Sequencing of DNA Lesions Facilitated by Site-Specific Excision via Base Excision Repair DNA Glycosylases Yielding Ligatable Gaps.

ABSTRACT: Modifications to nucleotides in the genome can lead to mutations or are involved in regulation of gene expression, and therefore, finding the site of modification is a worthy goal. Robust methods for sequencing modification sites on commercial sequencers have not been developed beyond the epigenetic marks on cytosine. Herein, a method to sequence DNA modification sites was developed that utilizes DNA glycosylases found in the base excision repair pathway to excise the modification. This approach yields a gap at the modification site that is sealed by T4-DNA ligase, yielding a product strand missing the modification. Upon sequencing, the modified nucleotide is reported as a deletion mutation, identifying its location. This approach was used to detect a uracil (U) or 8-oxo-7,8-dihydroguanine (OG) in codon 12 of the KRAS gene in synthetic oligodeoxynucleotides. Additionally, an OG modification site was placed in the VEGF promoter in a plasmid and sequenced. This method requires only commercially available materials and can be put into practice on any sequencing platform, allowing this method to have broad potential for finding modifications in DNA.

SUBMITTER: Riedl J 

PROVIDER: S-EPMC4828909 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature