ELECTROPHORESIS OF DNA-SEQUENCING FRAGMENTS AT ELEVATED-TEMPERATURE IN CAPILLARIES FILLED WITH POLY(N-ACRYLOYLAMINOPROPANOL) GELS

The performance of poly(N-acryloylaminopropanol) (poly AAP) gel column s, proved to be stable during electrophoresis at elevated temperature, was investigated. The column manufacturing procedure included the pre paration of a coating of the inner wall of the fused silica capillary column with linear poly(AAP). Then, a mixture of the AAP monomer, the cross-linker dihydroxyethylenebisacrylamide (DHEBA) and linear poly(AA P) was introduced into the column and in situ polymerized (for prepara tion of linear gel columns, the addition of DHEBA was omitted). The po ly(AAP) columns were first evaluated by electrophoresis of oligonucleo tides at room temperature and at 50 degrees C, utilizing 260 nm UV-abs orbance detection. In a further evaluation of column performance, samp les of T-terminated DNA Sanger fragments from the bacteria Moraxella w ere separated at 200 V/cm electrical field strength, utilizing a 488 n m argon ion laser and a confocal optical setup for laser-induced fluor escence (LIF) detection. A temperature increase from 25 degrees C to 5 0 degrees C effectively released a compression of DNA bands. However, for cross-linked poly(AAP) gel columns, the elevated temperature resul ted in a considerable reduction of the DNA sequence reading length. Wh en a linear poly(AAP) column was utilized, no detrimental effect of el evated temperature on the separation could be observed.