Christoph Heller

Principles of DNA separation with capillary electrophoresis.

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.

SEPARATION OF DOUBLE-STRANDED AND SINGLE-STRANDED DNA IN POLYMER SOLUTIONS: I. MOBILITY AND SEPARATION MECHANISM

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.

Free-solution electrophoresis of DNA

We report the first experimental evidence that double-stranded DNA fragments can be electrophoretically separated in free-solution (i.e., in the absence of a sieving matrix) when either a single or two streptavidin molecules are attached to the end(s) using biotinylated nucleotides. As previously predicted, higher resolution is obtained at higher electric fields or when two streptavidin molecules are attached to each DNA fragment. The resolution is also affected by the diameter and coating of the capillaries.

A FULLY AUTOMATED MULTICAPILLARY ELECTROPHORESIS DEVICE FOR DNA ANALYSIS

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.

FINDING A UNIVERSAL LOW VISCOSITY POLYMER FOR DNA SEPARATION (II)

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.

Separation of double-stranded and single-stranded DNA in polymer solutions: II. Separation, peak width and resolution.

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.

Influence of electric field strength and capillary dimensions on the separation of DNA

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.

Electrohydrodynamically induced aggregation during constant and pulsed field capillary electrophoresis of DNA

We present a study aimed at understanding the factors affecting the separation of large DNA molecules by capillary electrophoresis in polymer solutions. In a first series of experiments, a systematic study of the effect of operational parameters on the development of an electrohydrodynamic instability resulting in DNA aggregation and spurious peaks in the electropherograms is presented. The results are discussed in regard to a recent theory of electrohydrodynamic instabilities in macroion suspensions, recently proposed by Isambert et al. Overall, the results provide strong support to the theory. Some situations of interest for applications, and not explicitly considered in the theory, such as asymmetric field pulsing and the use of polymer additives in the buffer, were also considered. Furthermore, robust optimized protocols for high resolution separation of DNA in the range of 100 base pairs to 160 kilobase pairs, are proposed. As predicted by the model, it is shown that using a concentrated isoelectric buffer (histidine) strongly reduces aggregation as compared to the use of a conventional buffer at the same concentration, and allows separation of DNA from 100 bp to 160 kbp in less than 10 min. We also present a systematic study of the dependence of the mobility vs DNA size, pulse frequency, and field strength. The results are discussed with respect to the Biased Reptation with Fluctuations model and a good agreement is obtained. Copyright 1999 John Wiley & Sons, Inc.

A FULLY AUTOMATED MULTI-CAPILLARY ELECTROPHORESIS DEVICE FOR DNA ANALYSIS

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.