14. 0.25 M HCl (Penta Technologies, catalog number: 77232 )
  15. 0.4 M NaOH (Penta Technologies, catalog number: 71691 )
  16. 100 mM PMSF (in isopropanol) (SERVA Electrophoresis GmbH, catalog number: 32395 )
  17. Proteinase K from Tritirachium album min. 8 DMC-U/mg (SERVA Electrophoresis GmbH, catalog number: 33752.02 )
  18. N-Lauroylsarcosine sodium salt (Sigma-Aldrich, catalog number: L5125 )
  19. D-mannitol (Duchefa, catalog number: M0803 )
  20. Nylon membrane (Hybond XL) (GE Healthcare, catalog number: RPN303 S )
  21. T4 polynucleotide kinase (New England Biolabs, catalog number: M0201L )
  22. DecaLabel DNA Labeling kit (Life Technologies, catalog number: K0622 )
    Note: Currently, it is “Thermo Fisher Scientific, catalog number: K0622”.
  23. 32P-γ-ATP [e.g., Institute of isotopes (Hungary, catalog number: FP-501 )]
  24. 32P-α-dATP (or 32P-α-dCTP) [Institute of isotopes (Hungary, catalog number: FP-203 )]
  25. Synthetic telomeric oligonucleotide (4 telomeric repeats (CCCTAAA)4 or (TTTAGGG)4), or telomeric concatemers
  26. NaCl
  27. Tris-HCl (pH 8)
  28. MgCl2
  29. CaCl2
  30. Glacial acetic acid
  31. Boric acid
  32. NaH2PO4
  33. Na2HPO4
  34. SDS
  35. SSC
  36. BAL31 nuclease buffer (see Recipes) or can be purchased (New England Biolabs, catalog number: B0213S )
  37. 50x TAE (see Recipes)
  38. 5x TBE (see Recipes)
  39. Hybridization buffer (see Recipes)
  40. Washing solution (see Recipes)
  41. TE buffer (see Recipes)
  42. TEM buffer (see Recipes)
  43. Proteinase buffer (see Recipes)

Equipment

  1. Apparatus for standard agarose gel electrophoresis (e.g. Bio-Rad Laboratories, AbD Serotec®)
  2. PFGE electrophoresis apparatus; for optimal resolution resultion, use system with hexagonal electrodes (e.g. CHEF DR, Bio-Rad Laboratories, AbD Serotec® or Gene Navigator, Amersham)
  3. Thermo block (e.g. Eppendorf, model: Thermomixer )
  4. Vacuum concentrator (e.g. Thermo Fisher Scientific, model: SpeedVac )
  5. Vacuum blotter (e.g. Bio-Rad Laboratories, AbD Serotec®)
  6. Gel documentation system (e.g. R&D Systems, FujiFilm, model: LAS3000 )
  7. Hybridization oven [e.g. HybriLinker (Analytik Jena, model: UVP )]
  8. Phosphoimager (e.g. GE Healthcare Dharmacon, model: FLA7000 )

Software

  1. Multi Gauge signal processing software (FujiFilm)

Procedure

  1. Restriction endonuclease(s) digestion
    Based on the expected telomere length, either DNA isolated by standard approaches (telomeres ˂ 10 kb) or high molecular weight DNA (telomeres > 10 kb) is digested.
    1. Digestion of genomic DNA isolated by a standard protocol
      1. Choose appropriate restriction endonuclease or mix of enzymes. These should (i) be frequently cutting with no recognition site inside the canonical telomeric repeat, (ii) cleave under similar reaction conditions (buffer, temperature optimum), (iii) show relatively long survival at working temperature, and (iv) be not sensitive to cytosine methylation.
        Examples of restriction enzymes and experimental conditions: MseI, 37 °C; AluI, 37 °C; HinfI + HaeIII, 37 °C; HinfI + RsaI, 37 °C; BstNI, 60 °C.
      2. Digest ~1 μg of genomic DNA with 20 U of enzyme (or mix of enzymes) for 3 h. Add next aliquot of enzyme(s) and digest O/N.
      3. If necessary for loading the sample on the agarose gel, reduce the volume of reaction mixture using the vacuum concentrator.
    2. Digestion of high molecular weight DNA
      1. Choose appropriate restriction endonuclease or mix of enzymes.
      2. Incubate DNA embedded in the agarose plug (Note 1) in 200-300 μl of the corresponding restriction buffer in 2 ml Eppendorf tube for 30 min at the temperature for the digestion (Note 2).
      3. Replace the restriction buffer by a fresh aliquot (200 μl per 1 agarose block, 300 μl per 2 agarose blocks), add the restriction enzyme(s) (30 U/1 agarose block), and incubate for 3 h. Add next aliquot of enzyme(s) and digest O/N.
      4. Analyze DNA in agarose plugs by PFGE. Fraction of low molecular weight DNA released in the course of digestion to the reaction buffer can be isolated by phenol-chloroform extraction and analyzed by conventional agarose gel electrophoresis.
    3. Optional: Digestion with BAL31 nuclease prior to restriction enzyme(s) treatment
      To determine the terminal position of the putative telomeric sequence, high molecular weight DNA (Note 1) is treated with BAL31 nuclease to degrade terminal sequences. In the TRF analysis, loss of telomere-specific hybridization signal in the course of BAL31 digestion is observed.
      1. Take six or more DNA agarose blocks (based on the number of BAL31 digestion time intervals, e.g., 0, 10, 20, 40, 60, 90 min), put each to a 2 ml Eppendorf tube, add 200 μl of BAL31 reaction buffer, incubate for 30 min at 30 °C.
      2. Replace the reaction buffer by the fresh aliquot (200 μl), incubate the tube in a thermo block (30 °C), add 3 U of BAL31 nuclease (except of zero-time sample) and digest for the respective time (digestion time e.g., 10, 20, 40, 60, 90 min).
      3. Stop the reaction by removing the restriction buffer and adding 500 μl of 50 mM EGTA (pH 8).
      4. Inactivate BAL31 by incubation at 55 °C for 30 min.
      5. Replace EGTA by 1.5 ml of 0.1x TE buffer, incubate at 4 °C for 15 min. Repeat the washing step at least twice more.
      6. Continue with digestion by restriction endonuclease (see step A2).

  2. Separation of DNA by agarose gel electrophoresis
    1. Conventional agarose electrophoresis (separation of terminal fragments of expected lengths ˂ 10 kb)
      1. Prepare 1% agarose gel in 1x TAE buffer.
      2. Cool the gel to ~60 °C, add ethidium bromide to a final concentration of 0.2 μg/ml.
      3. Add DNA loading buffer to DNA samples, load on the gel. Load DNA marker (1 kb DNA ladder).
      4. Run electrophoresis (1.2-2.6 V/cm), check positions of the DNA marker fragments.
      5. Document the gel using a documentation system (in the mode for ethidium bromide-excitation at 312 nm). An example of the Arabidopsis thaliana DNA separated using conventional agarose gel electrophoresis is presented in Figure 1A.
      6. Transfer DNA to the nylon membrane by capillary or vacuum alkali blotting (depurination in 0.25 M HCl, denaturation and transfer in 0.4 M NaOH), hybridize with radioactively labeled telomeric probe (see step C3).
    2. Pulsed field gel electrophoresis (separation of terminal restriction fragments of expected lengths > 10 kb)
      1. Prepare 1% agarose gel in 0.5x TBE buffer.
      2. Pour 0.5 TBE buffer to the PFGE unit, precool to 14 °C.
      3. Set the electrophoresis parameters either using a machine software or manually (e.g., for a DNA fragment range between 10 and 200 kb, appropriate parameters are 6 V/cm, switch time ramped from 1 to 12 sec for 15 h).
      4. Load the DNA samples in agarose plugs into the gel, load DNA marker.
      5. After separation, stain the gel with 0.5 μg/ml ethidium bromide in 0.5x TBE for 15 min, document using documentation system. An example of the Nicotiana tabacum DNA separated using PFGE is presented in Figure 1B.
      6. Transfer DNA to the nylon membrane by capillary or vacuum alkali blotting, hybridize with radioactively labeled telomeric probe (see step C3).

  3. Radioactive labeling of telomeric probe and hybridization
    1. End-labeling of telomeric oligonucleotide (Note 3)
      1. Prepare the end-labeling reaction mix at room temperature: 6.5 μl of water, 5 μl of 10 μM synthetic telomeric oligonucleotide, 2 μl of 10×concentrated T4-polynucleotide kinase buffer, 5 μl of 32P-γ-ATP (1.85 MBq), 1 μl of T4-polynucleotide kinase.
      2. Incubate for 30 min at 37 °C. Heat-inactivate the enzyme (2 min / 94 °C).
      3. Add the mixture directly to the hybridization solution (see step C3c).
    2. Labeling of telomeric concatemers by random priming (Note 3)
      1. Prepare telomeric concatemers by non-template PCR as described in (Ijdo et al., 1991).
      2. Perform end-labeling of telomeric concatemers using 32P-α-dATP or 32P-α-dCTP (1.85 MBq) according to instructions in DecaLabel DNA Labeling kit.
      3. Denature the probe for 10 min at 95 °C, put on ice. Add the probe to pre-heated hybridization solution (see step C3c).
    3. Hybridization
      1. Pre-hybridize the membrane in Hybridization buffer for 1 h at 55 °C in slowly rotating cylinders in hybridization oven. The volume of Hybridization buffer used for pre-hybridization depends on the size of the cylinder; common volume is 30- 50 ml.
      2. Replace Hybridization buffer by the pre-heated fresh aliquot. Keep the volume of Hybridization buffer as low as possible; common volume ranges between 10-20 ml.
      3. Add all volume of the radioactively labeled probe (from the C1 or C2) to the Hybridization buffer.
      4. Hybridize at 55 °C O/N (or at least 16 h).
      5. Pour out the hybridization solution (to the radioactive waste vessel), add the Washing solution (volume of the Washing solution depends on the size of the cylinder; common volume is 50-80 ml), incubate in the hybridization oven for 15 min at 55 °C. Repeat washing step twice.
      6. Discard Washing solution, rinse the membrane by 2x SSC and by water and pack the wet membrane to the Saran wrap.
      7. Expose the membrane to the phosphoimager screen. The time of the exposure depends on the radioactive signal intensity and ranges from 6 h to 24 h.

  4. Evaluation of hybridization signals
    1. Visualize hybridization signals using phosphoimager.
    2. Evaluate the data using available tools, e.g., TeloTool (Gohring et al., 2014), Telometric (Grant et al., 2001), or TELORUN (Baur et al., 2004). Choosing the appropriate processing tool depends namely on distribution of telomere hybridization signals-see Note 4 for details.
      Examples of the TRF analysis are presented in Figure 2A (Arabidopsis thaliana telomeres) and Figure 2B (Nicotiana tabacum telomeres).


