Direct Detection and Sequencing of Damaged DNA Bases

DNA is under constant stress from both endogenous and exogenous sources. As the carrier of genetic information, DNA relies on the maintenance and repair of existing molecules and is the only biological molecule to do so. The bases exhibit limited chemical stability and are vulnerable to chemical modifications through different types of damage, including oxidation, alkylation, radiation damage, and hydrolysis. DNA base modifications resulting from these types of DNA damage are wide-spread and play important roles in affecting physiological states and disease phenotypes (reviewed in [1-3]). Examples include 8-oxoguanine, 8-oxoadenine (oxidative damage; aging, Alzheimer's, Parkinson's), 1-methyladenine, 6-O-methylguanine (alkylation; gliomas and colorectal carcinomas), benzo[a]pyrene diol epoxide (BPDE), pyrimidine dimers (adduct formation; smoking, industrial chemical exposure, UV light exposure; lung and skin cancer), and 5-hydroxycytosine, 5-hydroxyuracil, 5-hydroxymethyluracil, and thymine glycol (ionizing radiation damage; chronic inflammatory diseases, prostate, breast and colorectal cancer).Currently, methods for detecting these and other products of DNA damage are limited to bulk measurements including chromatographic techniques, polymerase chain reaction assays, the Comet assay, mass spectrometry, electrochemistry, radioactive labeling and immunochemical methods (reviewed in [4]). To our knowledge, the integration of DNA damage detection into a high-throughput DNA sequencing technique has not been reported. Because base damage can occur at random DNA template positions, sequencing capabilities reaching the level of individual DNA molecules are highly desirable.Recently, single-molecule, real-time (SMRT) DNA sequencing has been described for the direct detection of methylated and hydroxymethylated DNA bases [5]. In SMRT sequencing, the progression of single molecules of DNA polymerase is monitored in real time during base incorporations using fluorescent phosph