Julia Khandurina - Academia.edu (original) (raw)

Papers by Julia Khandurina

Research paper thumbnail of System-level studies of a cell-free transcription-translation platform for metabolic engineering

bioRxiv (Cold Spring Harbor Laboratory), Aug 3, 2017

Current methods for assembling biosynthetic pathways in microorganisms require a process of repea... more Current methods for assembling biosynthetic pathways in microorganisms require a process of repeated trial and error and have long design-build-test cycles. We describe the use of a cell-free transcription-translation (TX-TL) system as a biomolecular breadboard for the rapid engineering of the 1,4-butanediol (BDO) pathway. We demonstrate the reliability of TX-TL as a platform for engineering biological systems by undertaking a careful characterization of its transcription and translation capabilities and provide a detailed analysis of its metabolic output. Using TX-TL to survey the design space of the BDO pathway enables rapid tuning of pathway enzyme expression levels for improved product yield. Leveraging TX-TL to screen enzyme variants for improved catalytic activity accelerates design iterations that can be directly applied to in vivo strain development. Cell-free systems simulate a controlled cellular environment that delivers repeatable results. Recent research has explored the application of cell-free systems for biological circuits, renewable energy, and medicine. The cell-free transcription-translation (TX-TL) system was first developed as a biomolecular breadboard to test genetic circuits, and many have been demonstrated since. 7-10 The synthesis of hydrogen and the development of enzymatic fuel cells in cell-free systems has charted new paths for renewable energy. 11-12 The high yield of therapeutic proteins in E. coli-based cell-free synthesis system also offered new methods for medicinal synthesis. 13-15 Using cell-free systems for prototyping metabolic pathways is an attractive alternative platform for the engineering of biosynthesis in microbial hosts. Lysate of engineered

Research paper thumbnail of Automated Agarose Gel Electrophoresis of dsDNA Fragments on a Commercial DNA Sequencer

Research paper thumbnail of Automated High-Throughput RNA Analysis by Capillary Electrophoresis

Research paper thumbnail of Analysis of Carbohydrates by Capillary Electrophoresis

Handbook of Capillary and Microchip Electrophoresis and Associated Microtechniques, Third Edition, 2007

Research paper thumbnail of Micropreparative Applications and On-Line Sample Treatment

Principles and Applications in Analytical Chemistry and Microchip Technology, 2003

Research paper thumbnail of Chapter 11 Microfabricated analytical devices

Journal of Chromatography Library, 2004

Publisher Summary Microfabricated analytical devices are referred to as “lab-on-a-chip systems” a... more Publisher Summary Microfabricated analytical devices are referred to as “lab-on-a-chip systems” and include microseparation units, miniaturized reactors, and microarrays. This chapter reviews recent developments and trends in microfluidic analyses of biological interest, including DNA, proteins, and complex carbohydrate analysis and high-throughput screening. The microfabrication methods comprise photolithography in rigid materials and fabrication in plastics and elastomers. Glass substrates are common because of their good optical properties and well-developed microfabrication technology and surface chemistry. For miniaturized analysis systems, sample handling and manipulation are of great importance. Specific problems resulting from the shrinking of macroscopic systems include the failure of samples to be representative and of manipulations to be reproducible. This makes it necessary to incorporate specific microscale techniques and components in such devices. The analytical performances of microfluidic devices are drastically affected by dead volumes in the system and also by the surface properties of the fluidic channels.

Research paper thumbnail of An Ultrafast Method to Evaluate Rna Quality

Research paper thumbnail of Monolithic photonics-microfluidics integration for micrototal analysis systems

We demonstrate the monolithic integration of waveguides and microfluidic channels using same mate... more We demonstrate the monolithic integration of waveguides and microfluidic channels using same material, PDMS (polydimethylsiloxane). We also developed two different schemes of integration. Both of them show precise alignment and robust structure. These techniques hold great promise of expanding functionality of micrototal analysis systems with photonic integrated circuits.

