Novel Single Cell Methods to Identify the Genetic Composition at a Single Nuclear Structure.

摘要

Reported here are two novel techniques that allow for the identification of DNA obtained from a nanoscale region of a single cell nucleus based on either direct DNA dissection with high-precision nano-tools operated inside a scanning electron microscope, or the ligation of oligo probes to subnuclear sites of double stranded breaks induced by 2-photon irradiation. It is demonstrated that these techniques afford the ability to identify loci occupying a shared site at a single nuclear substructure, and are thus uniquely suited for identifying specific sets of loci that converge non-randomly at a shared nuclear body (NB; the "gene hub" model of nuclear organization). The techniques are applied towards determining DNA in the vicinity of single PML nuclear bodies and yield novel loci that show significant frequency of association with PML bodies in a population of cells. As the "gene hub" model may be a general feature of nuclear organization, these techniques, applied to other NBs, may reveal specific sets of convergent loci that reflect novel gene co-regulatory relationships.