P. Lindberg et al., ELECTROPHORESIS OF DNA-SEQUENCING FRAGMENTS AT ELEVATED-TEMPERATURE IN CAPILLARIES FILLED WITH POLY(N-ACRYLOYLAMINOPROPANOL) GELS, Electrophoresis, 18(15), 1997, pp. 2909-2914
Citations number
20
Categorie Soggetti
Biochemical Research Methods","Chemistry Analytical
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.