DNA斑点杂交修复试验

Jeong-Min Park; Tae-Hong Kang

doi:10.21769/BioProtoc.1453

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Peer-reviewed

DNA Slot Blot Repair Assay

DNA斑点杂交修复试验

Jeong-Min Park

Tae-Hong Kang email

发布: 2015年04月20日第5卷第8期 DOI: 10.21769/BioProtoc.1453 浏览次数: 21090

评审: HongLok LungPinchas TsukermanAnonymous reviewer(s)

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The authors used this protocol in:

Cover of Oncogene, featuring study using the protocol.

Jan 2014

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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)

Nucleotide excision repair (核苷酸切除修复)

UV damage (紫外线的伤害)

CPD (CPD)

6-4PP (6-4PP)

Materials and Reagents

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

Equipment

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