      Figure 1. Conventional electrophoresis of Arabidopsis thaliana DNA (A) and pulse field gel electrophoresis of Nicotiana tabacum DNA (B). A. DNA isolated from A. thaliana (ecotype Columbia) leaves was digested with MseI and run on 1% agarose gel in 1x TAE buffer. B. High molecular weight DNA isolated from N. tabacum leaves was digested with TaqI (lane 1), MseI (lane 2) and HinfI + HaeIII (lane 3) and analyzed by PFGE in 0.5x TBE buffer. N-non-digested DNA; M - DNA marker (Low Range PFG Marker).


      Figure 2. Analysis of telomeres of Arabidopsis thaliana (A) and Nicotiana tabacum (B) plant species by the TRF method. A. DNA isolated from A. thaliana (ecotype Columbia) leaves was digested with MseI, analyzed by conventional agarose electrophoresis and hybridized using radioactively labeled telomeric oligonucleotide. Telomeres of this species are about 3 kb long. Arrows indicate the position of ITR bands; N-non-digested DNA; M-DNA marker (GeneRuler 1 kb DNA Ladder). B. High molecular weight DNA isolated from N. tabacum leaves was digested with TaqI (lane 1), MseI (lane 2) and HinfI + HaeIII (lane 3), analyzed by PFGE and hybridized using radioactively labeled telomeric oligonucleotide. Terminal restriction fragments of tobacco are highly heterogeneous in length (20-160 kb). N-non-digested DNA; M-DNA marker (Low Range PFG Marker).

Notes

  1. To obtain accurate and reproducible data, the integrity of DNA is essential. For analysis of telomere length by conventional electrophoresis (expected telomere length ˂ 10 kb, BAL31 analysis is not performed), use any standard method of DNA isolation (e.g., phenol-chloroform extraction or column-based purification using commercial kits). Check carefully the length of DNA by agarose gel electrophoresis; a sharp band in the compression zone (length > 20 kb) should be visible.
    To avoid shearing during isolation, preparation of DNA in agarose plugs is recommended for pulsed-field gel electrophoresis (expected telomere length > 10 kb) and for analysis of the terminal position of telomeric repeats by BAL31 nuclease digestion.
    Preparation of high molecular weight DNA in agarose plugs: 1 g of plant tissue is homogenized in liquid nitrogen. Homogenate is mixed with 1 ml of TEM buffer (buffer is preheated in water bath to 45 °C), in the case of thick suspension more TEM buffer is added. Then one volume of 2% low melt agarose in TEM buffer (preheated in a water bath to 45 °C) is added and mixed by pipetting. The mixture is transferred into the disposable plug molds. DNA in agarose plugs is purified by the proteinase K treatment (3x 24 h at 37 °C in the Proteinase buffer, fresh proteinase K is added every 24 h). Proteinase K is inactivated by the PMSF treatment (1 mM PMSF in 1x TE buffer, 10 °C, 3x 30 min) and plugs are washed by 0.1x TE buffer (3x 30 min at room temperature). Before digestion by restriction enzyme or BAL31, high molecular weight DNA in agarose plugs is stored at 4 °C in 0.25 M EDTA.
  2. Be careful and do not use high temperature when handling with DNA embedded in a low melting agarose block. E.g., digestion temperature of commonly used enzyme in TRF, TaqI or Tru1I restriction endonucleases (recognition sequence TCGA and TTAA, respectively), is 65 °C. Incubation of agarose plugs at this temperature leads to their melting and experiment debasement. Temperatures below the optimum (e.g., 55-58 °C) may be used with these enzymes, or such enzymes should be replaced by enzymes with lower temperature optimum (e.g., instead of Tru1I, use its isoschizomer MseI with reaction optimum at 37 °C).
  3. Two different kinds of probes have been used for probing telomeres in TRF analysis: synthetic telomeric oligonucleotides and concatenated telomeric oligonucleotides (concatemers) (Fajkus et al., 1995; Fajkus et al., 2005; Ijdo et al., 1991; Neplechova et al., 2005; Peska et al., 2015; Sykorova et al., 2003). The advantage of synthetic telomeric oligonucleotides is that the target sequence is well defined. Another advantage is the possibility to use synthetic telomeric oligonucleotides to obtaining strand-specific information, such as the specific detection of overhangs of G-rich telomeric single-stranded DNA (Polanska et al., 2012). The second kind of telomeric probe-concatemers is prepared using PCR and synthetic oligonucleotides designed against both strands of the putative telomere sequence. These are concatenated into a complex probe with extensive heterogeneity in probe length (Ijdo et al., 1991; Peska et al., 2015) but with surprising homogeneity in sequence (Neplechova et al., 2005).
  4. Telomere lengths (TL) can be evaluated using the following approaches: TeloTool, Telometric 1.2, TELORUN. Each has its own strengths and weaknesses. TeloTool (Gohring et al., 2014) provides very fast, accurate, reproducible and easy-to-use measurements of telomeric signals with automatic detection of sample lanes and markers. After establishing the intensity profiles of sample lanes and markers and probe correction, TL can be computed immediately. This method is based on fitting of Gaussian function to hybridization signal intensity. Fitted Gauss curve is used to compute relevant statistics (mean, standard deviation and range of TL). Hence, TeloTool is suitable for evaluation of TL with normally distributed intensity of hybridization signal. The quality of TL evaluation - how well the curve describes sample data - is described by coefficient of determination. In case of asymmetrically distributed data it is recommended to use Telometric 1.2 (Grant et al., 2001), which allows to compute median of TL, that reflects the real lengths of telomeres more appropriately. On the other hand, Telometric 1.2 may suffer from overestimation of TL. Detailed comparison of both methods is referred in (Gohring et al., 2014). TL can also be measured manually by Multi Gauge signal processing software (FujiFilm) followed by computing statistics. Briefly, using DNA marker as a standard, all sample lanes in hybridization pattern are sectioned into rectangles corresponding to the same molecular weight intervals and unweighted mean telomere length is calculated as Ʃ(ODi × Li)/Ʃ(ODi), where ODi is the hybridization signal intensity (above background) within the interval i, and Li is the molecular weight at the mid-point of the interval i. This method is quite accurate but results may be biased by human factor, namely during rectangle lane sectioning and background subtraction. The computing statistics and its graphical output may be easily generated through TELORUN, an excel sheet with preset computing formulas which is freely available at URL (http://www.swmed.edu/home_pages/cellbio/shaywright/research/sw_lab_methods.htm) (Baur et al., 2004).

