Electrophoresis of DNA sequencing fragments at elevated temperature in capillaries filled with poly(N‐acryloylaminopropanol) gels

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Abstract
The performance of poly(N‐acryloylaminopropanol) (poly AAP) gel columns, proved to be stable during electrophoresis at elevated temperature, was investigated. The column manufacturing procedure included the preparation 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(AAP) was introduced into the column and in situ polymerized (for preparation of linear gel columns, the addition of DHEBA was omitted). The poly(AAP) columns were first evaluated by electrophoresis of oligonucleotides at room temperature and at 50°C, utilizing 260 nm UV‐absorbance detection. In a further evaluation of column performance, samples of T‐terminated DNA Sanger fragments from the bacteria Moraxella were separated at 200 V/cm electrical field strength, utilizing a 488 nm argon ion laser and a confocal optical setup for laser‐induced fluorescence (LIF) detection. A temperature increase from 25°C to 50°C effectively released a compression of DNA bands. However, for cross‐linked poly(AAP) gel columns, the elevated temperature resulted in a considerable reduction of the DNA sequence reading length. When a linear poly(AAP) column was utilized, no detrimental effect of elevated temperature on the separation could be observed.