Discrimination between 8-oxo-2'-deoxyguanosine and 2'-deoxyguanosine in DNA by the single nucleotide primer extension reaction with adap triphosphate.
The adenosine derivative of 2-oxo-1,3-diazaphenoxazine (Adap) exhibits a superb ability to recognize and form base pairs with 8-oxo-2'-deoxyguanosine (8-oxo-dG) in duplex DNA. In this study, the triphosphate of Adap (dAdapTP) was synthesized and tested for single nucleotide incorporation into primer strands using the Klenow Fragment. The efficiency of dAdapTP incorporation into 8-oxo-dG-containing templates was more than 36-fold higher than with dG-containing templates, and provides better discrimination than does the incorporation of natural 2'-deoxyadenosine triphosphate (dATP). The selective incorporation of dAdapTP into 8-oxo-dG templates was therefore applied to the detection of 8-oxo-dG in human telomeric DNA sequences extracted from H2 O2 -treated HeLa cells. The enzymatic incorporation of dAdapTP into 8-oxo-dG-containing templates may provide a novel basis for sequencing oxidative DNA damage in the genome.
Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan). taniguch@phar.kyushu-u.ac.jp.
Yosuke
Y
Taniguchi
Yoshiya
Y
Kikukawa
Shigeki
S
Sasaki
D000080242:8-Hydroxy-2'-Deoxyguanosine; D020029:Base Pairing; D004247:DNA / Q000032:analysis* / Q000378:metabolism; D004249:DNA Damage; D004256:DNA Polymerase I / Q000378:metabolism; D017931:DNA Primers / Q000378:metabolism; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000032:analysis*; D005456:Fluorescent Dyes / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D021141:Nucleic Acid Amplification Techniques; D010084:Oxidation-Reduction; D011122:Polyphosphates / Q000138:chemical synthesis / Q000378:metabolism; D016615:Telomere / Q000235:genetics
D017931:DNA Primers; D005456:Fluorescent Dyes; D011122:Polyphosphates; D000080242:8-Hydroxy-2'-Deoxyguanosine; D004247:DNA; D006861:Hydrogen Peroxide; D004256:DNA Polymerase I; D003849:Deoxyguanosine; C005692:triphosphoric acid
DNA damage; enzymes; nucleotides; oxidation; sequence determination