A Standardized Protocol for Sample Preparation for Scanning Electron Microscopy (SEM) to Visualize Extrachromosomal DNA (ecDNA)

Extrachromosomal DNA (ecDNA) are large ([~]kilo to megabase) acentric, atelomeric, circular DNAs that are established cytogenetic markers for malignancy, hard-to-treat tumors and drug resistance. Often referred to as double minute chromosomes, ecDNA have been studied since the 1960s, primarily through molecular biology techniques, such as cytogenetics and fluorescence microscopy. More recently, next generation sequencing technologies present novel opportunities for identifying ecDNA. However, none of these approaches adequately address the architecture, size and composition of ecDNA within single cells. Developing an approach to systematically visualize ecDNA, confirm their circular architecture and determine their ultrastructure and composition is an urgent, unmet need. This work presents a protocol for visualizing ecDNA at high resolution using scanning electron microscopy (SEM). To this end, we have optimized an end-to-end procedure that involves preparing, processing and visualizing metaphase spread samples. This protocol was tested on five human cancer cell lines (COLO320DM, NCIH716, NCIH2170, SKGT2, SNU16), four of which express ecDNA in various amounts and one amplifies DNA via a homogeneous staining region (HSR). This work presents a standardized approach to preparing samples and visualizing ecDNA using SEM. SignificanceExtrachromosomal DNA (ecDNA) are proposed to have unique molecular traits, which include acentric, atelomeric and circular DNA. Standardized, high resolution microscopy approaches are in high demand to better understand structure-function relationships of ecDNA.