Research paper thumbnail of High Precision Micropreparative Separation System Based on Plastc Microfluidic Module — Capillary Coupling

Micro Total Analysis Systems 2002, 2002

We report on a new approach for high precision fraction collection of electrophoretically separat... more We report on a new approach for high precision fraction collection of electrophoretically separated DNA molecules. The fraction collection system is based on a microfluidic cross-connector module coupled to four fused silica capillaries. The cross-connector was fabricated of an acrylic substrate by microdrilling flat end and through channels to ensure close to zero dead volume connections with capillary tubing. DNA fragments were size separated and physically isolated by appropriate electrokinetic manipulations. Selected portions of analyte were redirected to the corresponding collection reservoirs, while maintaining the rest of the sample virtually stopped or slowly reversed within the separation capillary. In this way, the spacing between consecutive bands of interest can be increased enabling high precision isolation of spatially close analyte zones. The separation/collection cycle was repeated until all desired sample zones are captured. The amounts of the collected DNA fractions were enough for further downstream sample processing, such as PCR amplification and analysis.

Research paper thumbnail of High-throughput genotyping by microchip electrophoresis

Journal of capillary electrophoresis and microchip technology

Easy applicability of modern microfabrication technology to electrophoresis microchips has initia... more 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.

Research paper thumbnail of UV/ozone modification of poly(dimethylsiloxane) microfluidic channels

Sensors and Actuators B: Chemical, 2004

UV/ozone method of oxidation of poly(dimethylsiloxane) (PDMS) was investigated to enhance microfl... more UV/ozone method of oxidation of poly(dimethylsiloxane) (PDMS) was investigated to enhance microfluidic device performance, i.e., increase electroosmotic flow (EOF) stability, minimize adsorption of analytes in microchannels, etc. PDMS microfluidic devices are easily fabricated at low cost, but high hydrophobicity and poor surface properties make this material inferior to glass or quartz in most high-performance microfluidic applications. PDMS can be converted into a silica-like material by UV/ozone treatment enabling much deeper oxidation of PDMS without crack failures compared to oxygen plasma treatment. In this study application of UV/ozone treatment to fabrication of microfluidic devices has been presented. Specifically, conversion of bulk PDMS by deep penetration and complete oxidation of thick-film membranes by UV/ozone were investigated. Changes of physical properties upon PDMS modification were monitored by FTIR spectroscopy, contact angle measurements, and channel cross-section visualization. Two microfabrication approaches were attempted and compared: microchannels molded in bulk PDMS were oxidized at the surface and sealed, as well as microchannels molded in a 15 m PDMS membrane followed by sealing against glass and complete UV/ozone oxidation. Improved wettability, enhanced electroosmotic flow, and reduced adsorption properties were observed in the modified PDMS channels.

Research paper thumbnail of Micromachined capillary cross-connector for high-precision fraction collection

Journal of Chromatography A, 2002

A new approach for high-precision fraction collection of double-stranded DNA fragments by capilla... more 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.

Research paper thumbnail of Differential gene expression analysis by micro-preparative capillary gel electrophoresis

Journal of Chromatography A, 2003

Differential display analysis by cDNA fractionation, collection of differentially expressed fract... more 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.

Research paper thumbnail of Bioanalysis in microfluidic devices

Journal of Chromatography A, 2002

Microfabricated bioanalytical devices (also referred to as laboratory-on-a-chip or micro-TAS) off... more 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.

Research paper thumbnail of DNA profiling by capillary array electrophoresis with non-covalent fluorescent labeling

Journal of Chromatography A, 2004

Increasing need for large-scale DNA profiling necessitated the development of automated electroph... more Increasing need for large-scale DNA profiling necessitated the development of automated electrophoresis based methods enabling rapid, high performance analysis of nucleic acids in a wide molecular-mass range. In this paper, we report on the adaptation of a commercial 96-capillary array electrophoresis (CAE) instrument for high-throughput DNA fragment analysis and the evaluation of the effects of different non-covalent DNA staining dyes on separation efficiency. The applicability of different color internal fluorescent standards is shown with mathematical spectral overlap correction algorithms. Large-scale quality control assessment of oligonucleotide probes using non-covalent fluorophore labeling is also demonstrated. The method requires small sample amounts, offers automation and quantification capabilities to accommodate modern biotechnology industry needs.

