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Author(s): Clark, Tyson and Luong, Khai and Spittle, Kim and Lee, Jessica and Bullard, James and Respuela, Patricia

DNA is under constant stress from both endogenous and exogenous sources. DNA base modifications resulting from various types of DNA damage are wide-spread and play important roles in affecting physiological states and disease phenotypes. Examples include oxidative damage (8- oxoguanine, 8-oxoadenine; aging, Alzheimer’s, Parkinson’s), alkylation (1-methyladenine, 6-O- methylguanine; cancer), adduct formation (benzo[a]pyrene diol epoxide (BPDE), pyrimidine dimers; smoking, industrial chemical exposure, chemical UV light exposure, cancer), and ionizing radiation damage (5-hydroxycytosine, 5- hydroxyuracil, 5-hydroxymethyluracil; cancer). Currently, these and other products of DNA damage cannot be sequenced with existing sequencing methods. In contrast, single molecule, real-time (SMRT) DNA sequencing can report on modified DNA bases through an analysis of the DNA polymerase kinetics that is affected by a modified base in the template. We demonstrate the DNA strand-resolved sequencing of over 8 different DNA-damage associated base modifications, with base pair resolution and single DNA molecule sensitivity. We also report on the application of this sequencing capability to biological samples and the development of a generic, open-source algorithm to analyze kinetic information from SMRT sequencing.

Organization: PacBio
Year: 2012

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