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Telomere Restriction Fragment (TRF) Analysis
端粒限制酶片段(TRF)分析   

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Abstract

While telomerase is expressed in ~90% of primary human tumors, most somatic tissue cells except transiently proliferating stem-like cells do not have detectable telomerase activity (Shay and Wright, 1996; Shay and Wright, 2001). Telomeres progressively shorten with each cell division in normal cells, including proliferating stem-like cells, due to the end replication (lagging strand synthesis) problem and other causes such as oxidative damage, therefore all somatic cells have limited cell proliferation capacity (Hayflick limit) (Hayflick and Moorhead, 1961; Olovnikov, 1973). The progressive telomere shortening eventually leads to growth arrest in normal cells, which is known as replicative senescence (Shay et al., 1991). Once telomerase is activated in cancer cells, telomere length is stabilized by the addition of TTAGGG repeats to the end of chromosomes, thus enabling the limitless continuation of cell division (Shay and Wright, 1996; Shay and Wright, 2001). Therefore, the link between aging and cancer can be partially explained by telomere biology. There are many rapid and convenient methods to study telomere biology such as Telomere Restriction Fragment (TRF), Telomere Repeat Amplification Protocol (TRAP) (Mender and Shay, 2015b) and Telomere dysfunction Induced Foci (TIF) analysis (Mender and Shay, 2015a). In this protocol paper we describe Telomere Restriction Fragment (TRF) analysis to determine average telomeric length of cells.
Telomeric length can be indirectly measured by a technique called Telomere Restriction Fragment analysis (TRF). This technique is a modified Southern blot, which measures the heterogeneous range of telomere lengths in a cell population using the length distribution of the terminal restriction fragments (Harley et al., 1990; Ouellette et al., 2000). This method can be used in eukaryotic cells. The description below focuses on the measurement of human cancer cells telomere length. The principle of this method relies on the lack of restriction enzyme recognition sites within TTAGGG tandem telomeric repeats, therefore digestion of genomic DNA, not telomeric DNA, with a combination of 6 base restriction endonucleases reduces genomic DNA size to less than 800 bp.

Keywords: Telomere Restriction Fragment(端粒限制性片段), Telomeres(端粒酶), Telomere length(端粒长度)

Materials and Reagents

  1. Whatman 3MM chromatography paper (46 x 57 cm) (Thermo Fisher Scientific, catalog number: 05-714-5 )
  2. 25 ml serological pipette (Thermo Fisher Scientific, catalog number: 13-668-2 )
  3. DNeasy Blood and Tissue Kit (QIAGEN, catalog number: 69504 )
  4. Proteinase K (QIAGEN, catalog number: 19131 or 19133 )
  5. Enzymes
    1. HhaI 20,000 units/ml (New England BioLabs, catalog number: R0139L )
    2. HinF1 10,000 units/ml (New England BioLabs, catalog number: R0155L )
    3. MspI 20,000 units/ml (New England BioLabs, catalog number: R0106S )
    4. HaeIII 10,000 units/ml (New England BioLabs, catalog number: R0108L )
    5. RsaI 10,000 units/ml (New England BioLabs, catalog number: R0167L )
    6. AluI 10,000 units/ml (New England BioLabs, catalog number: R0137L )
    7. NE Buffer2 10x concentrate (New England BioLabs, catalog number: B7002S )
  6. Uracil DNA Glycosylase (UDG) 5,000 units/ml (New England BioLabs, catalog number: M0280S )
  7. Klenow Fragment (3’→5’ exo-) 5,000 units/ml (New England BioLabs, catalog number: M0212S )
  8. DEPC-treated water (Life Technologies, catalog number: AM9906 )
    Note: Currently, it is “Thermo Fisher Scientific, AmbionTM, catalog number: AM9906”.
  9. Phosphate Buffered Saline (PBS) (Santa Cruz Biotechnology, ChemCruz, catalog number: sc-24947 )
  10. Tris-Acetate-EDTA (TAE) buffer (Thermo Fisher Scientific, catalog number: BP1332-1 )
  11. Tris-Base Ultrapure (Research Products International Corp., catalog number: T60040-5000.0 )
  12. Ethylenediamine Tetraacetic Acid (EDTA), Disodium Salt Dihydrate (Thermo Fisher Scientific, catalog number: BP120-1 )
  13. Boric acid (Sigma-Aldrich, catalog number: B6768 )
  14. UltraPureTM Agarose (Thermo Fisher Scientific, InvitrogenTM, catalog number: 16500-500 )
  15. GelRed Nucleic Acid Stain (PHENIX Research Products, catalog number: RGB-4102-1 )
  16. Radiolabelled TRF Marker (Herbert et al., 2003)
  17. DNA marker (Bionexus, catalog number: BN2050 )
  18. Sodium Chloride (NaCl) (Thermo Fisher Scientific, catalog number: S271-10 )
  19. Sodium Hydroxide (NaOH) (Thermo Fisher Scientific, catalog number: BP359-212 )
  20. Ficoll-Paque Plus (Thermo Fisher Scientific, catalog number: 45-001-749 )
  21. Polyvinylpyrrolidone (Sigma-Aldrich, catalog number: PVP40 )
  22. Bovine Serum Albumin (BSA), Fraction V (Gemini Bio-Products, catalog number: 700-106P )
  23. dCTP, [α-32P]-6,000 Ci/mmol 20 mCi/ml EasyTide Lead, 500 µCi (PerkinElmer, catalog number: NEG513Z500UC )
  24. 20x Saline-Sodium Citrate (SSC) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15557-036 )
  25. Sodium Dodecyl Sulfate (SDS) (Sigma-Aldrich, catalog number: L4509 )
  26. 10x Buffer M (Roche Diagnostics, catalog number: 11417983001 )
    Note: Currently, it is “Sigma-Aldrich, catalog number: 11417983001”.
  27. 10x PBS buffer-phosphate buffer saline (see Recipes)
  28. 1x TAE buffer-Tris-Acetate-EDTA (see Recipes)
  29. 5x TBE buffer-Tris-Borate-EDTA (see Recipes)
  30. Hybridization solution (see Recipes)
  31. 100x Denhardt solution (see Recipes)
  32. 6x Glycerol and Bromophenol blue Loading Dye (see Recipes)

