Jan Sudor

High-resolution studies of hyaluronic acid mixtures through capillary gel electrophoresis

Hyaluronic acid is a negatively charged polysaccharide with a high degree of polydispersity that makes the separation of its oligomers extremely difficult. Through the use of columns filled with a highly viscous polyacrylamide matrix, the unit resolution of hyaluronate oligomers could be achieved, up to at least 80 kDa of mass, through capillary electrophoresis. As analytical application examples, the fractions of enzymatically or ultrasonically degraded hyaluronates were monitored through this method. Because of the very high resolving power, peaks additional to the regular oligomers can be observed that are assumed to be conformers of this regular, unbranched biopolymer.

The mobility minima in pulsed-field capillary electrophoresis of large DNA.

Pulsed-field capillary electrophoresis represents a new tool for rapid and highly efficient separations of large biopolymers. The method has been utilized here to study dependencies of the electrophoretic mobility upon the frequency and pulse shape of applied voltage for large, double-stranded DNA molecules (5-100 kb) migrating in neutral polymer solutions. Two different shapes of alternating electric field (sine- and square-wave impulses) were examined with the frequency values ranging from 1 to 30 Hz. The linear dependence between duration of the forward pulse (at which the DNA molecule experiences a minimum mobility) and the product N.In(N) (where N is the number of base pairs) was experienced in field-inversion gel electrophoresis, while exponential dependence was found with the sinusoidal electric field. The mobility minima were lower in field-inversion electrophoresis than with the biased sinusoidal-field technique. The DNA (5 kb concatamers) was adequately separated using a ramp of frequency in the square-wave electric field, in approximately 1 h. The migration order of DNA fragments was referenced through adding a monodisperse DNA (48.5 kb) into the sample. The band inversion phenomena were not observed under any experimental conditions used in this work.

Pulsed-Field Capillary Electrophoresis of Large DNA

Over its many years of existence, electrophoresis in slab gels has become a very useful technique in biochemistry and biology. It has been commonly applied to a wide range of problems [1]: mostly to the separation of proteins and nucleic acids and, to a lesser degree, of sugars and other poly electrolytes. Research directed toward the separation of nucleic acids has been driven by the explosion of molecular biology and genomic projects; these activities have been particularly visible during the last several years. Conventional DNA separations were initially restricted to the sizes of up to approximately 20–30 kbp (kilo-base pairs) [2]. The reason for this restriction has been the reptative motion of large DNAs in the direction of an applied electric field, and a subsequent molecular orientation [3–6]. At a large molecular weight, the migration velocity of an oriented DNA chain does not scale with N -1 (where N is the DNA size; for abbreviations, see Chapter 2) and becomes size-independent. In other words, the oriented and stretched DNAs of different sizes migrate with the same velocity and will not separate from each other in the continuous gel electrophoresis.