Recipes

  1. BAL31 nuclease buffer
    600 mM NaCl
    20 mM Tris-HCl (pH 8)
    1 mM EDTA
    12 mM MgCl2
    12 mM CaCl2
  2. 50x TAE
    242 g Tris base
    18.61 g EDTA
    57.1 ml glacial acetic acid
    Water to 1 L
  3. 5x TBE
    54 g Tris base, 27.5 g boric acid, 20 ml 0.5 M EDTA (pH 8), water to 1 L
  4. Hybridization buffer
    0.25 M NaH2PO4+Na2HPO4 (to get final pH 7.5)
    7% SDS, 0.016 M
    EDTA
  5. Washing solution
    2x SSC [1x SSC: 150 mM NaCl, 15 mM sodium citrate(pH 7)]
    0.1% SDS
  6. TE buffer
    10 mM Tris-HCl (pH 8)
    1 mM EDTA (pH 8)
  7. TEM buffer
    1 mM Tris-HCl (pH 8)
    50 mM EDTA
    0.4 M D-mannitol
  8. Proteinase buffer
    0.25 M EDTA (pH 8)
    10 mMTris-HCl (pH 8)
    1% N-lauroylsarcosine

Acknowledgments

This protocol was adapted from previous work (Fajkus et al., 2008). The work was supported by the Grant Agency of the Czech Republic (13-06595S, 13-06943S) and by the project “CEITEC - Central European Institute of Technology” (CZ.1.05/1.1.00/02.0068) from the European Regional Development Fund.

References

  1. Baur, J. A., Wright, W. E. and Shay, J. W. (2004). Analysis of mammalian telomere position effect. Methods Mol Biol 287: 121-136.
  2. Fajkus, J., Dvorackova, M. and Sykorova, E. (2008). Analysis of telomeres and telomerase. In: Hancock, R. (ed). The Nucleus. Humana Press, 267-296.
  3. Fajkus, J., Kovarik, A., Kralovics, R. and Bezdek, M. (1995). Organization of telomeric and subtelomeric chromatin in the higher plant Nicotiana tabacum. Mol Gen Genet 247(5): 633-638.
  4. Fajkus, J., Sykorova, E. and Leitch, A. R. (2005). Techniques in plant telomere biology. Biotechniques 38(2): 233-243.
  5. Gohring, J., Fulcher, N., Jacak, J. and Riha, K. (2014). TeloTool: a new tool for telomere length measurement from terminal restriction fragment analysis with improved probe intensity correction. Nucleic Acids Res 42(3): e21.
  6. Grant, J. D., Broccoli, D., Muquit, M., Manion, F. J., Tisdall, J. and Ochs, M. F. (2001). Telometric: a tool providing simplified, reproducible measurements of telomeric DNA from constant field agarose gels. Biotechniques 31(6): 1314-1316, 1318.
  7. Ijdo, J. W., Wells, R. A., Baldini, A. and Reeders, S. T. (1991). Improved telomere detection using a telomere repeat probe (TTAGGG)n generated by PCR. Nucleic Acids Res 19(17): 4780.
  8. Neplechova, K., Sykorova, E. and Fajkus, J. (2005). Comparison of different kinds of probes used for analysis of variant telomeric sequences. Biophys Chem 117(3): 225-231.
  9. Peska, V., Fajkus, P., Fojtova, M., Dvorackova, M., Hapala, J., Dvoracek, V., Polanska, P., Leitch, A. R., Sykorova, E. and Fajkus, J. (2015). Characterisation of an unusual telomere motif (TTTTTTAGGG)n in the plant Cestrum elegans (Solanaceae), a species with a large genome. Plant J 82(4): 644-654.
  10. Polanska, E., Dobsakova, Z., Dvorackova, M., Fajkus, J. and Stros, M. (2012). HMGB1 gene knockout in mouse embryonic fibroblasts results in reduced telomerase activity and telomere dysfunction. Chromosoma 121(4): 419-431.
  11. Richards, E. J. and Ausubel, F. M. (1988). Isolation of a higher eukaryotic telomere from Arabidopsis thaliana. Cell 53(1): 127-136.
  12. Sykorova, E., Lim, K. Y., Kunicka, Z., Chase, M. W., Bennett, M. D., Fajkus, J. and Leitch, A. R. (2003). Telomere variability in the monocotyledonous plant order Asparagales. Proc Biol Sci 270(1527): 1893-1904.

