Electrophoresis of DNA in novel thermoreversible matrices.

Abstract

We demonstrate the feasibility of temperature-sensitive polymers as novel matrices for capillary and slab electrophoresis of DNA. These matrices combine the case-of-use of agarose with resolution properties of polyacrylamide. Two classes of matrices are used: (i) aqueous suspensions of gel microspheres and (ii) solutions containing uncross-linked temperature-sensitive polymers. When heated, the viscosity of these solutions drops dramatically because of the phase transition behavior of these polymers, and therefore these formulations are easy to pour or load. Results are presented for separation of double-stranded DNA fragments (< 2000 bp) in capillary, tube, and slab electrophoresis. Preliminary results are also presented for separation of single-stranded sequencing fragments by capillary electrophoresis. In the tube format, good resolution was obtained for phi X174/HaeIII fragments. In the slab format, excellent resolution of double-stranded DNA was obtained, including simultaneous separation of a 10 bp ladder up to 150 base pairs. In the capillary format, pBR322/MspI fragments were completely resolved, and single-base resolution of sequencing fragments was obtained up to 150 bases. We believe that temperature-sensitive polymers represent a new and promising class of electrophoretic media.