HIGH-SPEED DNA SEQUENCING BY USING MIXED POLY(ETHYLENE OXIDE) SOLUTIONS IN UNCOATED CAPILLARY COLUMNS

摘要

We describe a new non-cross-linked polymer matrix and a new column treatment procedure for high-speed DNA sequencing by using capillary electrophoresis. A solution of commercially available poly(ethylene oxide) (PEO) in two size ranges is used as the sieving matrix under denaturing conditions. When compared to a commercial 6% T polyacrylamide solution, the mixed PEO matrix offers identical separation performance for the small DNA fragments, better performance for the large DNA fragments, increased separation speed, and a lower viscosity. To avoid degradation of the standard bonded coating on the capillary column over many runs, we evaluated the resolution of DNA fragments in the mixed PEO matrix in bare fused-silica capillaries, in a dry, fresh column, the performance is identical to that observed in coated (bonded) capillaries. To regenerate the favorable surface characteristics after each run, 0.1 N HCl was used to retitrate the surface silanol groups back to the fully protonated form. The sequencing runs in fact proceeded much faster, with bases 28-420 from the Sanger ladder eluting in a span of only 16 min, We observed little degradation of the performance over tens of sequencing runs.