简介

染色体末端 - 端粒是密集研究的焦点,因为它们对维持染色体稳定性的重要性。它们由于不完全复制而缩短作为计数细胞分裂数目的分子时钟,最终导致细胞周期停滞和细胞衰老。端粒长度的测定是端粒生物学中用于研究和诊断应用的基本方法。末端限制性片段(TRF)分析是分析端粒长度的最古老的方法,并且即使在目前的研究中仍然是"金标准"。该技术依赖于重复的小卫星端粒单元不含有限制酶的靶位点的事实。因此,端粒保持相对长的片段(TRF),而基因组DNA被消化成短片段。然后通过与放射性标记的端粒探针杂交显现端粒DNA的片段。由于TRF除了端粒外还包括直到第一限制性位点的端粒相关DNA的短区域,结果稍微偏向更高的TRF值。因此,建议使用频繁的刀具或其混合物,以尽量减少这种差异。此外,通过使用TRF分析,可以区分真正(末端)端粒与间质端粒重复(ITR)(Richards和Ausubel,1988)。在该方法中,首先将BAL31消化应用于高分子量DNA。酶从其末端逐渐降解线性DNA。然后用一种或多种限制酶消化降解的DNA,并通过凝胶电泳分离片段。印迹后,用末端标记序列或端粒序列探测膜。真正的TRF可以区别于ITR,因为它们随着BAL31消化时间的增加而逐步缩短,而ITR是BAL31抗性的。在时间零时的TRF BAL31消化模式表示近似端粒长度(Fajkus等人,2005)。

关键字:端粒, 分析, 评价, 末端限制性片段, Southern杂交

材料和试剂

  1. 从植物或动物组织中分离的DNA(注1)
  2. 栓模具(Bio-Rad Laboratories,目录号:170-3713)
  3. 限制酶 - 频繁切割酶限制性酶例如Mse I(New England Biolabs,目录号:R0525L),Alu I(New England Biolabs,目录号:R0137S),Bst NI(New England Biolabs,目录号:R0168S),Hae III(New England Biolabs,目录号:R0108S) I(New England Biolabs,目录号:R0155L),Rsa I(New England Biolabs,目录号:R0167S)
    注意:反应缓冲液由制造商提供。
  4. BAL31核酸酶(New England Biolabs,目录号:M0213L)
  5. 50mM乙二醇双(2-氨基乙醚)-N,N,N',N'-四乙酸(EGTA)(pH8.0)(SERVA Electrophoresis GmbH,目录号:11 290.02)
  6. 0.5M EDTA(pH8.0)(Duchefa Biochemie,目录号:E0511)
  7. 电泳用琼脂糖(SERVA Electrophoresis GmbH,目录号:11404.05)
  8. 用于脉冲场凝胶电泳(PFGE)的琼脂糖(Bio-Rad Laboratories,目录号:162-0138)
  9. 低熔点琼脂糖(Bio-Rad Laboratories,目录号:161-3112)
  10. 溴化乙锭水溶液1%w/v(10mg/ml)(SERVA Electrophoresis GmbH,目录号:2125.01)
  11. DNA装载缓冲液(6倍浓缩)(Thermo Fisher Scientific,目录号:R0611)
  12. 用于常规琼脂糖电泳(例如GeneRuler 1kb DNA Ladder)(Thermo Fisher Scientific,目录号:SM0311)的DNA标记物
  13. 用于PFGE的DNA标记[例如中档或低范围PFG标记(New England Biolabs,目录号:N3551S或N0350S)]
  14. 0.25 M HCl(Penta Technologies,目录号:77232)
  15. 0.4 M NaOH(Penta Technologies,目录号:71691)
  16. 100mM PMSF(异丙醇)(SERVA Electrophoresis GmbH,目录号:32395)
  17. 来自Tritirachium的蛋白酶K 8 DMC-U/mg(SERVA Electrophoresis GmbH,目录号:33752.02)
  18. N-月桂酰肌氨酸钠盐(Sigma-Aldrich,目录号:L5125)
  19. D-甘露醇(Duchefa,目录号:M0803)
  20. 尼龙膜(Hybond XL)(GE Healthcare,目录号:RPN303S)
  21. T4多核苷酸激酶(New England Biolabs,目录号:M0201L)
  22. DecaLabel DNA标记试剂盒(Life Technologies,目录号:K0622)
    注意:目前,它是"Thermo Fisher Scientific,目录号:K0622"。
  23. 32 P-γ-ATP [例如同位素研究所(匈牙利,目录号:FP-501)]
  24. 同位素研究所(匈牙利,目录号:FP-203)] P-α-dATP(或 32 P-α-dCTP)
  25. 合成端粒寡核苷酸(4端粒重复(CCCTAAA)4或(TTTAGGG)4)或端粒连接子
  26. NaCl
  27. Tris-HCl(pH 8)
  28. MgCl 2
  29. CaCl <2>
  30. 冰醋酸
  31. 硼酸
  32. NaH 2 PO 4 sub
  33. Na HPO 4
  34. SDS
  35. SSC
  36. BAL31核酸酶缓冲液(参见Recipes)或可购买(New England Biolabs,目录号:B0213S)
  37. 50x TAE(请参阅配方)
  38. 5x TBE(参见配方)
  39. 杂交缓冲液(参见配方)
  40. 洗涤液(见配方)
  41. TE缓冲区(参见配方)
  42. TEM缓冲液(参见配方)
  43. 蛋白酶缓冲液(参见配方)

