Sequencing 5-Hydroxymethyluracil at Single-Base Resolution.
- Abstract:
- 5-hydroxymethyluracil (5hmU) is formed through oxidation of thymine both enzymatically and non-enzymatically in various biological systems. Although 5hmU has been reported to affect biological processes such as protein-DNA interactions, the consequences of 5hmU formation in genomes have not been yet fully explored. Herein, we report a method to sequence 5hmU at single-base resolution. We employ chemical oxidation to transform 5hmU to 5-formyluracil (5fU), followed by the polymerase extension to induce T-to-C base changes owing to the inherent ability of 5fU to form 5fU:G base pairing. In combination with the Illumina next generation sequencing technology, we developed polymerase chain reaction (PCR) conditions to amplify the T-to-C base changes and demonstrate the method in three different synthetic oligonucleotide models as well as part of the genome of a 5hmU-rich eukaryotic pathogen. Our method has the potential capability to map 5hmU in genomic DNA and thus will contribute to promote the understanding of this modified base.
- Authors:
- F Kawasaki, S Martínez Cuesta, D Beraldi, A Mahtey, RE Hardisty, M Carrington, S Balasubramanian
- Journal:
- Angew Chem Int Ed Engl
- Citation info:
- 57(31):9694-9696
- Publication date:
- 26th Jul 2018
- Full text
- DOI