Reference - Detail
| 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 | 6.765 |
| Times Cited | 77 |
| WOS Category | BIOLOGY |
| Resource | |
| General Microbes | JCM 9153 |