Investigation of the neutral filter elution technique: I. Effects of pore density and pore diameter on elution rate at pH 9.6

To understand better the biophysical mechanism of neutral filter elution (pH 9.6), we eluted genomes of known size and shape: coliphage T4c (M(r) 1.15 X 10⁸), E. coli (M(r) 2.7 X 10⁹), and Chinese hamster lung fibroblasts (V79, M(r) 2-4 X 10¹⁰). DNA eluted through 15% sucrose atop the filter in a biphasic pattern. The elution rate of the initial component correlated (r > 0.97) exponentially with 1/M(r) for monodisperse samples of DNA eluted through pore sizes 0.1-3.0 μm. Using this relationship between elution rate and M(r), we estimated M(n) of polydisperse, X-irradiated (253 Gy) samples of DNA from E. coli or V79 cells to be 3.15 ± 1.46 and 1.42 ± 0.33, respectively, compared to expected values of 2.93 and 3.52 (10⁸ Da). The best predictor of elution rate for DNA from T4c and intact and X-irradiated V79 cells was pore density, and pore diameter for DNA from X-irradiated E. coli. The rate of elution of DNA from unirradiated E. coli was unrelated to pore density or diameter. While the mechanism of neutral filter elution remains unknown, its use for linear DNAs with M(n) ca. 10⁸ Da appears to be valid quantitatively.