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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

SV Sona Valuchova *
ED Elisa Derboven *
KR Karel Riha

 (*contributed equally to this work) 发布: 2016年04月05日第6卷第7期 DOI: 10.21769/BioProtoc.1775 浏览次数: 9299

评审: Tie LiuShyam SolankiTzvetina Brumbarova

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

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

English
中文翻译

文章信息

版权信息

© 2016 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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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分类

植物科学 > 植物分子生物学 > DNA

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