Biomedical subjects

Andrei Mirzabekov

Publications and source records attributed to Andrei Mirzabekov.

5 recordsLinked to original sources

PubMed2002

Analysis of SNPs and other genomic variations using gel-based chips.

Application of microarrays for the analysis of point mutations and SNPs in genomic DNAs is currently under intensive development. Various technologies are being investigated, employing enzymatic, chemical, and physical tools [for review, see Tillib and Mirzabekov, 2001]. Our current approach is based on the use of IMAGE chips (immobilized microarrays of gel elements) consisting of an array of gel pads attached to a hydrophobic glass surface. The gel pads range in size from picoliters to nanoliters and are used for immobilization of oligonucleotide probes, as well as miniature test tubes for chemical or enzymatic reactions with tethered compounds. Nucleic acids are hybridized, fractionated, modified, and subjected to enzymatic reactions inside the pads. All steps of sequence analysis (PCR-amplification, activation or release of primers and products, DNA extension, hybridization, and reading of the results) can be performed within the same pad. A flexible and inexpensive technology platform enables one to monitor processes in the arrays in both real time and steady-state. Identification of SNPs, microsequencing, and other specific tasks are easily performed. In particular, stacking interactions with short oligonucleotides enhance the capability of high-throughput screening. The IMAGE chips can be analyzed using a variety of equipment, from a dedicated multi-color fluorescent microscope or MALDI-spectrometer to an inexpensive portable analyzer suitable for field conditions. Customized gel-based chips were successfully used for screening of SNPs in a broad range of biologically meaningful genes.

Alleles↗

PubMed2002

Emerging array-based technologies in proteomics.

Microarray format is the method of choice for high-throughput hybridization of nucleic acids. Lately, microarray strategy has been applied to broad studies of interactions of proteins with other molecules. The diversity of these interactions and variations of the properties of proteins present new challenges to microchip technology. Significant achievements have been reported. In particular, three-dimensional (gel-based) chips can be manufactured with limited resources and offer flexibility, capacity, homogeneous solvent environment and the ability to record processes in real time and in variable conditions.

Animals↗

PubMed2002

Species-level identification of orthopoxviruses with an oligonucleotide microchip.

A method for species-specific detection of orthopoxviruses pathogenic for humans and animals is described. The method is based on hybridization of a fluorescently labeled amplified DNA specimen with the oligonucleotide DNA probes immobilized on a microchip (MAGIChip). The probes identify species-specific sites within the crmB gene encoding the viral analogue of tumor necrosis factor receptor, one of the most important determinants of pathogenicity in this genus of viruses. The diagnostic procedure takes 6 h and does not require any sophisticated equipment (a portable fluorescence reader can be used).

Animals↗

PubMed2002

Fluorescence data analysis on gel-based biochips.

A series of biochip readers developed for gel-based biochips includes three imaging models and a novel nonimaging biochip scanner. The imaging readers, ranging from a research-grade versatile reader to a simple portable one, use wide-field objectives and 12-bit digital large-coupled device cameras for parallel addressing of multiple array elements. This feature is valuable for monitoring the kinetics of sample interaction with immobilized probes. Depending on the model and the label used, the sensitivity of these readers approaches 0.3 amol of a labeled sample per gel element. In the selective scanner, both the spot size of the excitation laser beam and the detector field of view match the size of the biochip array elements so that the whole row of the array can be read in a single scan. The portable version reads 50-mm long, 150-element, one-dimensional arrays in 5 s. With a dynamic range of 4000:1, a sensitivity of 1-5 amol of a labeled sample per gel element, and a data format facilitating online processing, the scanner is an attractive, inexpensive solution for biomedical diagnostics. Fluorophores for sample labeling were compared experimentally in terms of detection sensitivity, influence on duplex stability, and suitability for multilabel analysis and thermodynamic studies. Texas Red and tetracarboxyphenylporphyn proved to be the best choice for two-wavelength analysis using the imaging readers.

Fluorescent Dyes↗

PubMed2002

Identification of chromosomal translocations in leukemias by hybridization with oligonucleotide microarrays.

BACKGROUND AND OBJECTIVES: Identification of chromosomal rearrangements is important for a precise risk-stratified diagnosis of hematologic malignancies. As the number of known translocations, specific for different types of leukemia increases, it takes ever more time and increasing amounts of patient's material to screen a single patient with individual polymerase chain reactions (PCR). The aim of this study was to develop a new approach combining specificity with high-throughput sufficient for rapid screening of clinical samples for the presence of numerous translocations. DESIGN AND METHODS: We designed an oligonucleotide microarray and used hybridization with microarrays in combination with multiplex reverse transcription-polymerase chain reaction (RT-PCR) assay for accurate and rapid identification of some major leukemias. The following translocations were used as prototypic: t(9;22) p210 and p190, t(4;11), t(12;21), and t(15;17). This approach was tested on five different cell cultures carrying translocations and on 22 clinical samples from leukemic patients. RESULTS: Distinctive hybridization signals were obtained for all types of chimeric transcripts from cell lines with translocations. Both the type of translocation and the splice variant were determined. The data demonstrated high specificity and reproducibility of the method. Analysis of the 22 clinical samples using the microarray-based approach showed complete agreement with standard PCR analysis. INTERPRETATION AND CONCLUSIONS: Our data suggest that oligonucleotide microarrays can be used as an efficient, alternative approach to the traditional post-PCR Southern blot analysis. The oligonucleotide microarray approach appears suitable for clinical screening of major risk-stratifying translocations in patients with leukemia.

Bone Marrow↗