ROUTINE DNA-SEQUENCING OF 1000 BASES IN LESS-THAN ONE-HOUR BY CAPILLARY-ELECTROPHORESIS WITH REPLACEABLE LINEAR POLYACRYLAMIDE SOLUTIONS

Long, accurate reads are an important factor for high-throughput de no vo DNA sequencing. In previous work from this laboratory, a separation matrix of high-weight-average molecular mass (HMM) linear polyacrylam ide (LPA) at a concentration of 2% (w/w) was used to separate 1000 bas es of DNA sequence in 80 min with an accuracy close to 97% (Carrilho, E.; et al. Anal. Chem. 1996, 68, 3305-3313). In the present work,signi ficantly improved speed and sequencing accuracy have been achieved by further optimization of factors affecting electrophoretic separation a nd data processing. A replaceable matrix containing a mixture of 2.0% (w/w) HMM (9 MDa) and 0.5% (w/w) low-weight-average molecular mass (50 kDa) LPA was employed to enhance the separation of DNA sequencing fra gments in CE, Experimental conditions, such as electric field strength and column temperature, as well as internal diameter of the capillary column, have been optimized for this mixed separation matrix. Under t hese conditions, in combination with energy-transfer (BigDye) dye-labe led primers for high signal-to-noise ratio and a newly developed exper t system for base calling, the electrophoretic separation of 1000 DNA sequencing fragments of both standard (M13mp18) and cloned single-stra nded templates from human chromosome 17 could be routinely achieved in less than 55 min, with a base-calling accuracy between 98 and 99%. Id entical read length, accuracy, and migration time were achieved in mor e than 300 consecutive runs in a single column.