[Multiplex Assay to Evaluate the Genetic Risk of Developing Human Melanoma].

A genotyping procedure based on single-step PCR and subsequent allele-specific hybridization on a hydrogel biochip was developed to address the polymorphisms of HERC2, OCA2, SLC24A4, SLC45A2, TYR, IRF4, MC1R,MITF, PIGU, MYH7B, NCOA6, and CDK10. Amplified gene fragments were fluorescently labeled in PCR, and fluorescent signals from biochip cells were detected to evaluate how efficiently the PCR product formed a perfect duplex with an immobilized probe. The analytical characteristics of hybridization analysis were estimated for several fluorophores with different optical spectra. Cyanine dyes fluorescing in the range of Cy5 and Cy7 were synthesized for the purpose and used as 5'-tags of universal primers in single-step PCR. A Cy7 analog fluorescing in the near infrared range was found to increase the sensitivity of hybridization analysis by producing a lower background signal in the cases where target gene amplification was low.

[Infrared fluorescent markers for microarray DNA analysis on biological microchip].

To expand the informational capabilities of molecular genetic research, on the biological microchips, new indotricarbocyanine dyes that fluoresce in the near infrared (IR) spectral region have been synthesized. The developed IR dyes were studied using a biochip-based test system for detection of mutations in the BRCA1/BRCA2 and CHECK2 genes associated with breast cancer. The fluorescent label was introduced to the analyzed DNA during PCR using primers labeled with the synthesized IR dyes. An analyzer that allows recording and processing of images of fluorescent microarrays in the IR spectral region was designed and manufactured. It has been shown that the use of the synthesized dyes enables to conduct analysis in the IR region and improve the reliability of medical diagnostic tests due to low fluorescence intensity of sample components as well as of a biochip substrate and the reagents used for analysis.

[Methods for Reducing Laser Speckles to Achieve Even Illumination of the Microscope Field of View in Biophysical Studies].

The aim of this work was to compare different speckle reduction techniques. It was shown that the use of devices based on liquid crystals only leads to partial reduction of speckle contrast. In quantitative luminescent microscopy an application of the mechanical devices when a laser beam is spread within the field of view turned out to be more efficient. Laser speckle noise was virtually eliminated with the developed and manufactured mechanical device comprising a fiber optic ring light guide and the vibrator that permits movement of optical fiber ends towards the laser diode during measurements. The method developed for the analysis of microarrays was successfully applied to the problem of speckle reduction.

[The Effect of the Chromophore Charge on the Efficiency of Incorporation of Fluorescently-labeled Nucleotides in Matrix Synthesis by Taq DNA Polymerase].

In order to study the effect of an electrical charge of the chromophore, on the efficiency of incorporation of fluorescently-labeled nucleotides into DNA during PCR, three fluorescently-labeled dUPT, one of which with electroneutral and other two with positively and negatively charged dyes (Cy5 analogs), were synthesized. It is shown that dUPT, labeled with electroneutral Cy 5 analog, is most effectively incorporated into DNA when Tag polymerase is used for PCR.

Kinetics of hybridization on surface oligonucleotide microchips: theory, experiment, and comparison with hybridization on gel-based microchips.

The optimal design of oligonucleotide microchips and efficient discrimination between perfect and mismatch duplexes strongly depend on the external transport of target DNA to the cells with immobilized probes as well as on respective association and dissociation rates at the duplex formation. In this paper we present the relevant theory for hybridization of DNA fragments with oligonucleotide probes immobilized in the cells on flat substrate. With minor modifications, our theory also is applicable to reaction-diffusion hybridization kinetics for the probes immobilized on the surface of microbeads immersed in hybridization solution. The main theoretical predictions are verified with control experiments. Besides that, we compared the characteristics of the surface and gel-based oligonucleotide microchips. The comparison was performed for the chips printed with the same pin robot, for the signals measured with the same devices and processed by the same technique, and for the same hybridization conditions. The sets of probe oligonucleotides and the concentrations of probes in respective solutions used for immobilization on each platform were identical as well. We found that, despite the slower hybridization kinetics, the fluorescence signals and mutation discrimination efficiency appeared to be higher for the gel-based microchips with respect to their surface counterparts even for the relatively short hybridization time about 0.5-1 hour. Both the divergence between signals for perfects and the difference in mutation discrimination efficiency for the counterpart platforms rapidly grow with incubation time. In particular, for hybridization during 3 h the signals for gel-based microchips surpassed their surface counterparts in 5-20 times, while the ratios of signals for perfect-mismatch pairs for gel microchips exceeded the corresponding ratios for surface microchips in 2-4 times. These effects may be attributed to the better immobilization efficiency and to the higher thermodynamic association constants for duplex formation within gel pads.

Hydrogel-based protein microchips: manufacturing, properties, and applications.

Here a simple, reproducible, and versatile method is described for manufacturing protein and ligand chips. The photo-induced copolymerization of acrylamide-based gel monomers with different probes (oligonucleotides, DNA, proteins, and low-molecular ligands) modified by the introduction of methacrylic groups takes place in drops on a glass or silicone surface. All probes are uniformly and chemically fixed with a high yield within the whole volume of hydrogel semispherical chip elements that are chemically attached to the surface. Purified enzymes, antibodies, antigens, and other proteins, as well as complex protein mixtures such as cell lysates, were immobilized on a chip. Avidin- and oligohistidine-tagged proteins can be immobilized within biotin- and Ni-nitrilotriacetic acid-modified gel elements. Most gel-immobilized proteins maintain their biological properties for at least six months. Fluorescence and chemiluminescence microscopy were used as efficient methods for the quantitative analysis of the microchips. Direct on-chip matrix-assisted laser desorption ionization-time of flight mass spectrometry was used for the qualitative identification of interacting molecules and to analyze tryptic peptides after the digestion of proteins in individual gel elements. We also demonstrate other useful properties of protein microchips and their application to proteomics and diagnostics.

