The use of elevated column temperature to extend DNA sequencing read lengths in capillary electrophoresis with replaceable polymer matrices.
Capillary electrophoresis with a replaceable linear polyacrylamide matrix operated at elevated column temperatures of 55 degrees and 60 degrees C was used to extend the separation of DNA sequencing fragments to lengths greater than 800 bases. A solid-state heater was employed to provide stable, uniform temperature control over a significant portion of the capillary. The polymer matrix, 3% w/v linear polyacrylamide in a denaturing buffer, was replaced in the capillary after each run. Using dye-labeled primers and Sequenase chemistry on an M13mp18 single-stranded template, four-color separations for the sequencing products were obtained, with read lengths in excess of 800 bases. This paper also briefly discusses the effects of buffer denaturants and capillary temperature on separation speed, resolution, and gel compression.
Klepárnik
K
K
Institute of Analytical Chemistry, Brno, Czech Republic.
Foret
F
F
Berka
J
J
Goetzinger
W
W
Miller
A W
AW
Karger
B L
BL
D000180:Acrylic Resins; D001483:Base Sequence; D004396:Coloring Agents; D004247:DNA / Q000737:chemistry*; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase; D019075:Electrophoresis, Capillary / Q000379:methods*; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA / Q000379:methods*; D013698:Templates, Genetic; D000099166:Polyacrylamides
D000180:Acrylic Resins; D004396:Coloring Agents; D004247:DNA; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase; D000099166:Polyacrylamides; C034970:bacteriophage T7 induced DNA polymerase