The read length for DNA sequencing using capillary electrophoresis and replaceable linear polyacrylamide (LPA) solutions has been extended to more than 1000 bases with a run time of 80 min. This result was successfully achieved through the combined use of cycle sequencing with dye-labeled primers, improved matrix and separation conditions, and enhanced base-calling software. The influences of LPA molecular weight and concentration on separation were investigated. Additionally, the separation buffer, column temperature, and electric field were adjusted to increase the number of resolvable DNA fragments per run while maintaining an enhanced separation speed. Using low concentrations [2% (w/v)] of high molecular weight LPA polymers (> 5.5 x 10(6) Da), elevated column temperature (50 degrees C) and moderately high field (150 V/cm), rapid sequencing analysis for more than 1000 bases on a model ssM13mp18 template was obtained with 96.8% accuracy.

This paper presents results on ultralong read DNA sequencing with relatively short separation times using capillary electrophoresis with replaceable polymer matrixes. In previous work, the effectiveness of mixed replaceable solutions of linear polyacrylamide (LPA) was demonstrated, and 1000 bases were routinely obtained in less than 1 h. Substantially longer read lengths have now been achieved by a combination of improved formulation of LPA mixtures, optimization of temperature and electric field, adjustment of the sequencing reaction, and refinement of the base-caller. The average molar masses of LPA used as DNA separation matrixes were measured by gel permeation chromatography and multiangle laser light scattering. Newly formulated matrixes comprising 0.5% (w/w) 270 kDa and 2% (w/w) 10 or 17 MDa LPA raised the optimum column temperature from 60 to 70 degrees C, increasing the selectivity for large DNA fragments, while maintaining high selectivity for small fragments as well. This improved resolution was further enhanced by reducing the electric field strength from 200 to 125 V/cm. In addition, because sequencing accuracy beyond 1000 bases was diminished by the low signal from G-terminated fragments when the standard reaction protocol for a commercial dye primer kit was used, the amount of these fragments was doubled. Augmenting the base-calling expert system with rules specific for low peak resolution also had a significant effect, contributing slightly less than half of the total increase in read length. With full optimization, this read length reached up to 1300 bases (average 1250) with 98.5% accuracy in 2 h for a single-stranded M13 template.

Long, accurate reads are an important factor for high-throughput de novo DNA sequencing. In previous work from this laboratory, a separation matrix of high-weight-average molecular mass (HMM) linear polyacrylamide (LPA) at a concentration of 2% (w/w) was used to separate 1000 bases 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, significantly improved speed and sequencing accuracy have been achieved by further optimization of factors affecting electrophoretic separation and 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 fragments 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 these conditions, in combination with energy-transfer (BigDye) dye-labeled primers for high signal-to-noise ratio and a newly developed expert system for base calling, the electrophoretic separation of 1000 DNA sequencing fragments of both standard (M13mp18) and cloned single-stranded templates from human chromosome 17 could be routinely achieved in less than 55 min, with a base-calling accuracy between 98 and 99%. Identical read length, accuracy, and migration time were achieved in more than 300 consecutive runs in a single column.

Replaceable linear polyacrylamide (LPA) has been utilized as a sieving matrix for DNA sequencing by capillary electrophoresis (CE). Difficulties associated with cross-linked polyacrylamide gel stability have been overcome for the routine application of CE to DNA sequencing. A simple laser-induced fluorescence (LIF) detection system based on a single laser and two photomultipliers (PMT) has been adopted for this work. Sequencing information for four bases has been obtained from two fluorescent dyes and two peak height ratios, detected in two optical channels. FAM- and JOE-labeled M13 (-21) primers have been chosen because both dyes are efficiently excited with a low-power argon ion laser, can be optically separated, and exhibit minimal dye-based shifts in DNA fragment mobilities. Addition of denaturants to the electrophoresis running buffer (1 x TBE, 3.5 M urea, 30% formamide) and column operation at 32 degrees C permitted the resolution of difficult compressed sites in the sequence of phage M13mp18. Careful examination of the polymerization reaction of LPA has led to methodology that has proven to be reproducible for obtaining DNA sequencing information of M13mp18 phage for 350 nucleotides in close to 30 min.