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Development of a ³²P-postlabeling assay for O⁶-methylguanine

University of British Columbia

Monitoring of the promutagenic DNA adduct, O⁶-methylguanine, in the exfoliated cells (e.g. oral mucosal) of tissues from individuals exposed to tobacco-specific N-nitrosamines may aid in the evaluation of tissue-specific risk of carcinogenesis. People with high levels of this adduct could be identified and the appropriate intervention taken. Current techniques for detection of O⁶-methylguanine are unsuitable for the measurement of low adduct levels in small tissue samples. This thesis describes the development of a ³²P-postlabeling method for detection of O⁶-methylguanine in microgram amounts of DNA. In the first part of the project I synthesized O⁶-methyldeoxyguanosine 3'-monophosphate (0⁶mdG3'p), needed as a chromatography marker. This was achieved using a two step approach; preparation of 0⁶-methyldeoxyguanosine (0⁶ mdG) followed by chemical phosphorylation with KH₂PO₄ in formamide. The identity of the compound was confirmed by U.V. spectroscopy and enzymatic analysis. This is the first reported synthesis of the modified nucleotide. Using the synthetic marker, high performance liquid chromatography (HPLC) procedures were then developed for isolation of 0⁶mdG3'p from digested DNA prior to postlabeling. The basic method for analysis of 0⁶-methylguanine by ³²P-postlabeling comprises five steps, a) digestion of DNA containing 0⁶-methylguanine to deoxynucleoside 3'-monophosphates using micrococcal nuclease and spleen phosphodiesterase, b) isolation of 0⁶mdG3'p from normal nucleotides using reverse-phase HPLC, c) ³²P-labeling of 0⁶mdG3'p to give 0⁶- methyldeoxyguanosine 3',5'³²P-bisphosphate (0⁶mdG3'5'p) using ³²P-ATP and polynucleotide kinase, d) 2-dimensional polyethyleneimine cellulose thin layer chromatography (PEI-TLC) to resolve 0⁶mdG3'5'p from other radioactive materials and e) autoradiography and scintillation counting to quantitate 0⁶mdG3'5'p. Several variations of the basic technique were then investigated with the goal of improving the sensitivity. One modification, encompassing a second HPLC purification of 0⁶mdG3'5'p showed the greatest sensitivity: 0.5 micromole 0⁶-methylguanine per mole normal nucleotide (0.5 µmole/mole) . Using this method, 0⁶-methylguanine was detected in the DNA of mammalian tissue-culture cells treated with an agent, N-methyl-N-nitrosourea (MNU) known through independent techniques to form this adduct. The development of ³²P-postlabeling methods for detection of other small DNA adducts is feasible using the approach described here for 0⁶- methylguanine. The performance of the ³²P-postlabeling method for 0⁶- methylguanine equals that of the best available methods when microgram quantities of DNA are assayed. This new method will complement existing techniques.

Text

2010-08-30

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http://hdl.handle.net/2429/27977

Genetics

University of British Columbia

Graduate