title: Detection and mapping of 5-methylcytosine and 5-hydroxymethylcytosine with nanopore MspA.
abstract: Precise and efficient mapping of epigenetic markers on DNA may become an important clinical tool for prediction and identification of ailments. Methylated CpG sites are involved in gene expression and are biomarkers for diseases such as cancer. Here, we use the engineered biological protein pore Mycobacterium smegmatis porin A (MspA) to detect and map 5-methylcytosine and 5-hydroxymethylcytosine within single strands of DNA. In this unique single-molecule tool, a phi29 DNA polymerase draws ssDNA through the pore in single-nucleotide steps, and the ion current through the pore is recorded. Comparing current levels generated with DNA containing methylated CpG sites to current levels obtained with unmethylated copies of the DNA reveals the precise location of methylated CpG sites. Hydroxymethylation is distinct from methylation and can also be mapped. With a single read, the detection efficiency in a quasirandom DNA strand is 97.5 ± 0.7% for methylation and 97 ± 0.9% for hydroxymethylation.
authors.affiliation: Department of Physics, University of Washington, Seattle, WA 98195-1560.
authors.forename: Andrew H
authors.initials: AH
authors.lastname: Laszlo
authors.forename: Ian M
authors.initials: IM
authors.lastname: Derrington
authors.forename: Henry
authors.initials: H
authors.lastname: Brinkerhoff
authors.forename: Kyle W
authors.initials: KW
authors.lastname: Langford
authors.forename: Ian C
authors.initials: IC
authors.lastname: Nova
authors.forename: Jenny Mae
authors.initials: JM
authors.lastname: Samson
authors.forename: Joshua J
authors.initials: JJ
authors.lastname: Bartlett
authors.forename: Mikhail
authors.initials: M
authors.lastname: Pavlenok
authors.forename: Jens H
authors.initials: JH
authors.lastname: Gundlach
mesh_terms: D044503:5-Methylcytosine / Q000302:isolation & purification / Q000378:metabolism* / Q000031:analogs & derivatives; D001499:Bayes Theorem; D003596:Cytosine / Q000031:analogs & derivatives* / Q000302:isolation & purification / Q000378:metabolism; D019175:DNA Methylation; D057890:Epigenomics / Q000379:methods; D008958:Models, Molecular; D015394:Molecular Structure; D058608:Nanopores; D018272:Porins / Q000378:metabolism*
chemical_list: D018272:Porins; C121838:mspA protein, Mycobacterium smegmatis; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine
keywords: DNA hydroxymethylation; DNA methylation; nanopore DNA sequencing; nanotechnology; next generation sequencing