An improved 32P-postlabelling assay for detection and quantitation of styrene 7,8-oxide-DNA adducts

Please always quote using this URN: urn:nbn:de:bvb:20-opus-86305
  • Using DNA modified with [7-3H]styrene 7,8-oxide (SO) in vitro we have standardized the 32P-postlabelling assay for detecting SO-DNA adducts. Nuclease P 1-enriched adducts were 32P-labelled and purified by high-salt ( 4.0 M ammonium formate, pH 6.1} C1s reverse-phase TLC. After elution from the layer with 2-butoxyethanol:H20 (4:6), adducts were separated by two-dimensional PEI cellulose TLC in non-urea solvents (2.0 M ammonium formate, pH 3.5, and 2.7 M sodium phosphate, pH 5.6). One major, three minor and several trace adducts were detected.Using DNA modified with [7-3H]styrene 7,8-oxide (SO) in vitro we have standardized the 32P-postlabelling assay for detecting SO-DNA adducts. Nuclease P 1-enriched adducts were 32P-labelled and purified by high-salt ( 4.0 M ammonium formate, pH 6.1} C1s reverse-phase TLC. After elution from the layer with 2-butoxyethanol:H20 (4:6), adducts were separated by two-dimensional PEI cellulose TLC in non-urea solvents (2.0 M ammonium formate, pH 3.5, and 2.7 M sodium phosphate, pH 5.6). One major, three minor and several trace adducts were detected. The efficiency of the kinase reaction depended on the ATP concentration. Use of standard labelling conditions (['Y· 32P]ATP, <3000 Ci/mmol; <2 Mikromol) resulted in poor ( 4-7%) adduct recovery. An ATP concentration of 40 Mikromol, however, increased the labeJling efficiency by a factor of 5-8 (35-55% based on 3H-SO labelied DNA). The results indicate that the new separation technique is suitable for the relatively polar SO-DNA adducts and that high labelling efficiency can be achieved.show moreshow less

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Metadaten
Author: S. Cantoreggi, R. C. Gupta, Werner K. Lutz
URN:urn:nbn:de:bvb:20-opus-86305
Document Type:Book article / Book chapter
Faculties:Medizinische Fakultät / Institut für Pharmakologie und Toxikologie
Language:English
Year of Completion:1993
Source:In: Postlabelling methods for detection of DNA adducts / ed. by D. H. Phillips. - Lyon: IARC [u.a.], 1993. - (Centre International de Recherche sur le Cancer <Lyon>: IARC scientific publications ; 124). - S. 201-205
Dewey Decimal Classification:6 Technik, Medizin, angewandte Wissenschaften / 61 Medizin und Gesundheit / 610 Medizin und Gesundheit
GND Keyword:Medizin
Release Date:2014/06/12
Licence (German):License LogoDeutsches Urheberrecht