Karel Klepárník

Injection bias of DNA fragments in capillary electrophoresis with sieving

The relative amounts of DNA fragments in a mixture injected into the capillary by electromigration or hydrodynamically by pressure were compared. Even if the electrophoretic mobilities of DNA fragments with different sizes are the same in a free solution in the sample vial, the size bias is brought about by the different mobilities in a sieving medium and by the electroosmosis. The experiments were performed in capillaries filled with a solution of liquified agarose, a replaceable sieving medium. The experimental results were compared with a theoretical model.

Ultrafast detection of microsatellite repeat polymorphism in endothelin 1 gene by electrophoresis in short capillaries

The methodology and instrumentation for fast denaturing electrophoresis in short capillaries was developed and exemplified by detection of short tandem repeat polymorphism in the endothelin 1 gene. The resolution of two nucleotides, which is required for the detection of a dinucleotide repeat polymorphism, was achieved in a capillary of an effective length of 2.5 cm at a temperature of 60°C and an electric field strength of 600 V/cm in 42 s. Thus, the use of denaturing electrophoresis in short capillaries with laser‐induced fluorescence detection resulted in a reduction of analysis time by a factor of 200 when compared to the conventional slab gel electrophoresis. The developed methodology and instrumentation is advantageous for an implementation in clinical diagnostics and genetic population screening where fast analytical instrumentation amenable to automation is of paramount importance.

Electromigration behaviour of DNA molecules at the free electrolyte-polymer solution interface

The velocity at which DNA molecules migrate across an interface between a free solution of an electrolyte and a sieving medium was investigated in capillaries. A model was proposed where the DNA molecules are supposed to be stacked at the interface and then, due to consecutive conformational changes, their velocities increase and reach the value of their effective electrophoretic mobilities in the polymer solution filling the rest of a capillary. This behaviour was shown by measuring the electromigration injection bias and by extrapolation of the migration times in capillaries of different lengths to the zero migration path. The experiments showed an extraordinarily high size-based separation selectivity of the electromigration across the interface, which seems to offer a potential for successful high resolution separation of DNA fragments in capillaries as short as several centimetres.

Detection of active apoptotic molecules with femtogram sensitivity in single cells: a pilot study

T epidermis is a dynamic epithelium with constant renewal throughout life. Epidermal homeostasis depends on two types of proliferative cells, keratinocyte stem cells (KSCs), and transit amplifying (TA) cells. In the case of chronologic aging, levels of KSCs tend to decrease and change functionally. However, little is known about the effect of photoaging on epidermal proliferative subtype populations. The aim of this study was to validate involucrin/beta1-integrin ratio as a molecular marker of epidermal photoaging, and to investigate the effects of photoaging caused by chronic UV exposure on the proliferative subtype populations. A total of 15 male volunteers (age range 20-24 and 77-85 years) provided sun-exposed and sun-protected skin samples. The expression of beta1-integrin was found to be significantly reduced in photoaged skin and ratios of the expressions of involucrin to beta1-integrin were increased 2.6-fold only in elderly subjects. Interestingly, immunostaining of the sun-exposed skins of elderly subjects showed aberrant beta1-integrin expression over the basal layer and greater numbers of Ki-67-positive cells than in sun-protected buttock skin. Flow cytometric analysis revealed that the proportion of KSCs to TA cells was reversed in sun-exposed and sun-protected skins of elderly subjects. Our results suggest that KSC numbers may be lower in photoaged skin than in chronologically aged skin and could be applied to hyperplastic pattern of photoaging. These findings suggest that the epidermis of photoaged skin is impaired in terms of its proliferative potential by attempting to repair chronic UV exposure and that photoaging may be associated with alteration in the two proliferative cell fractions.N exhibit modified chemical and physical properties that give them the ability to interact with the biological systems at the cellular and molecular levels. These interactions enhance the biomedical applications of nanotechnology in the field of tissue regeneration. A wide range of nanomaterials made of organic and inorganic composites could be self assembled in nanoscale size that simulates more accurately the dimensions of the natural human tissues such as nanoparticles, nanosurfaces and nanoscaffolds. These novel nanomaterials significantly influence the behavior and development of stem cells. The applications of nanotechnology in specific tissue regeneration, such as bone, cartilage, cardiovascular and neural tissues were investigated by several researchers. Nanostructures have been used to promote stem cell viability, proliferation and differentiation. Nanotechnology provides biodegradable and biocompatible biomimetic fabricates that restore and improve the tissue functions. Nanocomposition and nanotopography of a tissue engineered material determine the implant fate providing 3 dimentional tissue culture systems that promote normal cell growth and differentiation without adverse tissue reaction. Recent progress in the synthesis allows the cultured cells to react to the internal and external stimuli and to exchange the signaling factors between those cells and the external environment. However, further understanding to the interactions of nanomaterials with the biological system and more investigations of the safety of these nanostructures are still required before their full application in human tissue repair

NUCLEIC ACIDS | Chromatographic and Electrophoretic Methods

Techniques for separation of DNA and RNA provide powerful tools for the analysis of genetic material and transcription products. Modern methods of analysis can answer most of the questions about the sequence, nucleotide substitutions (mutations, polymorphisms, or single nucleotide polymorphisms—SNPs), and structure of nucleic acids. Crucial techniques in the analysis of DNA or RNA are the traditional separation methods—chromatography and electrophoresis—and, more recently, microfluidics and chip-based DNA arrays and mass spectrometric methods.