Norman J. Dovichi, Jian-Zhong Zhang, JuYing Yan, Jiang Rong, Rong Liu, Sue Bay, Pieter Roos, Karl Voss, Scott Dellinger
Department of Chemistry, University of Alberta, Edmonton, Alberta (CANADA T6G 2G2)
We have developed multiple capillary DNA sequencers. These instruments have several important attributes. First, by operation at electric fields greater than 100 V/cm, we are able to separate DNA sequencing fragments rapidly and efficiently. Second, the separation is performed with 3%T 0%C polyacrylamide. This low viscosity, noncrosslinked matrix can be pumped from the capillary and replaced with fresh material when required. Third, we operate the capillary at elevated temperature. High temperature operation eliminates compressions, speeds the separation, and increases the read length. Fourth, our fluorescence detection cuvette is manufactured locally by means of microlithography technology. These detection cuvettes provide robust and precise alignment of the optical system.
Currently, 5, 16, and 90 capillary instruments are in operation in our lab; 32 and 576 capillary devices are under development. Fourth, we use both avalanche photodiode photodetectors and CCD cameras for high sensitivity detection. We have obtained detection limits of 120 fluorescein molecules injected onto the capillaries. High sensitivity is important in detecting the low concentration fragments generated in long sequencing reads. This combination of low concentration acrylamide, high temperature operation, and high sensitivity detection allows separation of fragments over 800 bases in length in 90 minutes.