Equipment

  1. Water bath (Thermo Fisher Scientific, model: Isotemp 205 )
  2. Microcentrifuge (Eppendorf, model: 5424 )
  3. Nanodrop (Thermo Fisher Scientific, Nanodrop Technologies, model: ND-1000 UV/Vis Spectrophotometer )
  4. Gel Tank (Thermo Fisher Scientific, model: OwlTM A2 Large Gel Systems for 20 x 25 cm gel size )
  5. Savant Slab Gel dryer (SibGene, model: SGD4050 )
  6. Hybridizer (Bibby Scientific, Techne, model: Hybrigene )
  7. Cylinder (Bibby Scientific, Techne, model: FHB16/FHB15 )
  8. Thermocycler (LABGENE Scientific, Biometra, model: T1 Thermoblock )
  9. G-BOX (Syngene, model: G-BOX F3 )
  10. Power supply (Whatman Biometra, model: 250 EX )
  11. Microwave oven (GE, model: 1540WW002 )
  12. Screen (Molecular Dynamics, model: Kodak Storage Phosphor Screen )
  13. Typhoon PhosphorImager scanner system (Amersham Biosciences, GE Healthcare, model: Typhoon TRIO )

Software

  1. Image Quant® software (Molecular Dynamics)
  2. Graph Pad Prism 6®

Procedure

  1. Cell pellet: Prepare cell pellets (1 x 106 - 2 x 106) in 2 ml polypropylene screw-cap tubes. After removing the supernatant, cell pellets can be frozen at -80 °C.
    Note: It is preferred to wash cell pellet with 1x PBS, but this step is not required because the small amount of medium in the cell pellet does not interfere with subsequent steps.
  2. Cell lysis: Re-suspend the cells that are fresh or just thawed on ice from -80 °C with 200 μl 1x PBS and add 20 μl proteinase K (20 mg/ml).
  3. DNA extraction: Use DNeasy Blood and Tissue Kit to get high DNA yield. Quantify DNA samples by Nanodrop.
    Note: Multiple methods are available to extract DNA from cells. We prefer the commercial kit since it can easily be performed to get high yield DNA.
  4. DNA digestion: Digest 2.5 μg DNA for 4 h to overnight at 37 °C with combination of restriction enzymes (HhaI, HinF1, MspI, HaeIII, RsaI, AluI).
    Prepare 2.5 μg DNA in dH2O to make 40 μl total volume.
    Add 10 μl enzyme digestion mix to DNA sample (40 μl), so total volume is 50 μl. Incubate in a water bath or heating block overnight at 37 °C.
    Enzyme digestion mix:

    1 sample
    HhaI
    0.25 μl
    HinF1
    0.5 μl
    MspI
    0.25 μl
    HaeIII
    0.5 μl
    RsaI
    0.5 μl
    AluI
    0.5 μl
    NE buffer 2
    5.0 μl
    H2O
    2.5 μl
    Total:
    10 μl
  5. Gel migration: Load radiolabeled TRF ladder and unlabeled molecular weight marker on either or both sides of the samples. Separate digested DNA on 0.7% (w/v) agarose gel stained with gel red (1/20,000x) for 18 h at 70 volts for a 25 cm long gel in 1x TAE or 0.5x TBE buffer. 18 h after samples run, check the gel under a UV source (e.g., at G-BOX) to make sure genomic DNA is completely digested and runs as a smear below the 800 bp molecular weight marker (Figure 1A). Anything above 800 bp shows incomplete genomic DNA digestion and this could interfere with the telomere signal (Figure 1B).


    Figure 1. Gel with DNA ladder marker. A. DNA samples run as a smear below 800 bp (yellow dashline), showing complete digestion of genomic DNA. B. DNA samples are above 800 bp (yellow dashline), showing incomplete genomic digestion. bp: base pair

    Notes:
    1. Add 5 μl loading dye to the samples. While 0.5x TBE can be used to analyze less than ~8 kb, 1x TAE buffer can be used for the ones that have longer telomeres (8 to 20 kb range) to have better separation.
    2. 400 ml volume is good enough for 0.7% (w/v) agarose gel in large gel system.
    3. Radiolabeled TRF marker can be visualized after hybridization with telomere sequence-specific probe. The unlabeled, digested plasmid DNA can be visualized with Gel Red, not with the telomere sequence specific probe.
    4. To prevent leaking of the gel from the gel tray: Wait 20 min after agarose is dissolved in microwave. During this time, seal the space between the gel tray and gaskets (edges of gel tray) with 5-10 ml agarose gel (Figure 2A). Wait 30 min following the pour of the gel (do not forget the put comb when you pour the gel).
    5. Higher concentration of agarose in buffer will cause poor separation of samples and TRF marker (Figure 2B) and also processing gel drying will take a much more time than lower concentration of agarose.


