Detection of Bulky Endogenous Oxidative DNA Lesions Derived from 8,5'-Cyclo-2'-deoxyadenosine by ³²P-Postlabeling Assay.
8,5'-Cyclopurine-2'-deoxynucleotides represent a class of oxidative DNA lesions that are specifically repaired by nucleotide excision repair but not by base excision repair or direct enzymatic reversion. The 32P-postlabeling assay is an ultrasensitive method that has been extensively used for the detection of carcinogen-DNA adducts in laboratory animal and epidemiological studies. This assay under modified chromatographic conditions is also a suitable and sensitive method for the detection of 8,5'-cyclo-2'-deoxyadenosine (cA). After enzymatic digestion of DNA, and enrichment of the oxidative products from the DNA digest, four dinucleotides containing cA, i.e., Ap-cAp, Cp-cAp, Gp-cAp, and Tp-cAp, are 5'-labeled with [32P]orthophosphate from [γ-32P]ATP, mediated by polynucleotide kinase (PNK). The 32P-labeled cA products are separated by two-dimensional thin-layer chromatography (TLC) and quantified by Instant Imager or by a scintillation counter. The assay only requires 1 to 10 μg of DNA sample and is capable of detecting cA lesions at frequencies as low as 1 in 1010 normal nucleotides. © 2015 by John Wiley & Sons, Inc.
Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, Texas.
Guo-Dong
GD
Zhou
Department of Pediatrics, Baylor College of Medicine, Houston, Texas.
Bhagavatula
B
Moorthy
D000818:Animals; D001345:Autoradiography / Q000379:methods; D002855:Chromatography, Thin Layer / Q000379:methods*; D018736:DNA Adducts / Q000032:analysis; D004249:DNA Damage; D004260:DNA Repair; D003839:Deoxyadenosines / Q000032:analysis*; D057230:Limit of Detection; D018384:Oxidative Stress; D010761:Phosphorus Radioisotopes
D018736:DNA Adducts; D003839:Deoxyadenosines; D010761:Phosphorus Radioisotopes; C056752:8,5'-cyclo-2'-deoxyadenosine
32P-postlabeling assay; 8,5′-cyclopurine-2′-deoxynucleotides; oxidative DNA damage; thin-layer chromatography