Open AccessDetection of Bulky Endogenous Oxidative DNA Lesions Derived from 8,5′‐Cyclo‐2′‐deoxyadenosine by 32 P‐Postlabeling Assay
Author(s) -
Zhou GuoDong,
Moorthy Bhagavatula
Publication year - 2015
Publication title -
current protocols in toxicology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.449
H-Index - 23
eISSN - 1934-9262
pISSN - 1934-9254
DOI - 10.1002/0471140856.tx1717s64
Subject(s) - dna , nucleotide , chemistry , deoxyadenosine , carcinogen , microbiology and biotechnology , dna damage , biochemistry , oxidative phosphorylation , biology , gene
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 32 P‐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 [ 32 P]orthophosphate from [γ‐ 32 P]ATP, mediated by polynucleotide kinase (PNK). The 32 P‐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 10 10 normal nucleotides. © 2015 by John Wiley & Sons, Inc.