Research paper thumbnail of Micropreparative capillary gel electrophoresis of DNA: Rapid expressed sequence tag library construction

ELECTROPHORESIS, 2003

Micropreparative capillary gel electrophoresis of DNA: Rapid expressed sequence tag library const... more 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.

Research paper thumbnail of Analysis of high‐mannose‐type oligosaccharides by microliquid chromatography‐mass spectrometry and capillary electrophoresis

ELECTROPHORESIS, 2004

Analysis of high-mannose-type oligosaccharides by microliquid chromatography-mass spectrometry an... more Analysis of high-mannose-type oligosaccharides by microliquid chromatography-mass spectrometry and capillary electrophoresis We report on microbore liquid chromatography (mLC) and capillary electrophoresis (CE) separation of glycopeptides and high-mannose-type oligosaccharides, digested from recombinant phospholipase C, expressed in Pichia pastoris. The glycopeptides were subject to mLC/electrospray ionization/mass spectrometry (ESI-MS) and mLC/ESI-tandem MS (MS/MS) analysis that revealed high-mannose structure size variation between Man 7 GlcNAc 2 and Man 14 GlcNAc 2. Then, high-performance CE was applied to identify possible positional isomers of the high-mannose structures. For the CE experiments, the oligosaccharides were released from the glycoproteins by peptide-Nglycosidase F and labeled with 1-aminopyrene-3,6,8-trisulfonic acid (APTS). Excellent separation of the possible positional isomers was attained, suggesting one for Man 9-GlcNAc 2 , two for Man 10 GlcNAc 2 , three for Man 11 GlcNAc 2 , Man 12 GlcNAc 2 , and Man 13-GlcNAc 2 , and two for Man 14 GlcNAc 2. The CE results provided complementary information to the mLC/ESI-MS and MS/MS data with respect to the possible number of positional isomers.

Research paper thumbnail of Transcription factor binding study by capillary zone electrophoretic mobility shift assay

ELECTROPHORESIS, 2003

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

Research paper thumbnail of Large-scale carbohydrate analysis by capillary array electrophoresis: Part 1. Separation and scale-up

ELECTROPHORESIS, 2004

Large-scale carbohydrate analysis by capillary array electrophoresis: Part 1. Separation and scal... more Large-scale carbohydrate analysis by capillary array electrophoresis: Part 1. Separation and scale-up A 96-capillary array electrophoresis (CAE) instrument has been adapted for large-scale mono-and oligosaccharide analysis and characterization. Operational protocols and data processing tools have been developed to optimize the CAE system for this application. Effects of different additives to the running buffer on efficiency and capillary-to-capillary performance reproducibility have been studied.

Research paper thumbnail of Automated carbohydrate profiling by capillary electrophoresis: A bioindustrial approach

ELECTROPHORESIS, 2004

Automated, high-resolution, quantitative, high-throughput analysis of mono-and oligosaccharides, ... more Automated, high-resolution, quantitative, high-throughput analysis of mono-and oligosaccharides, produced by enzymatic digestion of cellohexaose (model substrate) and lignocellulosic biomass, is demonstrated using high-performance capillary electrophoresis in conjunction with a single-step fluorophore labeling strategy for sensitive laser-induced fluorescence detection. Unattended batch sample processing from 96-well plates enabled reliable industrial-scale carbohydrate analysis. Excellent resolution of mono-and oligosaccharides was achieved under suppressed electroosmotic flow conditions, using either covalently or dynamically coated fused-silica capillary columns. The proposed approach readily supports the demands of bioindustrial operation environments with respect to high-throughput carbohydrate profiling.