设备

  1. 用于标准琼脂糖凝胶电泳的装置(例如Bio-Rad Laboratories,AbD Serotec )
  2. PFGE电泳仪;为了获得最佳分辨率结果,使用具有六边形电极的系统(例如,CHEF DR,Bio-Rad Laboratories,AbD Serotec 或Gene Navigator,Amersham)
  3. 热块(例如:Eppendorf,型号:Thermomixer)
  4. 真空浓缩器(例如 Thermo Fisher Scientific,型号:SpeedVac)
  5. 真空吸印器(例如Bio-Rad Laboratories,AbD Serotec )
  6. 凝胶文件系统(例如,R& D Systems,FujiFilm,型号:LAS3000)
  7. 杂交炉[例如 HybriLinker(Analytik Jena,型号:UVP)]
  8. Phosphoimager(例如GE Healthcare Dharmacon,型号:FLA7000)

软件

  1. 多量程信号处理软件(FujiFilm)

程序

  1. 限制性内切酶消化
    基于预期的端粒长度,消化通过标准方法(端粒10kb)或高分子量DNA(端粒> 10kb)分离的DNA。
    1. 通过标准方案分离的基因组DNA的消化
      1. 选择 适当的限制性内切核酸酶或酶的混合物。这些应该(i) ?经常切割,在规范内没有识别网站 端粒重复,(ii)在类似的反应条件下裂解(缓冲液, ?温度最优),(iii)在工作中显示相对长的存活 温度,和(iv)对胞嘧啶甲基化不敏感。
        限制酶和实验条件的实例:Mse I,37 C; Alu I,37℃; I + Hae III,37°C; Hinf I + RsaI,37℃; Bst NI,60 ℃。
      2. 消化?1μg基因组DNA与20 U酶(或酶混合物)3小时。加入下一等份的酶和消化O/N。
      3. 如果需要将样品加载到琼脂糖凝胶上,使用真空浓缩器减少反应混合物的体积。
    2. 高分子量DNA的消化
      1. 选择合适的限制性内切核酸酶或酶的混合物。
      2. 孵育DNA嵌入琼脂糖塞(注1)在200-300微升 相应的限制性缓冲液在2ml Eppendorf管中30分钟 在消化温度下(注2)
      3. 更换 通过新鲜的等分试样(200μl每1个琼脂糖块,300μl) μl每2个琼脂糖块),加入限制酶(30U/l琼脂糖 块),并孵育3小时。加入下一等份的酶并消化 O/N。
      4. 通过PFGE在琼脂糖塞中分析DNA。分数低 在消化过程中释放到反应中的分子量DNA ?缓冲液可以通过苯酚 - 氯仿提取分离并分析 常规琼脂糖凝胶电泳。
    3. 可选:在限制酶处理之前用BAL31核酸酶消化
      为了确定推定的端粒序列的末端位置, 高分子量DNA(注1)用BAL31核酸酶处理 降解末端序列。在TRF分析中,损失 在BAL31消化过程中的端粒特异性杂交信号 。
      1. 取6个或更多DNA琼脂糖块(基于 BAL31消化时间间隔的数目,例如,0,10,20,40,60,90 min),将每个置于2ml Eppendorf管中,加入200μlBAL31反应物 缓冲液,在30℃温育30分钟。
      2. 更换反应 缓冲液通过新鲜等分试样(200μl),将管在热中孵育 块(30℃),加入3U的BAL31核酸酶(零时间样品除外) 并消化各自的时间(消化时间例如<10ms,20℃,40℃,60℃, ?90分钟)。
      3. 通过去除限制性缓冲液并加入500μl50mM EGTA(pH 8)停止反应
      4. 通过在55℃孵育30分钟灭活BAL31
      5. 用1.5ml的0.1×TE缓冲液替换EGTA,在4℃下孵育15分钟。重复洗涤步骤至少两次。
      6. 继续用限制性内切酶消化(参见步骤A2)。

  2. 通过琼脂糖凝胶电泳分离DNA
    1. 常规琼脂糖电泳(分离预期长度为10kb的末端片段)
      1. 准备在1X TAE缓冲液中的1%琼脂糖凝胶。
      2. 将凝胶冷却至约60℃,加入溴化乙锭至终浓度为0.2μg/ml。
      3. 向DNA样品中加入DNA上样缓冲液,加载到凝胶上。加载DNA标记(1kb DNA梯)。
      4. 运行电泳(1.2-2.6 V/cm),检查DNA标记片段的位置
      5. 使用文档系统记录凝胶(在模式中 溴化乙锭 - 在312nm激发)。拟南芥的一个例子 使用常规琼脂糖凝胶电泳分离的拟南芥DNA ?如图1A所示
      6. 转移DNA到尼龙膜 毛细管或真空碱吸印(在0.25M HCl中脱嘌呤, 变性和在0.4M NaOH中转移),与放射性杂交 标记的端粒探针(参见步骤C3)。
    2. 凝胶电泳(分离期望长度> 10kb的末端限制性片段)
      1. 在0.5x TBE缓冲液中制备1%琼脂糖凝胶
      2. 将0.5 TBE缓冲液倒入PFGE装置中,预冷至14℃
      3. 使用机器软件设置电泳参数 或手动(例如,),对于范围在10和200kb之间的DNA片段, 适当的参数为6V/cm,开关时间从1至12秒 15小时)。
      4. 加载DNA样品在琼脂糖插入凝胶,加载DNA标记。
      5. 分离后,用0.5μg/ml溴化乙锭染色凝胶 0.5x TBE 15分钟,使用文档系统的文档。一个例子 图中显示了使用PFGE分离的烟草(Nicotiana tabacum)DNA 1B。
      6. 通过毛细管或真空转移DNA到尼龙膜 碱性印迹,与放射性标记的端粒探针杂交 (参见步骤C3)。

