Precise genomic mapping of 5-hydroxymethylcytosine via covalent tether-directed sequencing
| Autor: | Saulius Klimašauskas, Zdislav Staševskij, Milda Narmontė, Povilas Gibas, Juozas Gordevičius, Edita Kriukienė |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Oligonucleotides Artificial Gene Amplification and Extension Biochemistry Polymerase Chain Reaction Polymerases Histones chemistry.chemical_compound Mice 0302 clinical medicine Sequencing techniques Genomic library DNA libraries DNA sequencing Biology (General) Polymerase Genome Mammalian Genomics Nucleotides General Neuroscience Methods and Resources Acetylation Genomics Bacteriophage lambda Nucleic acids DNA methylation 5-Methylcytosine General Agricultural and Biological Sciences QH301-705.5 Computational biology Biology Research and Analysis Methods General Biochemistry Genetics and Molecular Biology Cell Line 03 medical and health sciences DNA-binding proteins Genetics Animals Sulfites Molecular Biology Techniques Molecular Biology Embryonic Stem Cells 5-Hydroxymethylcytosine General Immunology and Microbiology Biology and life sciences Oligonucleotide Lysine Reproducibility of Results Computational Biology Proteins Oxidative DNA demethylation Sequence Analysis DNA DNA DNA Methylation Genome Analysis Genomic Libraries 030104 developmental biology DNA demethylation chemistry Animal Genomics biology.protein 5-hydroxymethylcytosine covalent tether-directed sequencing 030217 neurology & neurosurgery |
| Zdroj: | PLoS Biology, San Fransisco : Public Library of Science, 2020, vol. 18, no. 4, e3000684, p. 1-19 PLoS Biology PLoS Biology, Vol 18, Iss 4, p e3000684 (2020) PLOS Biology |
| ISSN: | 1544-9173 1545-7885 |
| Popis: | 5-hydroxymethylcytosine (5hmC) is the most prevalent intermediate on the oxidative DNA demethylation pathway and is implicated in regulation of embryogenesis, neurological processes, and cancerogenesis. Profiling of this relatively scarce genomic modification in clinical samples requires cost-effective high-resolution techniques that avoid harsh chemical treatment. Here, we present a bisulfite-free approach for 5hmC profiling at single-nucleotide resolution, named hmTOP-seq (5hmC-specific tethered oligonucleotide–primed sequencing), which is based on direct sequence readout primed at covalently labeled 5hmC sites from an in situ tethered DNA oligonucleotide. Examination of distinct conjugation chemistries suggested a structural model for the tether-directed nonhomologous polymerase priming enabling theoretical evaluation of suitable tethers at the design stage. The hmTOP-seq procedure was optimized and validated on a small model genome and mouse embryonic stem cells, which allowed construction of single-nucleotide 5hmC maps reflecting subtle differences in strand-specific CG hydroxymethylation. Collectively, hmTOP-seq provides a new valuable tool for cost-effective and precise identification of 5hmC in characterizing its biological role and epigenetic changes associated with human disease. This study describes hmTOP-seq, a bisulfite-free approach for profiling of the epigenetic mark 5-hydroxymethylcytosine (5hmC) at single-nucleotide resolution, based on direct sequence readout primed at an in situ tethered DNA oligonucleotide. |
| Databáze: | OpenAIRE |
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