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DNA Slot Blot Repair Assay

DNA斑点杂交修复试验

Jeong-Min Park Jeong-Min Park
Tae-Hong Kang Tae-Hong Kang

发布: 2015年04月20日第5卷第8期 DOI: 10.21769/BioProtoc.1453 浏览次数: 22414

评审: HongLok LungPinchas TsukermanAnonymous reviewer(s)

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Abstract

Ultraviolet (UV) irradiation induces helix distorting photolesions such as cyclobutane pyrimidine dimers (CPD) and pyrimidine-pyrimidone (6-4) photoproducts (6-4PP) which threaten genomic integrity if unrepaired. In mammals, nucleotide excision repair (NER) is the only pathway that removes UV-induced DNA damages. Here we describe DNA slot blot repair assay for quantitative detection of NER activity using DNA damage specific antibodies such as anti-CPD and anti-6-4PP. Briefly, genomic DNA irradiated with UV was isolated from cells, and the genomic DNA was vacuum-transferred to a nitrocellulose membrane using a Bio-Dot SF microfiltration apparatus (Bio-Rad). A monoclonal antibody that recognizes CPD or 6-4PP was applied to detect the remaining amount of photolesions in the genomic DNA. For loading control of even loading, DNA onto the membrane can be further analyzed by SYBR-gold staining.

Keywords: DNA repair (DNA修复) search Nucleotide excision repair (核苷酸切除修复) search UV damage (紫外线的伤害) search CPD (CPD) search 6-4PP (6-4PP) search

Materials and Reagents

  1. QIAamp® DNA Mini Kit (QIAGEN, catalog number: 51306 )
  2. Nitrocellulose membrane (GE Healthcare, catalog number: 10600003 )
  3. Whatman filter paper 3 MM Chr (Whatman, catalog number: 3030-917 )
  4. Mouse monoclonal antibody to CPD (Kamiya, catalog number: MC-062 )
  5. Mouse monoclonal antibody to 6-4PPs (COSMO BIO, catalog number: NMDND002 )
  6. Peroxidase affinipure goat anti-mouse IgG (H+L) (Jackson Laboratories, catalog number: 115-035-003 )
  7. SYBR® Gold Nucleic Acid Gel Stain (Life Technologies, catalog number: S-11494 )
  8. RNase A (100 mg/ml) (Bio Basic Canada Inc., catalog number: RB0473 )
  9. Ethanol (Burdick & Jackson, catalog number: RP090-1 )
  10. PBS (Bio Basic Canada Inc., catalog number: PD8117 )
  11. Sodium azide (Junsei Chemical co., catalog number: 26628-22-8 )
  12. NaOH (Bio Basic Canada Inc., catalog number: SB0617 )
  13. EDTA (USB Corporation, catalog number: 6381-92-6 )
  14. Ammonium acetate (NH4Ac) (Katayama Chemical, catalog number: 01-4400 )
  15. Acetic acid (CH3COOH) (Merck Millipore, catalog number: 64-19-7 )
  16. NaCl (Bio Basic Canada Inc., catalog number: DB0483 )
  17. Sodium citrate (Bio Basic Canada Inc., catalog number: CB0035 )
  18. HCl (Duksan PureChemicals, catalog number: 1129 )
  19. Tris base (Bio Basic Canada Inc., catalog number: TB0196 )
  20. Tween-20 (Bio Basic Canada Inc., catalog number: 9005-65-5 )
  21. Non-fat dry milk (Sigma-Aldrich, catalog number: M7409-1BTL )
  22. PierceTM ECL Plus Western Blotting Substrate (Life Technologies, catalog number: 32132 )
  23. 0.4 M NaOH + 10 mM EDTA buffer (see Recipes)
  24. 2 M ammonium acetate (see Recipes)
  25. SSC (see Recipes)
  26. TE buffer (see Recipes)
  27. PBS-T (see Recipes)
  28. Blocking buffer (see Recipes)
  29. Buffer for primary antibody dilution (see Recipes)
  30. Buffer for secondary antibody (see Recipes)

Equipment

  1. Ultraviolet irradiator (UVP, catalog number: CL-1000 )
  2. UV-C sensor (UVP, catalog number: 97-0016-01 )
  3. Bio-Dot SF microfiltration apparatus (Bio-Rad Laboratories, catalog number: 170-6542 )
  4. Vacuum pressure pump (Gardner Denver Welch Vacuum Technology Inc., catalog number: 2522C-10 )
  5. Vacuum drier (JS Research Inc., catalog number: JSVO-30T )
  6. Orbital shaker (Wisd Laboratory Instruments, catalog number: SHO-1D )
  7. NanoDrop (Beckman Coulter, catalog number: DU-730 )
  8. Heat block (Wealtec Corp., catalog number: HB-1 )
  9. Gel doc (Bio-Rad Laboratories, catalog number: 75S/01085 )
  10. Centrifuge (Hanil Science Industrial, catalog number: Smart R17 )
  11. Microcentrifuge tube (SPL, catalog number: 60015 )
  12. 12-channel multi pipette (HTL LAB Solutions, catalog number: 5127 )
  13. Paper forcep (USBECK, catalog number: 3110)
  14. X-ray film 8 x 10 inch (Fujifilm Corporation, catalog number: HR-U30 )
  15. X-ray Cassette-AFAB TYPE 8 x 10 inch (JPI, catalog number: AFAB8x10 )

Procedure

English
中文翻译

文章信息

版权信息

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

如何引用

Park, J. and Kang, T. (2015). DNA Slot Blot Repair Assay. Bio-protocol 5(8): e1453. DOI: 10.21769/BioProtoc.1453.

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分类

分子生物学 > DNA > DNA 损伤和修复

分子生物学 > DNA > DNA 结构

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