      Figure 2. Preparation of agarose gel electrophoresis. A. This figure shows how to seal between the gaskets and gel tray with agarose gel to prevent leaking. B. The separation difference between 0.7% and 1.4% agarose gel. While 0.7% agarose gel shows good separation on the TRF ladder, TRF ladder on 1.4% agarose gel is not separated well. 25 μl and 12.5 μl ladder was loaded in each lane of 0.7% and 1.4% gels, respectively. TRF ladder 1 and 2 in 1.4% gel are the same ladders. TRF ladder units are kilobase (kb).

  6. DNA Hybridization:
    1. Denature the gel for 20 min in 1.5 M NaCl and 0.5 M NaOH solution (pH 13.2) in a Pyrex® container (slowly shake). Make sure that denaturing solution covers the gel during shaking.
    2. Rinse gel with MilliQ® water to remove NaOH.
    3. Put the gel upside down on 2 sheets of 3MM Whatman® paper and wrap on the top of the gel (Figure 3A and B).


      Figure 3. The method for drying the gel (A and B). Whatman® papers are located at the bottom, gel is located in between Whatman® papers and plastic wrap is on top of the gel.

    4. Dry the gel using a gel dryer (56 °C for ~3 h).
    5. Transfer the gel to a Pyrex® container, rinse with MilliQ® water and remove the Whatman® paper.
    6. Neutralize the gel for 30 min with 0.5 M Tris-HCl and 1.5 M NaCl solution (pH 8). Make sure that neutralization solution covers the gel during shaking.
    7. Wrap gel around 25 ml pipette and transfer gel to a cylindrical hybridization tube.
      Note: Make sure that caps are sealed well and don’t leak.
    8. Prehybridize the gel with 10 ml hybridization solution for at least 10 min at 42 °C in hybridization oven.
    9. Add 12.5 μl hot probe (see step 7 at below) to a fresh 10 ml hybridization solution and let it hybridize overnight at 42 °C rotating hybridization oven.
      Notes:
      1. Be careful when you are working with radioactive material. Protect yourself with a shield and try to avoid any contamination to the work area.
      2. Remove hot hybridization buffer and keep it for next use (it can be used two times or place into radioactive liquid waste).

  7. Hot probe (C-rich) preparation
    1. Preannealed template
      3.4 μl of 10 pmol/μl (10 μM) GTU4 oligonucleotide (GTU4 primer: 5’ -GGG UUA GGG UUA GGG UUA GGG AAA- 3’)
      15.6 μl of 100 pmol/μl (100 μM) T3C3 + 9 oligonucleotide (T3C3 + 9 primer: 5’- TTT CCC TAA CCC TAA-3’)
      1 μl of 1M NaCl (50 mM final concentration)
      Cycler program:
      Heat to 99 °C 1 min
      37 °C 15 min
      25 °C 15 min
      Stored at -20 °C
    2. 8x Adjusted Buffer M
      500 μl 10x Buffer M
      100 μl 2 M Tris.HCl (pH 7.4-7.6)
      25 μl BSA (10 mg/ml)
    3. Reaction
      3.125 μl 8x Adjusted Buffer M
      1.0 μl pre-annealed template oligo
      2.5 μl dATP (0.5 mM)
      2.5 μl dTTP (0.5 mM)
      9.88 μl H2O (DEPC)
      5.0 μl α-P32 dCTP
      1.0 μl Klenow Exo:
      25 °C for 30 min
      98 °C for 5 min
      25 °C for 5 min
      Add 0.5 μl Uracil DNA glycosylase 1 U/μl (UDG)
      37 °C for 10 min
      95 °C for 10 min
      Store the probe for no more than 2 weeks at -20 °C.
  8. Washing: Wash the gel once in 2x SSC, 0.1% SDS solution for 15 min at 42 °C, then wash the gel twice in 0.5x SSC, 0.1% SDS solution for 15 min at 42 °C. Finally, wash the gel twice in 0.5x SSC, 1% SDS for 15 min at 42 °C. 10-15 ml washing solution can be used for each washing step at 42 °C rotating hybridization oven.
    Note: Prepare the washing solutions in the following order: SSC, water, SDS so they dissolve easily.
  9. Exposure: Prepare the gel for scanning. Briefly, wrap the gel with plastic (Saran type) wrap in the cassette and put the screen on the gel (Figure 4). Expose it at least 4 h, preferably overnight. Scan the screen on Typhoon PhosphorImager.


    Figure 4. Illustrates the preparation of the gel for scanning step by step. Prepare the saran plastic wrap in an appropriate cassette size and spread it into the cassette. Then, put gel onto this plastic wrap and cover the gel by wrapping with additional plastic wrap. Place screen on top of the gel that is surrounded plastic wrap, then incubate it in a dark place. All steps should be done behind a protective shield.

