Single Molecule Analysis of Chromatin Structure

In the last years a new approach emerged for genome mapping which does not require sequencing. The genome mapping approach uses nicking enzymes to form patterns along the DNA which can be imaged using fluorescence microscopy after the incorporation of fluorescent nucleotides into the nicked sites. The extraction of long DNA molecules (> 150 kb) and their labeling allow rapid de-novo assembly of large genomes with a high capacity for the detection of structural variations. The long DNA molecules are electrophoreted through an ordered array of nanochannels, yielding uniformly stretched molecules ready for imaging using fluorescent microscope. The images are processed automatically, revealing a unique barcode pattern formed by the nicking enzyme. The barcode patterns from multiple single molecules are then assembled to build de-novo genomic maps.Based on the platform for fluorescent mapping of genomic content we have recently developed a method for analyzing chromatin structure. We treated DNA of isolated nuclei from GM12878 cells under mild conditions of DNase I, yielding nicks in exposed areas of the chromatin. The nicks were subsequently labeled using red fluorescent-modified nucleotides. Next, long DNA molecules were extracted and a unique DNA barcode was formed using the BspQI nicking enzyme and green fluorescent-labeled nucleotides. The green-labeled barcode was used to align single long DNA molecules to a reference map and the red-labeled sites indicated the presence of DHS at specific positions. Comparison of our data with DNase I sequencing data from the ENCODE project showed a great agreement between the two methods. This method is easier to perform and can be done on new genomes with unknown sequences.