Sequencing DNA for the Oxidatively Modified Base 8-Oxo-7,8-Dihydroguanine.

Abstract

The DNA base guanine (G) can be oxidatively modified to 8-oxo-7,8-dihydroguanine (OG). Extraction of genomic DNA followed by nuclease digestion and mass spectrometry analysis has found OG is present at background levels of ~1 out of 106 Gs; however, this approach cannot determine the locations for the OGs in the genome. Thus, in this methods report, we outline three different methods (A, B, and C) for sequencing OG in DNA. Method A sequences OG by utilizing the base excision repair pathway to delete the OG nucleotide from the DNA that is then detected by Sanger sequencing as a deletion signature. Method B sequences OG by harnessing the base excision repair pathway to convert OG to an unnatural DNA base pair followed by Sanger sequencing to locate the unnatural base pair indicating where OG was located. Method C (i.e., OG-Seq) takes genomic DNA sheared to ~150bps followed by selectively biotinylating the OG-containing fragments for affinity purification and enrichment of the OG-modified strands. The OG-modified fragments are sequenced on a next-generation sequencing platform to locate OG on the genomic scale with a resolution of ~150bps. The methods outlined are then compared and contrasted allowing researchers to select the one that best suits their experimental goals.