POLYACRYLAMIDE SOLUTIONS FOR DNA-SEQUENCING BY CAPILLARY ELECTROPHORESIS - MESH SIZES, SEPARATION AND DISPERSION

Two preparations of linear polyacrylamide with average molecular weigh ts of 0.37 million and 1.14 million Da, and a deuterated preparation w ith an average molecular weight of 1.71 million Da, were used to study the effects of molecular weight, polydispersity, and concentration on the mesh size of entangled polymers in a DNA sequencing buffer soluti on and their ability to resolve DNA sequencing reactions by capillary electrophoresis. The polyacrylamide concentrations were above the over lap threshold C, the concentration above which an entangled polymer n etwork is expected to form. Small angle neutron scattering experiments showed that between 1% and 8% polyacrylamide, the mesh size (xi) can be expressed by the relation xi = 2.09C(-0.76), where xi is in Angstro m and C is the polymer concentration in g/mL. The mesh size depended o nly on the concentration and was independent of the average molecular weight of the polyacrylamide. Consistent with this result, electrophor etic mobilities of DNA moving through the polymer network depended alm ost entirely on the polyacrylamide concentration and not on its molecu lar weight or polydispersity. Although separation was little affected, band sharpness persisted to longer DNAs when the polymer network cont ained a higher fraction of larger polyacrylamide molecules. We postula te a dispersive effect that depends on the size of the DNA and the res iliency of the polymer network. This interpretation provides a rationa le for optimizing the design of polymer solutions to sieve DNA for seq uencing by capillary electrophoresis.