Capillary electrophoresis of DNA in uncross-linked polymer solutions

Abstract

We have used dilute and semi-dilute uncross-linked hydroxyethyl cellulose (HEC) solutions as separation matrices for capillary electrophoresis of DNA restriction fragments. In these experiments, we investigated the effects of HEC molecular weight and concentration on resolution, attempting to relate these parameters to the polymer entanglement threshold concentration. The entanglement thresholds of seven molecular weight fractions of hxdroxyethyl cellulose were determined from viscosity-concentration data; the entanglement threshold was found to scale as N −1.2, where N = number of HEC monomers. This finding is not in agreement with classical scaling arguments. We present a relationship to predict the observed entanglement threshold of HEC in solution as a function of number average molecular weight. It was found that excellent separation of ΦX174/ HaeIII DNA restriction fragments (72–1353 base pairs) by capillary electrophoresis in HEC solutions can be achieved significantly below the entanglement threshold, depending on DNA size and HEC molecular weight. The mechanism of separation in these uncross-linked polymer solutions must therefore be reexamined. Our experiments show that the entanglement threshold is a useful parameter in predicting a range of HEC concentrations which will separate certain DNA fragments for a given HEC molecular weight. However, the presence of a fully entangled network is not a prerequisite for separation.