Abstract
Abstract The dextran-polyethylene glycol, aqueous two-phase system enables large scale fractionation of homologous nucleic acids on the basis of their degree of double-helical structure, as well as rapid separation of DNA from RNA, and gentle removal of proteins from either nucleic acid. In agreement with theoretical predictions, substances of low molecular weight are generally confined to partition coefficients near 1.0 in this system, and their separation consequently requires multiple reextractions. On the other hand, two macromolecular species of high molecular weight will often partition strongly into opposite phases if the proper conditions are found. Due to variability in the behavior of different batches of the phase system polymers, it is not possible to forecast precisely the polymer concentrations and ionic environment that will optimize any given separation. Instead, routine calibrations will have to be performed for each application. The investigator might best begin by estimating a suitable ionic environment by reference to earlier literature. With the sample dialyzed into this solution, several extractions are made in which the phase system stock is added in increasing proportions to a fixed volume of sample. If the partitioned species all favor the same phase regardless of the concentration of phase system stock, then the ionic environment will have to be changed to move these substances more toward the least favored phase. Once the optimal proportion of phase system stock and the ionic environment have been determined for a given fractionation, they should be carefully controlled. Relatively small changes in these two parameters can drastically effect the partition coefficients observed for nucleic acids.
Published Version
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