PubMed Health⌕ Search

Biomedical subjects

Julia Khandurina

Publications and source records attributed to Julia Khandurina.

12 recordsLinked to original sources
PubMed2004

Multidimensional separations in the pharmaceutical arena.

The introduction of novel, powerful and rapid multidimensional separation and characterization methods has produced revolutionary global changes at the genome, proteome and metabolome level, bringing about a radical transition in our views of living systems, at the molecular level. The age of proteomics and metabolomics demands high-resolution multidimensional separation techniques. Multidimensional gas and liquid chromatography techniques, in addition to capillary and microchip electrophoresis methods, offer increased resolution and sensitivity, while also affording adequate throughput and reproducibility to meet the demands of the modern pharmaceutical industry. Coupled with MS, these techniques provide not only separation but also reliable identification of the sample components. The resolving power of these methods has proved to be superior over individual one-dimensional approaches, enabling the comprehensive separation of complex biological mixtures, with excellent resolution and reproducibility. High capacity computer systems that are capable of rigorous qualitative and quantitative analysis of the separation profiles allow the establishment and mining of large databases. Examples of various modern multidimensional separation techniques, and their integration with MS, are reviewed, here, with respect to pharmaceutical analysis.

Chromatography, Gas↗
PubMed2003

Differential gene expression analysis by micro-preparative capillary gel electrophoresis.

Differential display analysis by cDNA fractionation, collection of differentially expressed fractions of interests and their downstream characterization is demonstrated. cDNA pools from two strains of Cochliobolus heterostrophus fungus were generated by specific restriction digestion and selective ligation. Micropreparative separation and isolation of differentially expressed transcript representatives were accomplished by high-performance capillary gel electrophoresis. The collected individual DNA molecules were polymerase chain reaction amplified and sequenced to create expressed sequence tags for the genes of interests. High resolving power and sensitivity of capillary gel electrophoresis enabled fast and automated processing of minute amounts of cDNA samples with high precision.

Base Sequence↗
PubMed2003

Micropreparative capillary gel electrophoresis of DNA: rapid expressed sequence tag library construction.

A capillary gel electrophoresis based automated DNA fraction collection technique was developed to support a novel DNA fragment-pooling strategy for expressed sequence tag (EST) library construction. The cDNA population is first cleaved by BsaJ I and EcoR I restriction enzymes, and then subpooled by selective ligation with specific adapters followed by polymerase chain reaction (PCR) amplification and labeling. Combination of this cDNA fingerprinting method with high-resolution capillary gel electrophoresis separation and precise fractionation of individual cDNA transcript representatives avoids redundant fragment selection and concomitant repetitive sequencing of abundant transcripts. Using a computer-controlled capillary electrophoresis device the transcript representatives were separated by their size and fractions were automatically collected in every 30 s into 96-well plates. The high resolving power of the sieving matrix ensured sequencing grade separation of the DNA fragments (i.e., single-base resolution) and successful fraction collection. Performance and precision of the fraction collection procedure was validated by PCR amplification of the collected DNA fragments followed by capillary electrophoresis analysis for size and purity verification. The collected and PCR-amplified transcript representatives, ranging up to several hundred base pairs, were then sequenced to create an EST library.

Animals↗
PubMed2003

Transcription factor binding study by capillary zone electrophoretic mobility shift assay.

Regulation of gene expression through interaction of proteins with specific DNA sequences is a central issue in functional genomics. Capillary electrophoretic mobility shift assay is an efficient novel method for the investigation of sequence specific protein-DNA interactions, allowing rapid and sensitive quantification of the complex formation. In this paper, we present a pilot study on capillary zone electrophoretic mobility shift assay (CZEMSA) to investigate the interaction between the transcription factors of HeLa nuclear extract and Sp1-specific fluorescein-labeled oligonucleotide, using the unlabeled probe as competitor. The mobility shift assay was accomplished by CZE in coated capillaries without polymeric buffer additives. Specificity of the DNA protein complex formation was verified by competition experiments, as well as by supershift assay with an anti-Sp1 antibody. The applied electric field strength did not affect the stability of DNA-protein complex during the electrophoretic analysis, allowing rapid identification and quantification of the protein DNA interaction. A practical application to study the interaction between Oryza sativa MADS-box transcription factor 4 (OsMADS4) and its consensus sequence is also reported.

Base Sequence↗
PubMed2003

Microscale separation and analysis.

There is a recent and growing interest in microscale separation and analysis, a result of advantages of miniaturization such as rapid separation times, high performance and throughput, reduced costs, and the possibility of system integration and multiplexing. Adopting the concepts of conventional capillary electrophoresis, capillary electrochromatography, micellar electrokinetic chromatography and various sample preparation techniques to microchip format, in conjunction with the integration of different analysis steps into a monolithic system, have opened new levels in performance, functionality and throughput. This review summarizes the recent advances in the field of microfabricated separation devices for genomics, proteomics and high-throughput screening applications, also addressing system integration and micropreparative functionalities.