  3. 端粒探针和杂交的放射性标记
    1. 端粒寡核苷酸的末端标记(注3)
      1. 准备 末端标记反应混合物在室温:6.5μl水,5μl 10μM合成端粒寡核苷酸,2μl10×浓缩 T4多核苷酸激酶缓冲液,5μl的32P-γ-ATP(1.85MBq),1μl的 T4多核苷酸激酶。
      2. 在37℃孵育30分钟。热灭活酶(2分钟/94℃)
      3. 将混合物直接加入杂交溶液中(参见步骤C3c)。
    2. 通过随机引发(注3)标记端粒连环体
      1. 通过非模板PCR制备端粒多联体,如(Ijdo等人,1991)中所述。
      2. 根据DecaLabel DNA中的说明,使用32 P-α-dATP或32 P-α-dCTP(1.85 MBq)对端粒连接子进行末端标记 标签工具包。
      3. 使探针在95℃变性10分钟,置于冰上。将探针加入预热的杂交溶液中(参见步骤C3c)。
    3. 杂交
      1. 在55℃下在杂交缓冲液中预杂交膜1小时 在杂交炉中缓慢旋转的圆筒中。体积 用于预杂交的杂交缓冲液取决于大小 气缸;共同体积为30-50ml。
      2. 更换杂交 缓冲液通过预热的新鲜等分试样。保持杂交体积 ?缓冲器尽可能低;共同体积范围在10-20ml之间
      3. 将所有体积的放射性标记的探针(来自C1或C2)加入杂交缓冲液
      4. 在55°C O/N(或至少16小时)杂交
      5. 倒出杂交溶液(放射性废物 容器),加入洗涤溶液(洗涤溶液的体积 取决于圆筒的尺寸;共同体积为50-80ml), 在杂交炉中在55℃孵育15分钟。重复洗涤 步骤两次
      6. 弃去洗涤液,用2x SSC和水冲洗膜,将湿膜包装在Saran包装上
      7. 将膜暴露于phosphoimager屏幕。时间的 暴露取决于放射性信号强度,范围为6 h ?至24小时。

  4. 杂交信号的评价
    1. 使用phosphoimager可视化杂交信号。
    2. 评估 使用可用工具的数据,例如,TeloTool(Gohring ,,2014) (Grant et al。,2001)或TELORUN(Baur et al。,2004)。 选择适当的处理工具取决于分布 的端粒杂交信号 - 详见注4。
      例子 的TRF分析示于图2A(拟南芥端粒)和图2B(烟草(Nicotiana tabacum)端粒)中。


      图1。 拟南芥(Arabidopsis thaliana)DNA(A)和脉冲的常规电泳 烟草的DNA凝胶电泳(B)。 A。被隔绝的DNA 来自拟南芥(生态型哥伦比亚)叶片用Mse I/I消化, 在1%TAE缓冲液中的1%琼脂糖凝胶上电泳。 B.高分子量DNA 分离自N。烟草叶叶用 Taq I(泳道1), Mse I (泳道2)和Hinf I + Hae III(泳道3),并在0.5×TBE中通过PFGE分析 缓冲。 N-未消化的DNA; M-DNA标记(低范围PFG标记)。


      图2.通过TRF方法分析拟南芥(A)和烟草(B)植物物种的端粒。 A.分离的DNA 来自 A。用Mse I消化拟南芥(哥伦比亚生态型)叶片, 通过常规琼脂糖电泳分析并使用杂交 放射性标记的端粒寡核苷酸。这端的端粒 物种约3kb长。箭头指示ITR条带的位置; N-未消化的DNA; M-DNA标记(GeneRuler 1kb DNA Ladder)。 B.高 分离自N的分子量DNA。用泳道1(泳道1),泳道2(泳道2)和泳道1(泳道2)和泳道4中的泳道1和泳道4消化tabacum Hae III(泳道3),通过分析 PFGE并使用放射性标记的端粒进行杂交 寡核苷酸。烟草的末端限制性片段高度 长度不均一(20-160kb)。 N-未消化的DNA; M-DNA标记 (低范围PFG标记)。

