| Popis: |
Epigenetic modifications of cytosines, such as 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in CpG sites of DNA, are both, inheritable and influenced by the environment. These nucleotide modifications have been shown to be important in gene regulation, cell programming, and carcinogenesis. It is therefore imperative that next-generation sequencing techniques are able to detect epigenetic modifications. Conventional methods for detection of 5-mCpG and 5-hmCpG, require chemical conversion of 5-mC to uracil or are limited to bulk analysis of relative ratios of C to 5-mC to 5-hmC. Here we demonstrate a technique for mapping individual 5-mCpG and 5-hmCpG sites within single molecules of ssDNA. We used nanopore sequencing whereby the phi29 DNA polymerase draws ssDNA through the porin MspA. An ion current passing through the pore directly detects and maps the location of such modifications in single molecules. We will present data on specific detection of both 5-mCpG and 5-hmCpG sites based on comparisons with unmodified sequences. |