  10. Calculate the TRF lengths from phosphor Imager scans.
    Description of telomere length measurements by using Image Quant® software and Graph Pad Prism 6® together.
    1. Measure the distance for each marker band from the top in Image Quant software (X: measured distance, Y: molecular weight) (Figure 5A).
    2. Draw a rectangle with 150 rows around the samples and background (empty lane) in Image Quant software (Figure 5B).
    3. Get the intensity of each 150 boxes for each sample (Volume reports from analysis tool give the intensity values) in Image Quant software. Export volume values from Image Quant to Excel.
    4. Open Graph Pad Prism and create a new data and table. Check “Y: Enter and plot a single Y value for each point”. First column (X): measured distance, second column (Y): molecular weight. Go to analysis, analyze, XY analyses, nonlinear regression (curve fit) and select one phase exponential decay. Go to tab “Range”: check “create a table of XY coordinates of 150 points that define the curve”. Nonlinear fit of Data1 contains molecular weight for each distance in the Y row.
    5. Create a new page with new data and table in Graph Pad Prism. Check “Y: Enter and plot a single Y value for each point”. Copy background volume values (intensity) from excel into first column and sample values into second column. Go to analysis, Analyze, double click “transform”. Check “Transform Y values using Y=Y-X”. This equality gives the transform of data 2 [new intensity (Y)= sample intensity (Y) – background intensity (X)]. Go to analysis, analyze, column analysis, double click “column statistics”, click ok. This gives sum: Σ(Inti).
    6. Create a new page with new data and table in Graph Pad Prism. Check “Y: Enter and plot a single Y value for each point”. Copy and paste nonlinear fit of Data 1 (Y) into X column. Copy and paste Transform of Data 2 (Y) into Y column. Go to analysis, Analyze, double click “transform”. Check “Transform Y values using Y=Y/X”. Go to analysis, analyze, column analysis, double click “column statistics”, click ok. This gives sum: Σ(Inti/MWi). MW: molecular weight.
    7. Calculate the average telomere length using following formula =Σ(Inti)/ Σ(Inti/MWi). Figure 5 C and D show an example for TRF gel and average telomeric lengths for some non-small cell lung cancer cell lines, respectively.



      Figure 5. The beginning steps for telomeric length measurement in Image Quant and some examples for non-small cell lung cancer cell lines on different TRF gels. A. Figure shows the distance measurement for each molecular weight (MW) from top baseline (red dash line) in Image Quant. B. Figure shows columns with 150 rows on each sample and empty lane (background) to calculate the intensity (volume) of each lane in Image Quant. C. HCC15, HCC515 and A549 nonsmall cell lung cancer cell lines on different TRF gels with molecular weight (MW) marker. D. Average telomere length for HCC15, HCC515, A549. The unit for ladder sizes is kilobase (kb).

Recipes

  1. 10x PBS buffer-phosphate buffer saline
    Powder for 5 L of 10x is ready to use for preparation of 5 L of concentrated 10x phosphate-buffered saline (PBS)
    Prepare 5 L milliQ® water and add PBS powder
    Next add large stir bar and place on stirrer until solids are dissolved
  2. 1x TAE buffer-Tris-Acetate-EDTA
    Prepare 2% 50x Tris-Acetate-EDTA in milliQ® water
  3. 5x TBE buffer-Tris-Borate-EDTA
    Prepare a 5x stock solution (pH 8.3) in 10 L of MilliQ® water
    540 g of Tris Base
    275 g of boric acid (use a mask)
    200 ml of 0.5 M EDTA (pH 8.0)
    Add large stir bar and place on stirrer until solids are dissolved
  4. Hybridization solution
    30% (v/v) 20x SSC
    5% 100x Denhardt
    2.5% SDS
  5. 100x Denhardt solution
    Ficoll 5 g
    Polyvinylpyrrolidone 5 g
    BSA 5 g
    dH2O 250 ml
  6. 6x Glycerol and Bromophenol blue Loading Dye (50 ml)
    15 ml glycerol (30% final con.)
    125 mg bromophenol blue
    34 ml DEPC H2O
    ~1 ml Tris HCl (pH 7.6)

Acknowledgments

Some of these protocols were adapted from previously published studies (Herbert et al., 2003). We thank Zeliha Gunnur Dikmen for her help in acquisition of TRAP gel and Abhijit Bugde from the Live Cell Imaging Facility at UT Southwestern for his assistance with the imaging and analysis part of Telomere dysfunction Induced Foci (TIF) analysis.

References

  1. Harley, C. B., Futcher, A. B. and Greider, C. W. (1990). Telomeres shorten during ageing of human fibroblasts. Nature 345(6274): 458-460.
  2. Hayflick, L. and Moorhead, P. S. (1961). The serial cultivation of human diploid cell strains. Exp Cell Res 25: 585-621.
  3. Herbert, B. S., Shay, J. W. and Wright, W. E. (2003). Analysis of telomeres and telomerase. Curr Protoc Cell Biol Chapter 18: Unit 18 16.
  4. Mender, I. and Shay, J. W. (2015a). Telomere dysfunction induced foci (TIF) analysis. Bio-protocol 5(22): e1656.
  5. Mender, I. and Shay, J. W. (2015b). Telomerase repeated amplification protocol (TRAP). Bio-protocol 5(22): e1657.
  6. Olovnikov, A. M. (1973). A theory of marginotomy. The incomplete copying of template margin in enzymic synthesis of polynucleotides and biological significance of the phenomenon. J Theor Biol 41(1): 181-190.
  7. Ouellette, M. M., Liao, M., Herbert, B. S., Johnson, M., Holt, S. E., Liss, H. S., Shay, J. W. and Wright, W. E. (2000). Subsenescent telomere lengths in fibroblasts immortalized by limiting amounts of telomerase. J Biol Chem 275(14): 10072-10076.
  8. Shay, J. W. and Wright, W. E. (1996). Telomerase activity in human cancer. Curr Opin Oncol 8(1): 66-71.
  9. Shay, J. W. and Wright, W. E. (2001). Telomeres and telomerase: implications for cancer and aging. Radiat Res 155(1 Pt 2): 188-193.
  10. Shay, J. W., Wright, W. E. and Werbin, H. (1991). Defining the molecular mechanisms of human cell immortalization. Biochim Biophys Acta 1072(1): 1-7.

