Reference - Detail
|Author||Clark TA, Lu X, Luong K, Dai Q, Boitano M, Turner SW, He C, Korlach J.|
|Title||Enhanced 5-methylcytosine detection in single-molecule, real-time sequencing via Tet1 oxidation.|
BACKGROUND:DNA methylation serves as an important epigenetic mark in both eukaryotic and prokaryotic organisms. In eukaryotes, the most common epigenetic mark is 5-methylcytosine, whereas prokaryotes can have 6-methyladenine, 4-methylcytosine, or 5-methylcytosine. Single-molecule, real-time sequencing is capable of directly detecting all three types of modified bases. However, the kinetic signature of 5-methylcytosine is subtle, which presents a challenge for detection. We investigated whether conversion of 5-methylcytosine to 5-carboxylcytosine using the enzyme Tet1 would enhance the kinetic signature, thereby improving detection.
RESULTS:We characterized the kinetic signatures of various cytosine modifications, demonstrating that 5-carboxylcytosine has a larger impact on the local polymerase rate than 5-methylcytosine. Using Tet1-mediated conversion, we show improved detection of 5-methylcytosine using in vitro methylated templates and apply the method to the characterization of 5-methylcytosine sites in the genomes of Escherichia coli MG1655 and Bacillus halodurans C-125.
CONCLUSIONS:We have developed a method for the enhancement of directly detecting 5-methylcytosine during single-molecule, real-time sequencing. Using Tet1 to convert 5-methylcytosine to 5-carboxylcytosine improves the detection rate of this important epigenetic marker, thereby complementing the set of readily detectable microbial base modifications, and enhancing the ability to interrogate eukaryotic epigenetic markers.
|MeSH||5-Methylcytosine / metabolism* DNA Modification Methylases / metabolism DNA-Binding Proteins / metabolism* Escherichia coli / enzymology Genome, Bacterial Kinetics Mixed Function Oxygenases Oxidation-Reduction Proto-Oncogene Proteins / metabolism* Sequence Analysis, DNA* Substrate Specificity|
|General Microbes||JCM 9153|