Formation of concentration gradient and its application to DNA capillary electrophoresis

A new method to introduce the concentration gradient into the capillary has been developed and its application to DNA capillary electrophoresis is pre sented. The concentration gradient produced by mixing 5% w/v polyacrylamide -co-poly(N-dimethylacrylamide) (PAM-co-PDMA) solution and 1 x Tris/N-tris(h ydroxymethyl)methyl-3-amino-propanesulfonic acid/EDTA (TT) + 5 M urea buffe r was successfully achieved by using two programmable syringe pumps with st rict control of dead volume, flow rate, and pressure balance. This method h as the advantages of high stability, reproducibility, and versatility. The column with concentration gradient greatly improved the resolution, especia lly for the large DNA fragments, due to a decrease in band width broadening with time. A column containing 2-4% w/v gradient in four steps had a longe r read length, shorter separation time and better resolution (after 380 bas e) than that of 4% w/v single concentration polymer solution. The number of steps in the gradient had almost no effect on the performance. The change in the average concentration by relocating the position of the same step gr adient, i.e., a combination of different low concentration to high concentr ation polymer solution ratios, resulted in a different migration time, read length and resolution.