A DNA sieving matrix with thermally tunable mesh size.

Abstract

We present a "proof-of-concept" study showing that a blend of thermo-responsive and nonthermo-responsive polymers can be used to create a DNA sieving matrix with a thermally tunable mesh size, or "dynamic porosity". Various blends of two well-studied sieving polymers for CE, including hydroxypropylcellulose (HPC), a thermo-responsive polymer, and hydroxyethylcellulose (HEC), a nonthermo-responsive polymer, were used to separate a double-stranded DNA restriction digest (Phi X174-HaeIII). HPC exhibits a volume-phase transition in aqueous solution which results in a collapse in polymer coil volume at approximately 39 degrees C. Utilizing a blend of HPC and HEC in a ratio of 1:5 by weight, we investigated the effects of changing mesh size on DNA separation, as controlled by temperature. High-resolution DNA separations were obtained with the blended matrix at temperatures ranging from 25 degrees C to 38 degrees C. We evaluated changes in the selectivity of DNA separation with increasing temperature for certain pairs of small and large fragments. A pure HEC (nonthermo-responsive) matrix was used over the same temperature range as a negative control. In the blended matrix, we observe a maximum in selectivity at approximately 31 degrees C for small DNA, while a significant increase in the selectivity of large-DNA separation occurs at approximately 36 degrees C as the polymer mesh "opens". We also demonstrate, through a temperature ramping experiment, that this matrix can be utilized to obtain high-resolution separation of both small and large DNA fragments simultaneously in a single CE run. Blended polymer matrices with "dynamic porosity" have the potential to provide enhanced genomic analysis by capillary array or microchip electrophoresis in microfluidic devices with advanced temperature control.