Portable system for microbial sample preparation and oligonucleotide microarray analysis.

We have developed a three-component system for microbial identification that consists of (i) a universal syringe-operated silica minicolumn for successive DNA and RNA isolation, fractionation, fragmentation, fluorescent labeling, and removal of excess free label and short oligonucleotides; (ii) microarrays of immobilized oligonucleotide probes for 16S rRNA identification; and (iii) a portable battery-powered device for imaging the hybridization of fluorescently labeled RNA fragments with the arrays. The minicolumn combines a guanidine thiocyanate method of nucleic acid isolation with a newly developed hydroxyl radical-based technique for DNA and RNA labeling and fragmentation. DNA and RNA can also be fractionated through differential binding of double- and single-stranded forms of nucleic acids to the silica. The procedure involves sequential washing of the column with different solutions. No vacuum filtration steps, phenol extraction, or centrifugation is required. After hybridization, the overall fluorescence pattern is captured as a digital image or as a Polaroid photo. This three-component system was used to discriminate Escherichia coli, Bacillus subtilis, Bacillus thuringiensis, and human HL60 cells. The procedure is rapid: beginning with whole cells, it takes approximately 25 min to obtain labeled DNA and RNA samples and an additional 25 min to hybridize and acquire the microarray image using a stationary image analysis system or the portable imager.

A micro-dissection approach for isolation of NotI linking clones from regions frequently deleted in RCC and SCLC.

We demonstrate that micro-dissection can be used for isolating NotI linking clones from the human 3p21-pter region. This approach is an improvement to positional cloning techniques, since NotI linking clones are directly linked with genes.

DNA sequencing by hybridization to microchip octa-and decanucleotides extended by stacked pentanucleotides.

The efficiency of sequencing by hybridization to an oligonucleotide microchip grows with an increase in the number and in the length of the oligonucleotides; however, such increases raise enormously the complexity of the microchip and decrease the accuracy of hybridization. We have been developing the technique of contiguous stacking hybridization (CSH) to circumvent these shortcomings. Stacking interactions between adjacent bases of two oligonucleotides stabilize their contiguous duplex with DNA. The use of such stacking increases the effective length of microchip oligonucleotides, enhances sequencing accuracy and allows the sequencing of longer DNA. The effects of mismatches, base composition, length and other factors on the stacking are evaluated. Contiguous stacking hybridization of DNA with immobilized 8mers and one or two 5mers labeled with two different fluorescent dyes increases the effective length of sequencing oligonucleotides from 8 to 13 and 18 bases, respectively. The incorporation of all four bases or 5-nitroindole as a universal base into different positions of the 5mers permitted a decrease in the number of additional rounds of hybridization. Contiguous stacking hybridization appears to be a promising approach to significantly increasing the efficiency of sequencing by hybridization.

DNA analysis and diagnostics on oligonucleotide microchips.

We present a further development in the technology of sequencing by hybridization to oligonucleotide microchips (SHOM) and its application to diagnostics for genetic diseases. A robot has been constructed to manufacture sequencing "microchips." The microchip is an array of oligonucleotides immobilized into gel elements fixed on a glass plate. Hybridization of the microchip with fluorescently labeled DNA was monitored in real time simultaneously for all microchip elements with a two-wavelength fluorescent microscope equipped with a charge-coupled device camera. SHOM has been used to detect beta-thalassemia mutations in patients by hybridizing PCR-amplified DNA with the microchips. A contiguous stacking hybridization technique has been applied for the detection of mutations; it can simplify medical diagnostics and enhance its reliability. The use of multicolor monitoring of contiguous stacking hybridization is suggested for large-scale diagnostics and gene polymorphism studies. Other applications of the SHOM technology are discussed.

Bone scintigraphy findings in Lisfranc joint injury.

Three-phase bone scintigraphy diagnosis of Lisfranc injury in a patient after foot trauma is discussed. Early diagnosis of Lisfranc joint injury is frequently missed and radionuclide bone scintigraphy may show a specific pattern where the x-rays are inconclusive.

7-Amino-actinomycin D as a specific fluorophore for DNA content analysis by laser flow cytometry.

A technique for DNA amount determination by flow cytometry based on the use of 7-amino-actinomycin D (7-amino-AMD), a fluorescent analogue of antibiotic actinomycin has been investigated, and a particular staining procedure has been developed. The procedure includes short fixation in 70% ethanol and staining for 20 min in 10(-5)M solution of 7-amino-AMD at pH7. The results of DNA content measurements are very reproducible. The histograms obtained have a coefficient of variation less than 3%. The absorption maximum of the complex of 7-amino-AMD with DNA is situated in the green spectrum region, making this stain particularly suitable for argon laser flow cytometry.

Localization of nucleoli in Drosophila melanogaster polytene chromosomes.

The majority of D. melanogaster salivary gland nuclei contains many nucleoli which vary in size and number. All nucleoli hybridize in situ with a cloned Drosophila DNA fragment containing 26S ribosomal gene. Autoradiographic analysis of preparations after pulse H3-uridine or H3-thymidine labelling of the salivary gland indicates an intensive transcription and replication of DNA within nucleoli. The nucleoli are bound to different sites of polytene chromosomes by chromatin fibers similar to strands of ectopic pairing and they are most often bound to regions which may be defined as intercalary heterochromatin.