简介

虽然端粒酶在约90%的原发性人类肿瘤中表达,但除了瞬时增殖的干细胞样细胞之外,大多数体细胞组织细胞不具有可检测的端粒酶活性(Shay和Wright,1996; Shay和Wright,2001)。由于末端复制(滞后链合成)问题和其它原因例如氧化损伤,端粒在正常细胞中的每个细胞分裂(包括增殖的干细胞样细胞)逐渐缩短,因此所有体细胞具有有限的细胞增殖能力(Hayflick极限) (Hayflick和Moorhead,1961; Olovnikov,1973)。渐进性端粒缩短最终导致正常细胞中的生长停滞,其被称为复制衰老(Shay等人,1991)。一旦端粒酶在癌细胞中被激活,通过在染色体末端添加TTAGGG重复来稳定端粒长度,从而使细胞分裂无限延续(Shay和Wright,1996; Shay和Wright,2001)。因此,衰老和癌症之间的联系可以部分地解释端粒生物学。有许多快速和方便的方法来研究端粒生物学,例如端粒限制性片段(TRF),端粒重复扩增方案(Telomere Repeat Amplification Protocol, TRAP)(Mender and Shay,2015b)和端粒功能障碍诱导Foci(TIF)分析(Mender and Shay,2015a)。在本协议书中,我们描述端粒限制性片段(TRF)分析以确定细胞的平均端粒长度。
端粒长度可以通过称为端粒限制性片段分析(TRF)的技术间接测量。该技术是修饰的Southern印迹,其使用末端限制性片段的长度分布测量细胞群中端粒长度的异质性范围(Harley等人,1990; Ouellette等人。,2000)。该方法可用于真核细胞。以下的描述集中于人癌细胞端粒长度的测量。该方法的原理依赖于在TTAGGG串联端粒重复序列内缺少限制性内切酶识别位点,因此用6个碱基限制性内切核酸酶的组合消化基因组DNA而不是端粒DNA,将基因组DNA大小减少至小于800bp。

关键字:端粒限制性片段, 端粒酶, 端粒长度

材料和试剂

  1. Whatman 3MM色谱纸(46×57cm)(Thermo Fisher Scientific,目录号:05-714-5)
  2. 25ml血清移液管(Thermo Fisher Scientific,目录号:13-668-2)
  3. DNeasy Blood and Tissue Kit(QIAGEN,目录号:69504)
  4. 蛋白酶K(QIAGEN,目录号:19131或19133)

    1. HhaI 20,000单位/ml(New England BioLabs,目录号:R0139L)
    2. HinF1 10,000单位/ml(New England BioLabs,目录号:R0155L)
    3. MspI 20,000单位/ml(New England BioLabs,目录号:R0106S)
    4. HaeIII 10,000单位/ml(New England BioLabs,目录号:R0108L)
    5. RsaI 10,000单位/ml(New England BioLabs,目录号:R0167L)
    6. AluI 10,000单位/ml(New England BioLabs,目录号:R0137L)
    7. NE Buffer2 10x浓缩物(New England BioLabs,目录号:B7002S)
  5. 尿嘧啶DNA糖基化酶(UDG)5,000单位/ml(New England BioLabs,目录号:M0280S)
  6. Klenow片段(3'→5'外切)5,000单位/ml(New England BioLabs,目录号:M0212S)
  7. DEPC处理水(Life Technologies,目录号:AM9906)
    注意:目前,"Thermo Fisher Scientific,Ambion TM ,目录号:AM9906"。
  8. 磷酸盐缓冲盐水(PBS)(Santa Cruz Biotechnology,ChemCruz,目录号:sc-24947)
  9. Tris-Acetate-EDTA(TAE)缓冲液(Thermo Fisher Scientific,目录号:BP1332-1)
  10. Tris-Base Ultrapure(Research Products International Corp.,目录号:T60040-5000.0)
  11. 乙二胺四乙酸(EDTA),二水合二钠(Thermo Fisher Scientific,目录号:BP120-1)
  12. 硼酸(Sigma-Aldrich,目录号:B6768)
  13. UltraPure TM琼脂糖(Thermo Fisher Scientific,Invitrogen TM ,目录号:16500-500)
  14. GelRed核酸染料(PHENIX Research Products,目录号:RGB-4102-1)
  15. 放射性标记的TRF标记物(Herbert et al。,2003)
  16. DNA标记(Bionexus,目录号:BN2050)
  17. 氯化钠(NaCl)(Thermo Fisher Scientific,目录号:S271-10)
  18. 氢氧化钠(NaOH)(Thermo Fisher Scientific,目录号:BP359-212)
  19. Ficoll-Paque Plus(Thermo Fisher Scientific,目录号:45-001-749)
  20. 聚乙烯吡咯烷酮(Sigma-Aldrich,目录号:PVP40)
  21. 牛血清白蛋白(BSA),组分V(Gemini Bio-Products,目录号:700-106P)
  22. dCTP,[α-32 P] -6,000 Ci/mmol 20mCi/ml EasyTide Lead,500μCi(PerkinElmer,目录号:NEG513Z500UC)
  23. 20x盐水 - 柠檬酸钠(SSC)(Thermo Fisher Scientific,Invitrogen TM,目录号:15557-036)
  24. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:L4509)
  25. 10x缓冲液M(Roche Diagnostics,目录号:11417983001) 注意:目前,它是"Sigma-Aldrich,目录号:11417983001"。
  26. 10x PBS缓冲液 - 磷酸盐缓冲盐水(见配方)
  27. 1x TAE缓冲液 - 三醋酸EDTA(见配方)
  28. 5x TBE缓冲液 - 三硼酸盐-EDTA(见配方)
  29. 杂交溶液(参见配方)
  30. 100x Denhardt解决方案(参见配方)
  31. 6x甘油和溴酚蓝加载染料(参见配方)

