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 mat rix operated at elevated column temperatures of 55 degrees and 60 degr ees 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. Us ing dye-labeled primers and Sequenase chemistry on an M13mp18 single-s tranded template, four-color separations for the sequencing products w ere obtained, with read lengths in excess of 800 bases. This paper als o briefly discusses the effects of buffer denaturants and capillary te mperature on separation speed, resolution, and gel compression.