Research paper thumbnail of System-level studies of a cell-free transcription-translation platform for metabolic engineering

bioRxiv (Cold Spring Harbor Laboratory), Aug 3, 2017

Current methods for assembling biosynthetic pathways in microorganisms require a process of repea... more Current methods for assembling biosynthetic pathways in microorganisms require a process of repeated trial and error and have long design-build-test cycles. We describe the use of a cell-free transcription-translation (TX-TL) system as a biomolecular breadboard for the rapid engineering of the 1,4-butanediol (BDO) pathway. We demonstrate the reliability of TX-TL as a platform for engineering biological systems by undertaking a careful characterization of its transcription and translation capabilities and provide a detailed analysis of its metabolic output. Using TX-TL to survey the design space of the BDO pathway enables rapid tuning of pathway enzyme expression levels for improved product yield. Leveraging TX-TL to screen enzyme variants for improved catalytic activity accelerates design iterations that can be directly applied to in vivo strain development. Cell-free systems simulate a controlled cellular environment that delivers repeatable results. Recent research has explored the application of cell-free systems for biological circuits, renewable energy, and medicine. The cell-free transcription-translation (TX-TL) system was first developed as a biomolecular breadboard to test genetic circuits, and many have been demonstrated since. 7-10 The synthesis of hydrogen and the development of enzymatic fuel cells in cell-free systems has charted new paths for renewable energy. 11-12 The high yield of therapeutic proteins in E. coli-based cell-free synthesis system also offered new methods for medicinal synthesis. 13-15 Using cell-free systems for prototyping metabolic pathways is an attractive alternative platform for the engineering of biosynthesis in microbial hosts. Lysate of engineered

Research paper thumbnail of Automated Agarose Gel Electrophoresis of dsDNA Fragments on a Commercial DNA Sequencer

Research paper thumbnail of Automated High-Throughput RNA Analysis by Capillary Electrophoresis

Research paper thumbnail of Analysis of Carbohydrates by Capillary Electrophoresis

Handbook of Capillary and Microchip Electrophoresis and Associated Microtechniques, Third Edition, 2007

Research paper thumbnail of Micropreparative Applications and On-Line Sample Treatment

Principles and Applications in Analytical Chemistry and Microchip Technology, 2003

Research paper thumbnail of Chapter 11 Microfabricated analytical devices

Journal of Chromatography Library, 2004

Publisher Summary Microfabricated analytical devices are referred to as “lab-on-a-chip systems” a... more Publisher Summary Microfabricated analytical devices are referred to as “lab-on-a-chip systems” and include microseparation units, miniaturized reactors, and microarrays. This chapter reviews recent developments and trends in microfluidic analyses of biological interest, including DNA, proteins, and complex carbohydrate analysis and high-throughput screening. The microfabrication methods comprise photolithography in rigid materials and fabrication in plastics and elastomers. Glass substrates are common because of their good optical properties and well-developed microfabrication technology and surface chemistry. For miniaturized analysis systems, sample handling and manipulation are of great importance. Specific problems resulting from the shrinking of macroscopic systems include the failure of samples to be representative and of manipulations to be reproducible. This makes it necessary to incorporate specific microscale techniques and components in such devices. The analytical performances of microfluidic devices are drastically affected by dead volumes in the system and also by the surface properties of the fluidic channels.

Research paper thumbnail of An Ultrafast Method to Evaluate Rna Quality

Research paper thumbnail of Monolithic photonics-microfluidics integration for micrototal analysis systems

We demonstrate the monolithic integration of waveguides and microfluidic channels using same mate... more We demonstrate the monolithic integration of waveguides and microfluidic channels using same material, PDMS (polydimethylsiloxane). We also developed two different schemes of integration. Both of them show precise alignment and robust structure. These techniques hold great promise of expanding functionality of micrototal analysis systems with photonic integrated circuits.

