Toward Epigenetic Mapping of Human Chromosomes in Nanochannel Arrays

Tslil Gabrieli, Chemistry, Tel Aviv University, Tel Aviv, Israel
Yael Michaeli, Chemistry, Tel Aviv University, Tel Aviv, Israel
Yuval Ebenstein, Chemistry, Tel Aviv University, Tel Aviv, Israel

5-hydroxymethyl-cytosine (5hmC) is a recently rediscovered epigenetic modification of DNA with tissue and cell type specific distribution in mammalian genomes. Recent studies of genomic DNA, found that a substantial fraction of 5-methyl-cytosine (5mC) in CpG dinucleotides is converted to 5hmC and is mainly abundant in the central nerves system. The dynamic nature of this modification implies that 5hmC patterns may exhibit a high degree of cell to cell variation and should be studied by single-cell or single-molecule analysis. We have recently developed a method for optical detection of the epigenetic modification 5hmC at the single-molecule level. We use a chemo-enzymatic reaction to covalently attach a fluorescent reporter molecule to 5hmC nucleotides. Labeled DNA is electrokinetically squeezed into an array of silicon nanochannels and imaged on a fluorescence microscope. 5hmC residues are visible as fluorescent spots along the DNA contour.  The human genomic DNA was labeled with an additional color at specific sequence motifs (GCTCTTC) to generate a locus specific pattern of dots that can be used in order to map the molecule onto the reference genome. We show first results of single-molecule epigenetic mapping of chromosomal DNA extracted from human peripheral blood cells. DNA molecules typically spanning several hundred Kbp (and up to 2Mbp) were visualized in the channels.