SEPARATION OF DNA RESTRICTION FRAGMENTS BY POLYMER-SOLUTION CAPILLARY ZONE ELECTROPHORESIS - INFLUENCE OF POLYMER CONCENTRATION AND ION-PAIRING REAGENTS

To achieve sensitive and fast separations of the DNA fragments, polymer-solution capillary zone electrophoresis (PS-CZE) is examined. A systematic study of the influence of the linear polymer concentration on the separation of restriction fragments indicates that the larger DNA fragments are resolved with greater efficiency by using a lower concentration of the polymer, while the smaller ones are better resolved with a higher polymer concentration, when added to the buffer. A specific concentration of the polymer provides a certain limit of dynamic ''porosity'' (sieving), which is appropriate for a given range of the DNA fragments. Several ion-pairing reagents are examined here to improve resolutions of both small and large DNA fragments in PS-CZE. These reagents cause no interactions between the ammonium cations (reagent) and the silanol groups (capillary surface). However, they interact with the linear polymer, perhaps by hydrophobic functions, and change its physical properties, such as dynamic viscosity and sieving ability. Enhanced retention (interactions) of the DNA fragments is observed by increasing the ion-pairing contents in the polymer solution. The increased peak retention strongly suggests presence of interactions between the reagent and the DNA fragments. Different ion-pairing reagents produce different degrees of peak retentions. The interactions results from relatively greater hydrophobic interactions and smaller ionic interactions between the reagent and the polynucleotides. The study of ethidium bromide (EdBr) additon in PS-CZE indicates that a small concentration of EdBr reduces peak widths and decreases retention of all the fragments, but has little influence on the electroosmotic flow. However, a larger EdBr concentration results in broader peak widths and causes little change in their retention. Ion-pairing and intercalating reagents interact with the DNA fragments by different mechanisms. An anion-pairing agent appears to interact predominantly by hydrophobic interactions with the purine and pyrimidine bases, while EdBr intercalates with G:C base pairs and alter the chain length. Satisfactory separations of both larger and smaller fragments can be achieved by adding an appropriate ion-pairing reagent along with the linear polymer to the buffer. Critical concentrations of the linear polymer and the ion-pairing reagent must be determined in order to achieve satisfactory separations for a given range (size) of the DNA fragments.