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During the last decade, capillary electrophoresis (CE) of DNA has undergone rapid development. This improvement was especially important for DNA sequencing, where CE has now become a standard method facilitating to decipher several genomes within a very short time. Here, we give a review of the fundamentals of DNA separation in CE and the major factors influencing the performance.

This work is a continuation of earlier studies on the influence of polymer concentration and polymer composition on the capillary electrophoretic separation of DNA. The focus is on the capillary dimensions and the electric field strength as factors influencing the resolution. The aim was to establish optimum conditions for the separation of single-stranded DNA in capillaries and derive strategies for the construction of micromachined separation devices.

We have studied the separation of single-stranded and double-stranded DNA in a matrix of entangled, linear poly-N,N-dimethylacrylamide. Our results give better insight into the mechanisms involved during separations in polymer solutions. The dependence of different parameters on DNA size, electric field, pore size and the polymer chain length are evaluated and compared to theoretical predictions. Striking differences between experimental data and predicted scaling laws are found. Our data should help to optimize DNA separation in capillary electrophoresis and to improve existing models for DNA separation in porous matrices.

The electrophoretic separation of single-stranded and double-stranded DNA has been examined, using a matrix of linear poly-N,N-dimethylacrylamide (pDMA). The dependence of peak spacing, peak width and resolution on important parameters such as polymer concentration, polymer chain length and electric field strength, has been studied. This work complements our systematic study on electrophoretic mobility under different conditions (C. Heller, Electrophoresis 1999, 20, 1962-1977), and will help to further optimize and improve high performance DNA separation in capillary electrophoresis with entangled polymer solutions.

We describe the construction and performance of a fully automated multicapillary electrophoresis system for the analysis of fluorescently labeled biomolecules. A special detection system allows the simultaneous spectral analysis of all 96 capillaries. The main features are true parallel detection without any moving parts, high robustness, and full compatibility to existing protocols. The device can process up to 40 microtiter plates (96 and 384 well) without human interference, which means up to 15,000 samples before it has to be reloaded.

When investigating the use of different polymers for capillary electrophoresis we found that poly-N,N-dimethylacrylamide (pDMA) has a very low viscosity compared to other polymers of comparable molecular mass and resolving power. This makes it a potentially useful matrix for DNA separation in multi-capillary electrophoresis, where short cycle times or low pressure for matrix replacement are preferred. We have characterized this matrix by systematic studies on concentration, chain length and field strength dependence. It is shown that pDMA performs well for the separation of oligonucleotides and double-stranded DNA fragments. Together with the application of DNA sequencing, pDMA is a universal polymer for the separation of biological macromolecules.

We have investigated the viscosity of different commercially available polymers in solution and found that dextran has a low viscosity compared to other polymers of comparable molecular weight and resolving power. This makes it a potentially useful matrix for DNA separation in capillary electrophoresis, where either short time or low pressure are preferred for matrix replacement. We showed that dextran performs well for the separation of oligonucleotides and double-stranded DNA fragments. Together with the well-known application for protein separation, this makes dextran a universal polymer for the separation of biological macromolecules.

We have developed a new technique for the collection of DNA fragments separated by capillary electrophoresis, by direct transfer from the capillary outlet to a positively charged membrane. Transfer and post-run detection of two different nonradioactivety labeled DNA standards, ranging in size from 150 bp to 2 kbp and 120 bp to 23 kbp are presented, and discussed. Capillary electrophoresis with direct blotting presents several advantages over the blotting from gels: the separation is faster and requires less manual steps, the resolution is higher, and each DNA fragment is collected into a very concentrated spot on the membrane due to the small surface of the capillary outlet and to a design of the collection device inducing a refocusing of field lines across the hybridization membrane. Therefore, very small amounts of DNA (in the pg range) can be detected. This fraction collection makes further analysis of the sample possible, e.g. by hybridization, thus suppressing one of the major present limitations of the capillary electrophoresis technique for DNA analysis 1 .

We have built an automated pulsed field capillary electrophoresis system on the basis of a commercially available device. Using entangled polymer solutions as separating matrix, we demonstrate a considerable improvement of separation of double-stranded DNA in the range of 1-50 kbp under pulsed field conditions. The influence of the main parameters, i.e. pulse frequency and electric field, is studied and the results are compared to existing electrophoresis theories.

Continuous flow electrophoresis is a method to separate ions contained in a sample continuously injected into a laminar flow of electrolyte as a cylindrical stream. Usually, the sample is more conductive than the electrolyte, and the charges created at the sample-electrolyte interface lead to electrohydrodynamic distortions which reduce the separation power of this technique. We demonstrate theoretically that the rate of electrohydrodynamic distortion of a cylindrical sample stream can be reduced to zero, by superimposing to the AC field responsible for the separation of a DC field transverse to it and to the flow direction, with an appropriate frequency, and an effective strength equal to that of the DC field. Using a continuous flow electrophoresis chamber, in which such a field is produced using capacitive electrodes, the major predictions of the theory are confirmed. In particular, it is shown that a sample stream more conductive than the carrying electrolyte, which was seriously deformed in the absence of a transverse AC field, recovers its cylindrical shape in presence of the field. The implications of this discovery for the separating power of continuous-flow electrophoresis are discussed.

We have separated double stranded DNA fragments by electrophoresis using a membrane instead of a gel as separating matrix. Such a membrane, containing separated DNA fragments, is suitable for subsequent hybridization analysis. The method combines electrophoretic separation and transfer onto a solid support and therefore eliminates the need for a transfer or blotting step.