文章基本信息

标题：32P-labeling test for DNA damage
本地全文：下载
作者：K Randerath ; M V Reddy ; R C Gupta 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1981
卷号：78
期号：10
页码：6126-6129
DOI：10.1073/pnas.78.10.6126
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Covalent adducts formed by the reaction of DNA with chemical carcinogens and mutagens may be detected by a 32P-labeling test. DNA preparations exposed to chemicals known to bind covalently to DNA [N-methyl-N-nitrosourea, dimethyl sulfate, formaldehyde, beta-propiolactone, propylene oxide, streptozotocin, nitrogen mustard, and 1,3-bis(2-chloroethyl)-1-nitrosourea] were digested to a mixture of deoxynucleoside 3'-monophosphates by incubation with micrococcal endonuclease (EC 3.1.31.1 ) and spleen exonuclease (EC 3.1.16.1 ). The digests were treated with [gamma-32P]ATP and T4 polynucleotide kinase (ATP:5'-dephosphopolynucleotide 5'-phosphotransferase, EC 2.7.1.78 ) to convert the monophosphates to 5'-32P-labeled deoxynucleoside 3',5'-bis-phosphates. These compounds were then separated on polyethyleneimine-cellulose thin layers in ammonium formate and ammonium sulfate solutions. Autoradiograms of the chromatograms obtained by this high-resolution procedure showed the presence of nucleotides derived from chemically altered, as well as normal, DNA constituents. Maps from DNA exposed to any of the chemicals used exhibited a spot pattern typical for the particular chemical. This method detected a single adduct in 10(5) DNA nucleotides without requiring that the compound under investigation be radioactive and thus provides a useful test to screen chemicals for their capacity to damage DNA by covalent binding.