Research paper thumbnail of High Precision Micropreparative Separation System Based on Plastc Microfluidic Module — Capillary Coupling

Micro Total Analysis Systems 2002, 2002

We report on a new approach for high precision fraction collection of electrophoretically separat... more We report on a new approach for high precision fraction collection of electrophoretically separated DNA molecules. The fraction collection system is based on a microfluidic cross-connector module coupled to four fused silica capillaries. The cross-connector was fabricated of an acrylic substrate by microdrilling flat end and through channels to ensure close to zero dead volume connections with capillary tubing. DNA fragments were size separated and physically isolated by appropriate electrokinetic manipulations. Selected portions of analyte were redirected to the corresponding collection reservoirs, while maintaining the rest of the sample virtually stopped or slowly reversed within the separation capillary. In this way, the spacing between consecutive bands of interest can be increased enabling high precision isolation of spatially close analyte zones. The separation/collection cycle was repeated until all desired sample zones are captured. The amounts of the collected DNA fractions were enough for further downstream sample processing, such as PCR amplification and analysis.

Research paper thumbnail of High-throughput genotyping by microchip electrophoresis

Journal of capillary electrophoresis and microchip technology

Easy applicability of modern microfabrication technology to electrophoresis microchips has initia... more 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.

Research paper thumbnail of UV/ozone modification of poly(dimethylsiloxane) microfluidic channels

Sensors and Actuators B: Chemical, 2004

UV/ozone method of oxidation of poly(dimethylsiloxane) (PDMS) was investigated to enhance microfl... more UV/ozone method of oxidation of poly(dimethylsiloxane) (PDMS) was investigated to enhance microfluidic device performance, i.e., increase electroosmotic flow (EOF) stability, minimize adsorption of analytes in microchannels, etc. PDMS microfluidic devices are easily fabricated at low cost, but high hydrophobicity and poor surface properties make this material inferior to glass or quartz in most high-performance microfluidic applications. PDMS can be converted into a silica-like material by UV/ozone treatment enabling much deeper oxidation of PDMS without crack failures compared to oxygen plasma treatment. In this study application of UV/ozone treatment to fabrication of microfluidic devices has been presented. Specifically, conversion of bulk PDMS by deep penetration and complete oxidation of thick-film membranes by UV/ozone were investigated. Changes of physical properties upon PDMS modification were monitored by FTIR spectroscopy, contact angle measurements, and channel cross-section visualization. Two microfabrication approaches were attempted and compared: microchannels molded in bulk PDMS were oxidized at the surface and sealed, as well as microchannels molded in a 15 m PDMS membrane followed by sealing against glass and complete UV/ozone oxidation. Improved wettability, enhanced electroosmotic flow, and reduced adsorption properties were observed in the modified PDMS channels.

Research paper thumbnail of Micromachined capillary cross-connector for high-precision fraction collection

Journal of Chromatography A, 2002

A new approach for high-precision fraction collection of double-stranded DNA fragments by capilla... more 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.

Research paper thumbnail of Differential gene expression analysis by micro-preparative capillary gel electrophoresis

Journal of Chromatography A, 2003

Differential display analysis by cDNA fractionation, collection of differentially expressed fract... more 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.

Research paper thumbnail of Bioanalysis in microfluidic devices

Journal of Chromatography A, 2002

Microfabricated bioanalytical devices (also referred to as laboratory-on-a-chip or micro-TAS) off... more 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.

Research paper thumbnail of DNA profiling by capillary array electrophoresis with non-covalent fluorescent labeling

Journal of Chromatography A, 2004

Increasing need for large-scale DNA profiling necessitated the development of automated electroph... more Increasing need for large-scale DNA profiling necessitated the development of automated electrophoresis based methods enabling rapid, high performance analysis of nucleic acids in a wide molecular-mass range. In this paper, we report on the adaptation of a commercial 96-capillary array electrophoresis (CAE) instrument for high-throughput DNA fragment analysis and the evaluation of the effects of different non-covalent DNA staining dyes on separation efficiency. The applicability of different color internal fluorescent standards is shown with mathematical spectral overlap correction algorithms. Large-scale quality control assessment of oligonucleotide probes using non-covalent fluorophore labeling is also demonstrated. The method requires small sample amounts, offers automation and quantification capabilities to accommodate modern biotechnology industry needs.

