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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

Miloslava Fojtová Miloslava Fojtová
Petr Fajkus Petr Fajkus
Pavla Polanská Pavla Polanská
Jiří Fajkus Jiří Fajkus

发布: 2015年12月05日第5卷第23期 DOI: 10.21769/BioProtoc.1671 浏览次数: 18920

评审: Tie LiuIgor Cesarino Anonymous reviewer(s)

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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) search Analysis (分析) search Evaluation (评价) search Terminal restriction fragments (末端限制性片段) search Southern hybridisation (Southern杂交) search

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

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文章信息

版权信息

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

如何引用

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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分类

植物科学 > 植物分子生物学 > 染色质

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