DNA↗
PubMed2002

Micromachined capillary cross-connector for high-precision fraction collection.

A new approach for high-precision fraction collection of double-stranded DNA fragments by capillary electrophoresis coupled to a micromachined plastic capillary cross-connector is presented. The system design integrates four fused-silica capillaries with an acrylic cross-channel connector. The cross-channel structure was introduced to enhance the efficiency of the fraction collection process by electrokinetic manipulations. Following the detection of the sample zone of interest at or slightly upstream of the cross during the separation mode, the potentials were reconfigured to collection mode to direct the selected analyte zone into the corresponding collection vial, while keeping the rest of the sample components virtually stopped within the separation capillary. In this way the spacing between consecutive bands of interest can be physically increased, allowing precise isolation of spatially close sample zones. After collection of the target fraction the separation mode is resumed, and the separation/collection cycle is repeated until all desired sample zones are separated and captured. The capillary cross-connector was fabricated of a transparent acrylic substrate by microdrilling flat end and through channels, matching precisely the O.D. and I.D. of the connected capillary tubing, respectively. This design provided a close to zero dead volume connection assembly for the separation and collection capillaries causing minimal extra band broadening during high-precision micropreparative DNA fractionation.

Electrophoresis, Capillary↗
PubMed2002

Micropreparative fraction collection in microfluidic devices.

Micropreparative fraction collection following microchip-based electrophoretic analysis of biomolecules is of major importance for a variety of biomedical applications. In this paper, we present a microfabricated device-based fraction collection system. Various size DNA fragments were separated and collected by simply redirecting the desired portions of the detected sample zones to corresponding collection wells using appropriate voltage manipulations. The efficiency of sampling and collection of the fractions was enhanced by placing a cross channel at or downstream of the detection point. Following the detection of the band of interest, the potentials were reconfigured to sampling/collection mode, so that the selected sample zone migrated to the appropriate collection well of the microdevice. The potential distribution assured that the rest of the analyte components in the separation column was retarded, stopped, or reversed, increasing in this way the spacing between the sample zone being collected and the immediately following one. By this means, a precise collection of spatially close consecutive bands could be facilitated. Once the target sample fraction reached the corresponding collection well, the potentials were switched back to separation mode. Alternation of the separation/detection and sampling/collection cycles was repeated until all required sample zones were physically isolated. The integrated device consists of a sample introduction, separation, fraction sampling, and fraction collection compartments. The feasibility of the fraction collection technique was tested on a mixture of dsDNA fragments. The amounts of DNA collected in this way were enough for further downstream sample processing, such as conventional PCR-based analysis.

Chemical Fractionation↗
PubMed2002

Bioanalysis in microfluidic devices.

Microfabricated bioanalytical devices (also referred to as laboratory-on-a-chip or micro-TAS) offer highly efficient platforms for simultaneous analysis of a large number of biologically important molecules, possessing great potential for genome, proteome and metabolome studies. Development and implementation of microfluidic-based bioanalytical tools involves both established and evolving technologies, including microlithography, micromachining, micro-electromechanical systems technology and nanotechnology. This article provides an overview of the latest developments in the key device subject areas and the basic interdisciplinary technologies. Important aspects of DNA and protein analysis, interfacing issues and system integration are all thoroughly discussed, along with applications for this novel "synergized" technology in high-throughput separations of biologically important molecules. This review also gives a better understanding of how to utilize these technologies as well as to provide appropriate technical solutions to problems perceived as being more fundamental.

Chemistry Techniques, Analytical↗
PubMed2002

Microchip-based high-throughput screening analysis of combinatorial libraries.

In recent years, there have been significant advances in biochemical assay miniturization and integration of microchip-based technologies with combinatorial library screening for high-throughput and large-scale applications. Small-molecule microarrays, protein arrays and cell-based arrays and conventional DNA arrays as well as microfluidic approaches in HTS are discussed in this review.

Animals↗
PubMedDate not supplied

High-throughput genotyping by microchip electrophoresis.

Easy applicability of modern microfabrication technology to electrophoresis microchips has initiated a rapidly moving interdisciplinary field in analytical chemistry. Electric field-mediated separations in microfabricated devices are significantly faster than conventional electrophoresis methods and are usually completed in seconds to minutes. The flexibility of fluidic manipulations in electrophoresis microchips allows the use of a variety of separation techniques and conditions. In this study, large-scale genotyping of the repeat polymorphism in the regulatory (promoter) region of the serotonin transporter gene 5-HTT linked polymorphic region (5-HTTLPR) was attempted using polymerase chain reaction (PCR) amplification followed by rapid microchip electrophoresis analysis of the amplicons.

DNA↗