Molecular dosimetry of 7-alkyl- and O6-alkylguanine in DNA by electrochemical detection.
A methodology is described for the quantitation of 7-alkyl- and O6-alkylguanine in DNA isolated from experimental animals exposed to alkylating agents. Following purification, the DNA is hydrolysed under acid conditions after which 7-alkyl- and O6-alkylguanine are separated from unmodified bases by HPLC using a strong cation exchange column. The fractions containing the methylated purines are subsequently analyzed by HPLC using a reverse phase column coupled to an electrochemical detector (amperometric). This method allows the detection of 10-20 fmoles 7-alkyl- and O6-alkylguanine, when pure markers are analyzed. In practice, the detection limit is 0.5 adducts per 10(6) nucleotides for the methylated and 1 adduct per 10(6) nucleotides for the ethylated form of 7-alkyl- and O6-alkylguanine using 25 micrograms DNA.
de Groot
A J
AJ
MGC-Department of Radiation Genetics and Chemical Mutagenesis, Leiden University, The Netherlands.
Jansen
J G
JG
van Valkenburg
C F
CF
van Zeeland
A A
AA
D000477:Alkylating Agents / Q000494:pharmacology; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000737:chemistry* / Q000187:drug effects / Q000302:isolation & purification; D004563:Electrochemistry; D006147:Guanine / Q000031:analogs & derivatives* / Q000032:analysis
D000477:Alkylating Agents; D006147:Guanine; C008450:7-methylguanine; D004247:DNA; C008449:O-(6)-methylguanine