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Telomere Restriction Fragment (TRF) Analysis

端粒限制酶片段（TRF）分析

Ilgen Mender Ilgen Mender
Jerry W. Shay Jerry W. Shay

发布: 2015年11月20日第5卷第22期 DOI: 10.21769/BioProtoc.1658 浏览次数: 21294

评审: HongLok LungVanesa Olivares-IllanaAnonymous reviewer(s)

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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) search Telomeres (端粒酶) search Telomere length (端粒长度) search

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

English
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文章信息

版权信息

© 2015 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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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癌症生物学 > 无限复制 > 细胞生物学试验

癌症生物学 > 无限复制 > 细胞生物学试验

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