Research

Direct Detection and Sequencing of Damaged DNA Bases

Tyson A Clark, Kristi E Spittle, Stephen W Turner and Jonas Korlach^*

• * Corresponding author: Jonas Korlach jkorlach@pacificbiosciences.com

Author Affiliations

Pacific Biosciences, 1380 Willow Rd, Menlo Park, California, USA

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Genome Integrity 2011, 2:10 doi:10.1186/2041-9414-2-10

Published: 20 December 2011

Abstract

Products of various forms of DNA damage have been implicated in a variety of important biological processes, such as aging, neurodegenerative diseases, and cancer. Therefore, there exists great interest to develop methods for interrogating damaged DNA in the context of sequencing. Here, we demonstrate that single-molecule, real-time (SMRT^®) DNA sequencing can directly detect damaged DNA bases in the DNA template - as a by-product of the sequencing method - through an analysis of the DNA polymerase kinetics that are altered by the presence of a modified base. We demonstrate the sequencing of several DNA templates containing products of DNA damage, including 8-oxoguanine, 8-oxoadenine, O6-methylguanine, 1-methyladenine, O4-methylthymine, 5-hydroxycytosine, 5-hydroxyuracil, 5-hydroxymethyluracil, or thymine dimers, and show that these base modifications can be readily detected with single-modification resolution and DNA strand specificity. We characterize the distinct kinetic signatures generated by these DNA base modifications.