设备

  1. 水浴(Thermo Fisher Scientific,型号:Isotemp 205)
  2. 微量离心机(Eppendorf,型号:5424)
  3. Nanodrop(Thermo Fisher Scientific,Nanodrop Technologies,型号:ND-1000UV/Vis分光光度计)
  4. 凝胶罐(Thermo Fisher Scientific,型号:Owl TM A2 Large Gel Systems for 20×25cm凝胶大小)
  5. Savant Slab Gel干燥机(SibGene,型号:SGD4050)
  6. Hybridizer(Bibby Scientific,Techne,型号:Hybrigene)
  7. 气缸(Bibby Scientific,Techne,型号:FHB16/FHB15)
  8. 热循环仪(LABGENE Scientific,Biometra,型号:T1 Thermoblock)
  9. G-BOX(Syngene,型号:G-BOX F3)
  10. 电源(Whatman Biometra,型号:250 EX)
  11. 微波炉(GE,型号:1540WW002)
  12. Screen(Molecular Dynamics,型号:Kodak Storage Phosphor Screen)
  13. Typhoon PhosphorImager扫描仪系统(Amersham Biosciences,GE Healthcare,型号:Typhoon TRIO)

软件

  1. 图像Quant ?软件(Molecular Dynamics)
  2. Graph Pad Prism 6 ?

程序

  1. 细胞沉淀:在2ml聚丙烯螺旋盖管中制备细胞沉淀(1×10 6 - 2×10 6/- )。除去上清液后,可将细胞沉淀在-80℃冷冻 注意:优选用1x PBS洗涤细胞沉淀,但不需要此步骤,因为细胞沉淀中的少量培养基不会干扰后续步骤。
  2. 细胞裂解:用200μl1×PBS从-80℃重新悬浮新鲜或刚刚在冰上融化的细胞,并加入20μl蛋白酶K(20mg/ml)。
  3. DNA提取:使用DNeasy血液和组织试剂盒获得高DNA产量。通过Nanodrop定量DNA样品。
    注意:有多种方法可以从细胞中提取DNA。我们更喜欢商业试剂盒,因为它可以很容易地进行获得高产量的DNA。
  4. DNA消化:在37℃下用限制性酶(Hha I,EmF 1,Msp )的组合消化2.5μgDNA 4小时至过夜, I, Hae III, Rsa I, Alu 在dH 2 O中制备2.5μgDNA,使总体积为40μl 加入10μl酶消化混合物到DNA样品(40μl),因此总体积为50μl。在37℃下在水浴或加热块中孵育过夜 酶消化混合物:

    1个样品
    Hha I
    0.25μl
    Hinf 1
    0.5μl
    Msp I
    0.25μl
    Hae III
    0.5μl
    Rsa I
    0.5μl
    Alu I
    0.5μl
    NE缓冲区2
    5.0微升
    H sub 2 O
    2.5μl
    总计:
    10微升
  5. 凝胶迁移:在样品的任一侧或两侧装载放射性标记的TRF梯和未标记的分子量标记。分离消化的DNA在0.7%(w/v)琼脂糖凝胶上,用凝胶红色(1/20,000x)染色18小时,在70伏下在1×TAE或0.5×TBE缓冲液中的25cm长凝胶。在样品运行后18小时,在UV源(例如在G-BOX)下检查凝胶(例如在G-BOX)。以确保基因组DNA被完全消化并作为低于800bp分子量标记(图1A)。高于800bp的任何物质显示不完全的基因组DNA消化,并且这可能干扰端粒信号(图1B)。


    图1.具有DNA梯形标记的凝胶 A. DNA样品以800bp以下的染色条(黄色虚线)运行,显示基因组DNA的完全消化。 B.DNA样品高于800bp(黄色虚线),显示不完全的基因组消化。 bp:碱基对

    注意:
    1. 向样品中加入5μl负载染料。虽然可以使用0.5x TBE 分析小于?8 kb,可以使用1个TAE缓冲区 具有更长的端粒(8至20kb范围)以具有更好的分离。
    2. 在大凝胶体系中,400ml体积足以用于0.7%(w/v)琼脂糖凝胶。
    3. 放射性标记的TRF标记物可以在杂交后显现 端粒序列特异性探针。未标记,消化的质粒DNA 可以用凝胶红可视化,而不是端粒序列特异性 探头。
    4. 为了防止凝胶从凝胶盘中泄漏:等待20分钟 琼脂糖在微波中溶解后。在此期间,密封 凝胶托盘和垫圈(凝胶托盘的边缘)之间的空间用5-10ml 琼脂糖凝胶(图2A)。等待30分钟后,凝胶(do 不要忘记当你倒凝胶时放的梳子)。
    5. 更高 琼脂糖在缓冲液中的浓度将导致样品分离不良 ?和TRF标记(图2B)并且也处理凝胶干燥将采取a 比低浓度的琼脂糖更多的时间。


