Hydrogel drop microchips with immobilized DNA: properties and methods for large-scale production

Although gel-based microchips offer significant advantages over two-dimensional arrays, their use has been impeded by the lack of an efficient manufacturing procedure. Here we describe two simple, fast, and reproducible methods of fabrication of DNA gel drop microchips. In the first, copolymerization method, unsaturated groups are chemically attached to immobilized molecules, which are then mixed with gel-forming monomers. In the second, simpler polymerization-mediated immobilization method, aminated DNA without prior modification is added to a polymerization mixture. Droplets of polymerization mixtures are spotted by a robot onto glass slides and the slides are illuminated with UV light to induce copolymerization of DNA with gel-forming monomers. This results in immobilization of DNA within the whole volume of semispherical gel drops. The first method can be better controlled while the second one is less expensive, faster, and better suited to large-scale production. The microchips manufactured by both methods are similar in properties. Gel elements of the chip are porous enough to allow penetration of DNA up to 500 nucleotides long and its hybridization with immobilized oligonucleotides. As shown with confocal microscope studies, DNA is hybridized uniformly in the whole volume of gel drops. The gels are mechanically and thermally stable and withstand 20 subsequent hybridizations or 30-40 PCR cycles without decrease in hybridization signal. A method for quality control of the chips by staining with fluorescence dye is proposed. Applications of hydrogel microchips in research and clinical diagnostics are summarized.     

Petunia hybrida S-proteins: ribonuclease activity and the role of their glycan side chains in self-incompatibility

Summary Self-incompatibility in flowering plants is controlled by the S-gene, encoding stylar S (allele-specific) glycoproteins. In addition to three previously characterized Petunia hybrida S-proteins, we identified by N-terminal sequence analysis another stylar S-protein, co-segregating with the S _b-allele. Purified S-proteins reveal biological activity, as is demonstrated for two of them by the allele-specific inhibition of pollen tube growth in vitro. Moreover, the four isolated S-proteins are ribonucleases (S-RNases). Specific activities vary from 30 (S₁) to 1000 (S₂) units per min per mg protein. We attempted to investigate the functionality of the carbohydrate portion of the S-RNases. Deglycosylation studies with the enzyme peptide-N-glycosidase F (PNGase F) reveals differences in the number of N-linked glycan chains present on the four S-RNases. Variability in the extent of glycosylation accounts for most of the molecular weight differences observed among these proteins. By amino acid sequencing, the positions of two of the three N-glycosylation sites on the S₂-RNase could be located near the N-terminus. Enzymic removal of the glycan side chains has no effect on the RNase activity of native S-RNases. This suggests another role of the glycan moiety in the self-incompatibility mechanism.     

Lectinomics II. A highway to biomedical/clinical diagnostics

The review assesses current status and attempts to forecast trends in the development of lectin biorecognition technology. The progressive trend is characterized scientometrically and reflects the current transient situation, when standard low-throughput lectin-based techniques are being replaced by a novel microarray-based techniques offering high-throughput of detection. The technology is still in its infancy (validation phase), but already shows promise as an efficient tool to decipher the enormous complexity of the glycocode that influences physiological status of the cell. Further enhancement in robustness and flexibility of lectin microarrays is predicted by using recombinant and artificial lectins that will render production of lectin microarrays cost-effective and more affordable. Mass spectrometry is expected to play an important role to characterize the binding profile of new lectins. Differences in glycan recognition by lectins and anti-carbohydrate antibodies are given on a molecular basis, and strong and weak points of both biorecognition molecules in diagnosis are briefly discussed.