RRC ID 27511
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.
Journal BMC Biol.
Abstract 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.
Volume 11
Pages 4
Published 2013-1-22
DOI 10.1186/1741-7007-11-4
PII 1741-7007-11-4
PMID 23339471
PMC PMC3598637
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
IF 5.77
Times Cited 43
General Microbes JCM 9153