      图2. A。 此图显示如何密封垫圈和凝胶托盘之间 琼脂糖凝胶以防止泄漏。 B.分离差异 0.7%和1.4%琼脂糖凝胶。而0.7%琼脂糖凝胶显示良好分离 在TRF梯度上,在1.4%琼脂糖凝胶上的TRF梯度不能很好地分离。 ?将25μl和12.5μl梯液装载在0.7%和1.4%凝胶的每个泳道中, ?分别。 1.4%凝胶中的TRF梯1和2是相同的梯子。 TRF 梯形单位是千碱基(kb)。

  6. DNA杂交:
    1. 在1.5 M NaCl和0.5 M NaOH溶液(pH值)中将凝胶变性20分钟 13.2)在Pyrex容器中(缓慢摇动)。确保变性 溶液在摇动期间覆盖凝胶
    2. 用MilliQ 水冲洗凝胶以除去NaOH
    3. 将凝胶倒置在2张3MM Whatman ?纸上,并包裹在凝胶的顶部(图3A和B)。

      span>
      图3.干燥凝胶的方法( A和B )。 Whatman ?论文 位于底部,凝胶位于Whatman 纸之间 塑料包装在凝胶的顶部
    4. 使用凝胶干燥器(56℃,?3小时)干燥凝胶
    5. 将凝胶转移到Pyrex 容器中,用MilliQ 水冲洗并取出Whatman ?纸。
    6. 用0.5M Tris-HCl和1.5M NaCl中和凝胶30分钟 溶液(pH 8)。确保中和溶液覆盖凝胶 在摇动过程中
    7. 将凝胶包裹在25ml移液管上,并将凝胶转移到圆柱形杂交管 注意:请确保瓶盖密封良好,不要泄漏。
    8. 在杂交炉中在42℃下用10ml杂交溶液预杂交凝胶至少10分钟
    9. 加入12.5μl热探针(见下面的步骤7)到新鲜的10ml 杂交溶液,并使其在42℃旋转下杂交过夜 杂交炉 注意:
      1. 工作时要小心 与放射性物质。用盾牌保护自己,尽力 避免对工作区域造成任何污染。
      2. 取出热杂交缓冲液并保存备用(可以使用两次,或放入放射性废液中)。

  7. 热探针(富C)制备
    1. 预退款模板
      3.4μl10pmol/μl(10μM)GTU4寡核苷酸(GTU4引物:5'-GGG UUA GGG UUA GGG UUA GGG AAA-3')
      15.6μl100pmol /μl(100μM)T3C3 + 9寡核苷酸(T3C3 + 9引物:5'-TTT CCC TAA CCC TAA-3')
      1μl1M NaCl(50mM终浓度)
      循环程序:
      加热至99℃1分钟
      37℃15分钟
      25℃15分钟
      储存于-20°C
    2. 8x调整缓冲区M
      500μl10x缓冲液M/B 100μl2M Tris缓冲液(pH 7.4-7.6)
      25μlBSA(10mg/ml)
    3. 反应
      3.125μl8x调整缓冲液M
      1.0μl预退火的模板寡聚体 2.5μldATP(0.5mM) 2.5μldTTP(0.5mM) 9.88μlH 2 O(DEPC)
      5.0μlα-P 32 dCTP
      1.0μlKlenow Exo:
      25℃,30分钟
      98℃5分钟
      25℃,5分钟
      加入0.5μl尿嘧啶DNA糖基化酶1 U /μl(UDG)
      37℃10分钟
      95℃10分钟
      将探针在-20°C下保存不超过2周。
  8. 洗涤:在2×SSC,0.1%SDS溶液中在42℃下洗涤凝胶一次15分钟,然后在42℃下在0.5×SSC,0.1%SDS溶液中洗涤凝胶两次15分钟。最后,在42℃下在0.5x SSC,1%SDS中洗涤凝胶两次15分钟。在42℃旋转杂交炉的每个洗涤步骤中可以使用10-15ml洗涤溶液 注意:按以下顺序准备洗涤溶液:SSC,水,SDS,以便它们容易溶解。
  9. 曝光:准备凝胶进行扫描。简言之,用塑料(Saran型)包装将凝胶包裹在盒中,并将筛子放在凝胶上(图4)。暴露至少4小时,优选过夜。在Typhoon PhosphorImager上扫描屏幕。


    图4.说明逐步扫描凝胶的准备工作。准备合适的样品盒尺寸的saran塑料包装,并将其铺展在盒子中。然后,将凝胶放在这个塑料包装上,并用额外的塑料包装包裹凝胶。将屏幕放置在被塑料包裹的凝胶顶部,然后在黑暗的地方孵育。所有步骤应在保护罩后面进行。

