Field-flow fractionation of chromosomes

The first topic of this project involved the preparation, fractionation by sedimentation/steric Field Flow Fractionation (FFF), and modeling of metaphase chromosomes. After numerous unsuccessful attempts to prepare chromosomes, we have implemented a procedure (in collaboration with Los Alamos National Laboratory) to prepare metaphase chromosomes from Chinese hamster cells. Extensive experimentation was necessary to identify a suitable FFF channel surface to minimize chromosome adsorption and a carrier liquid to stabilize and disperse the chromosomes. Under suitable operating conditions, the Chinese hamster chromosomes were purified from cell debris and partially fractionated. The purified, preenriched chromosomes that can be prepared by sedimentation/steric FFF or produced continuously by continuous SPLITT fractionation provide an enriched feed material for subsequent flow cytometry. In the second project component, flow FFF permitted successful separations of single- from double-stranded circular DNA, double-stranded circular DNAs of various sizes, and linear double-stranded DNA fragments of various lengths. Diffusion coefficients extracted from retention data agreed well with literature data as well as predictions of major polymer theories. The capacity of FFF separations was evaluated to examine potential applications to long DNA chains.