笔记

  1. 为了获得准确和可重复的数据,DNA的完整性至关重要。对于通过常规电泳分析端粒长度(预期的端粒长度为10kb,不进行BAL31分析),使用任何标准的DNA分离方法(例如,苯酚 - 氯仿萃取或基于柱的纯化商业试剂盒)。通过琼脂糖凝胶电泳仔细检查DNA的长度;在压缩区域(长度> 20kb)中的尖锐带应该是可见的 为了避免在分离期间的剪切,建议在脉冲场凝胶电泳(预期的端粒长度> 10kb)中用于在琼脂糖塞中制备DNA,并且用于通过BAL31核酸酶消化分析端粒重复的末端位置。 在琼脂糖塞中制备高分子量DNA:将1g植物组织在液氮中匀浆。将匀浆与1ml TEM缓冲液(缓冲液在水浴中预热至45℃)混合,在厚悬浮液的情况下,加入更多的TEM缓冲液。然后加入1体积的在TEM缓冲液中的2%低熔点琼脂糖(在水浴中预热至45℃),并通过吸移混合。将混合物转移到一次性塞模中。通过蛋白酶K处理(在蛋白酶缓冲液中在37℃下3×24小时,每24小时添加新鲜蛋白酶K)纯化琼脂糖栓塞中的DNA。通过PMSF处理(1mM PMSF,在1x TE缓冲液中,10℃,3×30分钟)使蛋白酶K失活,并通过0.1×TE缓冲液(室温下3×30分钟)洗涤栓塞。在通过限制酶或BAL31消化之前,将琼脂糖栓中的高分子量DNA在4℃下储存在0.25M EDTA中。
  2. 小心,当使用嵌入低熔点琼脂糖块中的DNA处理时,不要使用高温。 (例如,分别为识别序列TCGA和TTAA)中的常用酶的消化温度,其中所述限制性内切核酸酶是分别为TRF,Taq或Tru 65℃。在该温度下孵育琼脂糖栓塞导致它们的解链和实验褪色。低于最佳温度(例如55-58℃)可以与这些酶一起使用,或者这些酶应该被具有较低温度最佳值的酶替换(例如,而不是 1I,使用其异构体 Mse I,反应最适宜在37℃)。
  3. 两种不同类型的探针已经用于在TRF分析中探测端粒:合成的端粒寡核苷酸和串联的端粒寡核苷酸(串联体)(Fajkus等人,1995; Fajkus等人, 2005; Ijdo等人,1991; Neplechova等人,2005; Peska等人,2015; Sykorova等人,2005; et al。,2003)。合成的端粒寡核苷酸的优点是靶序列被明确定义。另一个优点是使用合成的端粒寡核苷酸获得链特异性信息的可能性,例如富含G的端粒单链DNA的突出端的特异性检测(Polanska等人,2012)。使用PCR和针对推定的端粒序列的两条链设计的合成寡核苷酸制备第二种类型的端粒探针多联体。这些被连接成具有广泛的探针长度异质性的复杂探针(Ijdo等人,1991; Peska等人,2015),但是具有令人惊奇的序列同质性(Neplechova et al。,2005)。
  4. 端粒长度(TL)可以使用以下方法评估:TeloTool,Telometric 1.2,TELORUN。每个都有自己的优势和弱点。 TeloTool(Gohring等人,2014)通过自动检测样品泳道和标记物,提供了非常快速,准确,可重复和容易使用的端粒信号测量。在建立样品泳道和标记物的强度分布和探针校正之后,可以立即计算TL。该方法基于高斯函数与杂交信号强度的拟合。拟合高斯曲线用于计算相关统计量(平均值,标准偏差和TL范围)。因此,TeloTool适合用于评估具有正态分布的杂交信号强度的TL。 TL评估的质量 - 曲线描述样本数据的程度 - 由确定系数描述。在不对称分布的数据的情况下,推荐使用Telometric 1.2(Grant等人,2001),其允许计算TL的中值,其更适当地反映端粒的实际长度。另一方面,直升机1.2可能遭受对TL的过度估计。两种方法的详细比较参考(Gohring等人,2014)。 TL也可以通过多仪表信号处理软件(FujiFilm)手动测量,然后计算统计。简言之,使用DNA标记作为标准,将杂交模式中的所有样品泳道划分为对应于相同分子量区间的矩形,并且未加权平均端粒长度计算为Σ(ODi×Li)/Σ(ODi),其中ODi是在区间i内的杂交信号强度(高于背景),Li是区间i的中点处的分子量。这种方法是相当准确的,但是结果可能由于人为因素,即在矩形车道分段和背景减除期间。计算统计及其图形输出可以通过TELORUN容易地生成,TELORUN是具有预设计算公式的excel表,其可以在URL( http://www.swmed.edu/home_pages/cellbio/shaywright/research/sw_lab_methods.htm )(Baur等人,2004)。

食谱

  1. BAL31核酸酶缓冲液
    600 mM NaCl
    20mM Tris-HCl(pH8)
    1mM EDTA
    12mM MgCl 2/
    12mM CaCl 2
  2. 50x TAE
    242克三碱基
    18.61g EDTA
    57.1ml冰醋酸 水至1 L
  3. 5x TBE
    54克Tris碱,27.5克硼酸,20毫升0.5M EDTA(pH8),水至1升
  4. 杂交缓冲区
    0.25M NaH 2 PO 4 + Na 2 HPO 4(为了得到最终pH 7.5)。
    7%SDS,0.016M/dm EDTA
  5. 洗涤溶液
    2×SSC [1×SSC:150mM NaCl,15mM柠檬酸钠(pH7)] 0.1%SDS
  6. TE缓冲区
    10mM Tris-HCl(pH8)
    1mM EDTA(pH8)
  7. TEM缓冲区
    1mM Tris-HCl(pH8)
    50 mM EDTA
    0.4 M D-甘露糖醇
  8. 蛋白酶缓冲液
    0.25 M EDTA(pH 8)
    10mM Tris-HCl(pH8)
    1%N-月桂酰肌氨酸

致谢

该协议改编自以前的工作(Fajkus等人,2008)。 这项工作得到了捷克共和国赠款机构(13-06595S,13-06943S)和来自欧洲区域发展中心的"CEITEC-中欧理工学院"项目(CZ.1.05/1.1.00/02.0068) 基金。

参考文献

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  2. Fajkus,J.,Dvorackova,M。和Sykorova,E。(2008)。端粒和端粒酶的分析。 In:Hancock,R。(ed)。 。 Humana Press,267-296。
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  6. Grant,J.D.,Broccoli,D.,Muquit,M.,Manion,F.J.,Tisdall,J.and Ochs,M.F。(2001)。 Telometric:一种工具,提供来自恒定琼脂糖凝胶的端粒DNA的简化,可重现的测量。 Biotechniques 31(6):1314-1316,1318.
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引用:Fojtová, M., Fajkus, P., Polanská, P. and Fajkus, J. (2015). Terminal Restriction Fragments (TRF) Method to Analyze Telomere Lengths. Bio-protocol 5(23): e1671. DOI: 10.21769/BioProtoc.1671.
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