Nano-dissection and sequencing of DNA at single sub-nuclear structures.

Abstract

The relative positioning of gene loci within a mammalian nucleus is non-random and plays a role in gene regulation. Some sub-nuclear structures may represent "hubs" that bring specific genetic loci into close proximity where co-regulatory mechanisms can operate. The identification of loci in proximity to a shared sub-nuclear structure can provide insights into the function of the associated structure, and reveal relationships between the loci sharing a common association. A technique is introduced based on the nano-dissection of DNA from thin sections of cells by high-precision nano-tools operated inside a scanning electron microscope. The ability to dissect and identify gene loci occupying a shared site at a single sub-nuclear structure is demonstrated here for the first time. The technique is applied to the nano-dissection of DNA in vicinity of a single promyelocytic leukemia nuclear body (PML NB), and reveals novel loci from several chromosomes that are confirmed to associate at PML NBs with statistical significance in a cell population. Furthermore, it is demonstrated that pairs of loci from different chromosomes congregate at the same nuclear body. It is proposed that this technique is the first that allows the de novo determination of gene loci associations with single nuclear sub-structures.