The use of elevated column temperature to extend DNA sequencing read lengths in capillary electrophoresis with replaceable polymer matrices

Abstract

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.

Novel quasi‐interpenetrating network/gold nanoparticles composite matrices for DNA sequencing by CE

“…It has been shown by Dovichi and co-workers [52] and Karger and co-workers [53] that elevated temperature increased the read length in DNA sequencing by CE using LPA as a separation medium [51]. In the same way, it is observed that the sequencing size limit increased with higher temperatures using quasi-IPN/GNPs-2 as matrices (Figs.…”

Ultra-high throughput rotary capillary array electrophoresis scanner for fluorescent DNA sequencing and analysis

“…The remaining substitution, insertion, and deletion errors were mostly made in the low resolution regions past 600 bases. The compressions can be eliminated by running the sequencing separations at higher temperatures [12,13,33] or by using alternate chemistries such as replacing guanine with inosine in the sequencing reactions. The use of elevated temperatures for DNA sequencing also results in improved resolution for longer fragments [33].…”

Ultrafast detection of microsatellite repeat polymorphism in endothelin 1 gene by electrophoresis in short capillaries

“…The elevated temperature has two other positive effects on the analysis time. The electrophoretic mobility increases due to lower viscosity and a higher electric field strength can be applied without risk of the separation selectivity loss, since thermal energy protects molecular stretching of DNA fragments [45,46]. Moreover, capillaries can easily be flushed with higher concentration separation media at elevated temperatures.…”