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.