Lev Kotler

DNA sequencing of close to 1000 bases in 40 minutes by capillary electrophoresis using dimethyl sulfoxide and urea as denaturants in replaceable linear polyacrylamide solutions

The goal of this work was to reduce the capillary electrophoresis (CE) separation time of DNA sequencing fragments with linear polyacrylamide solutions while maintaining the previously achieved long read lengths of 1000 bases. Separation speed can be increased while maintaining long read lengths by reducing the separation matrix viscosity and/or raising the column temperature. As urea is a major contributor to the separation buffer viscosity, reducing its concentration is desirable both for increase in the separation speed and easier solution replacement from the capillary. However, at urea concentrations below 6 M, the denaturing capacity of the separation buffer is not sufficient for accurate base‐calling. To restore the denaturing properties of the buffer, a small amount of an organic solvent was added to the formulation. We found that a mixture of 2 M urea with 5% v/w of dimethyl sulfoxide (DMSO) resulted in 975 bases being sequenced at 70°C in 40 min with 98.5% accuracy. To achieve this result, the software was modified to perform base‐calling at a peak resolution as low as 0.24. It is also demonstrated that the products of thermal decomposition of urea had a deleterious effect on the separation performance at temperatures above 70°C. With total replacement of urea with DMSO, at a concentration of 5% v/w in the same linear polyacrylamide (LPA)‐containing buffer, it was possible to increase the column temperature up to 90°C. At this temperature, up to 951 bases with 98.5% accuracy could be read in only 32 min of separation. However, with DMSO alone, some groups of C‐terminated peaks remained compressed, and column temperature at this level cannot at present be utilized with existing commercial instrumentation.

DNA sequencing with hydrophilic and hydrophobic polymers at elevated column temperatures

Read length in DNA sequencing by capillary electrophoresis at elevated temperatures is shown to be greatly affected by the extent of hydrophobicity of the polymer separation matrix. At column temperatures of up to 80°C, hydrophilic linear polyacrylamide (LPA) provides superior read length and separation speed compared to poly(N,N‐dimethylacrylamide) (PDMA) and a 70:30 copolymer of N,N‐dimethylacrylamide and N,N‐diethylacrylamide (PDEA30). DNA‐polymer and polymer intramolecular interactions are presumed to be a major cause of band broadening and the subsequent loss of separation efficiency with the more hydrophobic polymers at higher column temperatures. With LPA, these interactions were reduced, and a read length of 1000 bases at an optimum temperature of 70°–75°C was achieved in less than 59 min. By comparison, PDMA produced a read length of roughly 800 bases at 50°C, which was close to the read length attained in LPA at the same temperature; however, the migration time was approximately 20% longer, mainly because of the higher polymer concentration required. At 60°C, the maximum read length was 850 bases for PDMA, while at higher temperatures, read lengths for this polymer were substantially lower. With the copolymer DEA30, read length was 650 bases at the optimum temperature of 50°C. Molecular masses of these polymers were determined by tandem gel permeation chromatography‐multiangle laser light scattering method (GPC‐MALLS). The results indicate that for long read, rapid DNA sequencing and analysis, hydrophilic polymers such as LPA provide the best overall performance.

Factors to be considered for robust high-throughput automated DNA sequencing using a multiple-capillary array instrument

The overall goal of our program is to develop a robust, high throughput, fully automated DNA sequencing instrument based on replaceable polymer solutions using a multicapillary array. Significant effort has already been devoted to column and polymer chemistry in order to obtain long read lengths per run in fast analysis time. In this paper we report on progress in instrument considerations and data processing software. A simple instrument design, based on no moving parts for continuous illumination of the capillaries and detection of the fluorescent light was used for this study. Our polymer solution replacement system with the permanent connection between the buffer/chamber manifold and capillary columns on the detector side is designed to prevent the trapping of air bubbles during matrix solution replacement. A special construction of a column-electrode couple on the injection side precludes air trapping during sample injection from small sample volumes. Our in-house software now features the significant reduction of the crosstalk signal from neighbor columns, which may be a potential problem in densely packed large capillary array sequencers.