Research paper thumbnail of Micropreparative capillary gel electrophoresis of DNA: Rapid expressed sequence tag library construction

ELECTROPHORESIS, 2003

Micropreparative capillary gel electrophoresis of DNA: Rapid expressed sequence tag library const... more 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.

Research paper thumbnail of Analysis of high‐mannose‐type oligosaccharides by microliquid chromatography‐mass spectrometry and capillary electrophoresis

ELECTROPHORESIS, 2004

Analysis of high-mannose-type oligosaccharides by microliquid chromatography-mass spectrometry an... more Analysis of high-mannose-type oligosaccharides by microliquid chromatography-mass spectrometry and capillary electrophoresis We report on microbore liquid chromatography (mLC) and capillary electrophoresis (CE) separation of glycopeptides and high-mannose-type oligosaccharides, digested from recombinant phospholipase C, expressed in Pichia pastoris. The glycopeptides were subject to mLC/electrospray ionization/mass spectrometry (ESI-MS) and mLC/ESI-tandem MS (MS/MS) analysis that revealed high-mannose structure size variation between Man 7 GlcNAc 2 and Man 14 GlcNAc 2. Then, high-performance CE was applied to identify possible positional isomers of the high-mannose structures. For the CE experiments, the oligosaccharides were released from the glycoproteins by peptide-Nglycosidase F and labeled with 1-aminopyrene-3,6,8-trisulfonic acid (APTS). Excellent separation of the possible positional isomers was attained, suggesting one for Man 9-GlcNAc 2 , two for Man 10 GlcNAc 2 , three for Man 11 GlcNAc 2 , Man 12 GlcNAc 2 , and Man 13-GlcNAc 2 , and two for Man 14 GlcNAc 2. The CE results provided complementary information to the mLC/ESI-MS and MS/MS data with respect to the possible number of positional isomers.

Research paper thumbnail of Transcription factor binding study by capillary zone electrophoretic mobility shift assay

ELECTROPHORESIS, 2003

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

Research paper thumbnail of Large-scale carbohydrate analysis by capillary array electrophoresis: Part 1. Separation and scale-up

ELECTROPHORESIS, 2004

Large-scale carbohydrate analysis by capillary array electrophoresis: Part 1. Separation and scal... more Large-scale carbohydrate analysis by capillary array electrophoresis: Part 1. Separation and scale-up A 96-capillary array electrophoresis (CAE) instrument has been adapted for large-scale mono-and oligosaccharide analysis and characterization. Operational protocols and data processing tools have been developed to optimize the CAE system for this application. Effects of different additives to the running buffer on efficiency and capillary-to-capillary performance reproducibility have been studied.

Research paper thumbnail of Automated carbohydrate profiling by capillary electrophoresis: A bioindustrial approach

ELECTROPHORESIS, 2004

Automated, high-resolution, quantitative, high-throughput analysis of mono-and oligosaccharides, ... more Automated, high-resolution, quantitative, high-throughput analysis of mono-and oligosaccharides, produced by enzymatic digestion of cellohexaose (model substrate) and lignocellulosic biomass, is demonstrated using high-performance capillary electrophoresis in conjunction with a single-step fluorophore labeling strategy for sensitive laser-induced fluorescence detection. Unattended batch sample processing from 96-well plates enabled reliable industrial-scale carbohydrate analysis. Excellent resolution of mono-and oligosaccharides was achieved under suppressed electroosmotic flow conditions, using either covalently or dynamically coated fused-silica capillary columns. The proposed approach readily supports the demands of bioindustrial operation environments with respect to high-throughput carbohydrate profiling.