  10. 计算磷光体成像仪扫描的TRF长度。
    通过使用Image Quant</sup>软件和Graph Pad Prism 6 一起描述端粒长度测量。
    1. 测量图像定量中每个标记带距离顶部的距离 软件(X:测量距离,Y:分子量)(图5A)
    2. 在Image Quant软件中(图5B),在样品和背景(空白泳道)周围绘制一个150行的矩形
    3. 获取每个样品的每个150箱的强度(体积报告 从分析工具给出的强度值)在Image Quant软件。 从图像定量到Excel导出体积值。
    4. 打开图形垫 Prism并创建一个新的数据和表。检查"Y:输入并绘制单个 ?每个点的Y值"。第一列(X):测量的距离,秒 柱(Y):分子量。进入分析,分析,XY分析, 非线性回归(曲线拟合)并选择一个相位指数衰减。 ?转到选项卡"范围":检查"创建一个XY坐标表150 定义曲线的点"。 Data1的非线性拟合包含分子 ?Y行中每个距离的权重。
    5. 使用创建新页面 新数据和表在Graph Pad Prism。检查"Y:输入并绘制单个 ?每个点的Y值"。从中复制背景音量值(强度) ?excel进入第一列,将样品值进入第二列。去 分析,分析,双击"变换"。选中"转换Y值" 使用Y = Y-X"。这种等式给出了数据2的变换[新强度 ?(Y)=样品强度(Y) - 背景强度(X)]。去分析, 分析,柱分析,双击"列统计",单击确定。 这给出和:Σ(Int )。
    6. 创建包含新数据和的新页面 在图垫Prism的表。检查"Y:输入并绘制单个Y值 每个点"。将数据1(Y)的非线性拟合复制并粘贴到X列中。 将数据2(Y)的转换复制并粘贴到Y列中。去分析, 分析,双击"变换"。选中"使用转换Y值" Y = Y/X"。转到分析,分析,列分析,双击"列 统计",点击确定。这给出和:Σ(Int /MW i)。 MW:分子 重量
    7. 使用以下计算平均端粒长度 公式=Σ(Int i)/Σ(Int /MW )。图5C和D示出了TRF的示例 凝胶和一些非小细胞肺癌的平均端粒长度 细胞系。




      图5.开始步骤 图像定量中的端粒长度测量和一些例子 非小细胞肺癌细胞系在不同TRF凝胶上。 A.图 显示了每个分子量(MW)距离顶部的距离测量 图像定量中的基线(红色虚线)。图中显示的列 每个样品150行,空行(背景)计算 Image Quant中每个泳道的强度(体积)。 C.HCC15,HCC515和 A549非小细胞肺癌细胞系在不同的TRF凝胶上 分子量(MW)标记。 D.HCC15的平均端粒长度, HCC515,A549。梯子大小的单位是千碱基(kb)。

食谱

  1. 10x PBS缓冲液 - 磷酸盐缓冲盐水
    5L 10x的粉末准备用于制备5L浓缩的10x磷酸盐缓冲盐水(PBS)
    准备5 L milliQ 水并加入PBS粉末
    接下来加入大搅拌棒,并放置在搅拌器上,直到固体溶解
  2. 1x TAE缓冲液 - 三 - 乙酸盐-EDTA
    制备2%50x Tris-乙酸盐-EDTA,在milliQ 水中
  3. 5x TBE缓冲液 - 三硼酸盐EDTA 在10升MilliQ 水中制备5x储备溶液(pH 8.3)
    540克Tris碱
    275克硼酸(使用面膜)
    200ml 0.5M EDTA(pH 8.0)
    加入大搅拌棒,放在搅拌器上,直到固体溶解
  4. 杂交溶液
    30%(v/v)20x SSC
    5%100x Denhardt
    2.5%SDS
  5. 100x Denhardt溶液
    Ficoll 5 g
    聚乙烯吡咯烷酮5克
    BSA 5g
    dH 2 <250ml
  6. 6x甘油和溴酚蓝加载染料(50ml)
    15ml甘油(30%最终浓度) 125mg溴酚蓝
    34ml DEPC H 2 O 2 / ?1ml Tris HCl(pH 7.6)

确认

其中一些方案改编自以前发表的研究(Herbert等人 ,2003)。我们感谢Zeliha Gunnur Dikmen帮助从UT西南的活细胞成像设备获得TRAP凝胶和Abhijit Bugde,他对端粒功能障碍诱导Foci(TIF)分析的成像和分析部分的帮助。

参考文献

  1. Harley,C.B.,Futcher,A.B.and Greider,C.W。(1990)。 人类成纤维细胞衰老期间端粒缩短。自然 em> e 345(6274):458-460。
  2. Hayflick,L。和Moorhead,P.S.(1961)。 人类二倍体细胞株的系列培养 Exp Cell Res < em> 25:585-621。
  3. Herbert,B.S.,Shay,J.W.and Wright,W.E。(2003)。 端粒和端粒酶的分析 Curr Protoc Cell Biol 第18章:第18单元16.
  4. Mender,I.和Shay,J.W。(2015a)。 端粒功能障碍诱导灶(TIF)分析 B io -prot ocol 5(22):e1656
  5. Mender,I.和Shay,J. W.(2015b)。 端粒酶重复扩增方案(TRAP) 生物协议 5(22 ):e1657。
  6. Olovnikov,A.M。(1973)。 边缘切除术的理论。在多核苷酸的酶合成中模板边缘的不完全复制和现象的生物学意义。 41 Theor Biol 41(1):181-190。
  7. Ouellette,M.M.,Liao,M.,Herbert,B.S.,Johnson,M.,Holt,S.E.,Liss,H.S.,Shay,J.W.and Wright,W.E。(2000)。 通过限制端粒酶量使永生化成纤维细胞中的端粒长度。生物Chem 275(14):10072-10076。
  8. Shay,J.W.and Wright,W.E。(1996)。 人类癌症中的端粒酶活性。Curr Opin Oncol 8 (1):66-71。
  9. Shay,J.W.and Wright,W.E。(2001)。 端粒和端粒酶:对癌症和衰老的影响 Radiat Res < em> 155(1 Pt 2):188-193。
  10. Shay,J.W.,Wright,W.E.and Werbin,H。(1991)。 定义人类细胞永生化的分子机制。生物化学生物物理学1072(1):1-7。
  • English
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Mender, I. and Shay, J. W. (2015). Telomere Restriction Fragment (TRF) Analysis. Bio-protocol 5(22): e1658. DOI: 10.21769/BioProtoc.1658.
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