Premium
Discrimination Between 8‐Oxo‐2′‐Deoxyguanosine and 2′‐Deoxyguanosine in DNA by the Single Nucleotide Primer Extension Reaction with Adap Triphosphate
Author(s) -
Taniguchi Yosuke,
Kikukawa Yoshiya,
Sasaki Shigeki
Publication year - 2015
Publication title -
angewandte chemie
Language(s) - English
Resource type - Journals
eISSN - 1521-3757
pISSN - 0044-8249
DOI - 10.1002/ange.201412086
Subject(s) - deoxyguanosine , chemistry , klenow fragment , nucleotide , primer extension , dna , primer (cosmetics) , duplex (building) , template , microbiology and biotechnology , base pair , biochemistry , stereochemistry , dna polymerase , biology , gene , exonuclease , nanotechnology , organic chemistry , materials science
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 H 2 O 2 ‐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.