DNA sequencing up to 1300 bases in two hours by capillary electrophoresis with mixed replaceable linear polyacrylamide solutions.

Abstract

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.