Gel electrophoresis of DNA fragments in narrow-bore capillaries

被引:16
作者
Lindberg, P [1 ]
Stjernstrom, M [1 ]
Roeraade, J [1 ]
机构
[1] ROYAL INST TECHNOL, DEPT ANALYT CHEM, S-10044 STOCKHOLM, SWEDEN
关键词
narrow-bore capillaries; capillary electrophoresis; gel electrophoresis; DNA separations; cross-linked polyacrylamide;
D O I
10.1002/elps.1150181116
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
In this work, we studied the behavior of electrophoretic columns, having an inner diameter (ID) of 2-10 mu m, filled with a cross-linked polyacrylamide gel matrix. The usefulness of these columns for DNA sequencing is discussed. Evaluation of column performance included tests of gel stability and migration time reproducibility. Confocal laser-induced fluorescence (LIF)-detection was employed utilizing a 488 nm argon ion laser for separations of C- and T-terminated DNA Sanger fragments. Reducing the inner diameter of the column from 50 mu m to 10 mu m resulted in an approximately eightfold increase in lifetime, under conditions in which the columns were subjected to a field strength of 1000 V/cm. The 10 mu m ID columns were utilized for separation of Sanger fragments, and adequate detection sensitivity was obtained by stacking of the fragments from a deionized sample solution. A linear algorithm for retention data synchronization between individual electropherograms was employed to provide a route towards a reliable automated base calling protocol.
引用
收藏
页码:1973 / 1979
页数:7
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