Monitoring γ-aminobutyric acid in human brain and plasma microdialysates using micellar electrokinetic chromatography and laser-induced fluorescence detection

Due to its low electrophoretic mobility, few studies have been able to measure gamma aminobutyric acid (GABA) in biological samples by means of capillary zone electrophoresis. Nevertheless, in micellar electrokinetic chromatography (MEKC) by adding a surfactant to the mobile phase separation can be carried out on the basis of the partition coefficient of the molecules rather than their electrophoretic mobility. In the present study microdialysis coupled to MEKC with laser induced fluorescence detection was used to successfully monitor GABA from cerebrospinal fluid and plasma dialysates. Moreover, we monitored changes in extracellular GABA from a human brain. Microdialysis samples were collected from a Parkinson’s disease patient undergoing a thallamotomy as part of her treatment. Significant decreases in extracellular GABA were detected during high frequency electrical stimulation and following a thermolesion of the thalamus. These results demonstrate the feasibility of MEKC coupled to laser-induced fluorescence detection in resolving neutral amino acids, specifically GABA, from different human body fluids.     

Fluorographic detection of [3H]thymidine-labeled deoxyribonucleic acid using agarose gels infused with 2,5-diphenyloxazole prior to electrophoresis

A fluorographic procedure for the detection of [3H]thymidine-labeled deoxyribonucleic acids electrophoresed in agarose gels was developed. 2,5-Diphenyloxazole (PPO) was added to the agarose solution before pouring of the gel for electrophoresis. This procedure did not interfere with the electrophoretic mobility of the DNA molecules. The radioactive detection efficiency was found to be improved over an existing procedure whereby the agarose gel was infused with PPO after electrophoresis with the aid of acetic acid.     

The influence of fluorescent dye structure on the electrophoretic mobility of end-labeled DNA.

Over the past 10 years, fluorescent end-labeling of DNA fragments has evolved into the preferred method of DNA detection for a wide variety of applications, including DNA sequencing and PCR fragment analysis. One of the advantages inherent in fluorescent detection methods is the ability to perform multi-color analyses. Unfortunately, labeling DNA fragments with different fluorescent tags generally induces disparate relative electrophoretic mobilities for the fragments. Mobility-shift corrections must therefore be applied to the electrophoretic data to compensate for these effects. These corrections may lead to increased errors in the estimation of DNA fragment sizes and reduced confidence in DNA sequence information. Here, we present a systematic study of the relationship between dye structure and the resultant electrophoretic mobility of end-labeled DNA fragments. We have used a cyanine dye family as a paradigm and high-resolution capillary array electrophoresis (CAE) as the instrumentation platform. Our goals are to develop a general understanding of the effects of dyes on DNA electrophoretic mobility and to synthesize a family of DNA end-labels that impart identically matched mobility influences on DNA fragments. Such matched sets could be used in DNA sequencing and fragment sizing applications on capillary electrophoresis instrumentation.     

Ultrasensitive protein-DNA binding assays

Protein-DNA binding assays have been used in a variety of fields from fundamental studies regarding the binding process itself, to serving as probes for the detection, quantification and separation of target analytes. These assays have been used for the study of protein-DNA complex stoichiometry, the detection of DNA damage, and real-time separation of free and bound complexes by electrophoretic mobility. Synthetic DNA oligonucleotides, known as aptamers, have been increasingly used for affinity binding assays to proteins, as well as for separation studies and as biosensors. Recent advances have been made in protein-DNA binding assays using capillary electrophoresis, laser-induced fluorescence, fluorescence polarization, molecular beacons, and affinity chromatography.     

A capillary electrophoretic method for monitoring the presence of alpha-tubulin in nuclear preparations

A sensitive capillary electrophoretic method was developed to detect the presence of alpha-tubulin, a microtubular cytoskeletal component, in isolated nuclear preparations. These preparations are treated with anti-alpha-tubulin primary mouse antibodies and then stained with a fluorescently labeled anti-mouse IgG antibody. The stained preparation is then analyzed by capillary electrophoresis with laser-induced fluorescence detection, a technique that allows for sensitive detection of fluorescently labeled species. Using this method, it is feasible to count individual subcellular aggregates containing alpha-tubulin (SATs), estimate the number of alpha-tubulin molecules per SAT, determine the cumulative intensity of all SATs as an estimate of the relative level of alpha-tubulin in a preparation, and obtain their apparent electrophoretic mobility distribution. The method was validated by comparing SATs from untreated cells with those from colchicine-treated cells. Since colchicine is a microtubule-disrupting agent, treatment reduced the number of SATs per cell as well as the cumulative intensity of all SATs in a preparation. In contrast, the apparent electrophoretic mobility distribution was not influenced by colchicine treatment, suggesting that this parameter is not strongly dependent on the alpha-tubulin content. Given the zeptomolar sensitivity of laser-induced fluorescence detection and the widespread availability of antibodies, the approach used here represents an improvement in the detection of cytoskeletal impurities in subcellular fractions.     

Combinatorial fluorescence energy transfer tags for multiplex biological assays

We report an approach for developing combinatorial fluorescence energy transfer (CFET) tags by tuning the tags' fluorescence emission signatures. The tags can all be excited at a single wavelength and analyzed by a simple optical system. We constructed eight CFET tags with unique fluorescence signatures, detected by a three-color capillary array electrophoresis (CAE) system with 488 nm excitation, using only three fluorescent dyes. A 1',2'-dideoxyribose phosphate spacer was used to separate the donor and acceptor to tune the energy transfer efficiency, generating unique fluorescence signatures. The spacer also served as an electrophoretic mobility tag to tune the mobility of CFET-labeled DNA for multiplex detection of single-nucleotide polymorphisms (SNPs). Six nucleotide variations were identified simultaneously using six CFET tags on synthetic DNA templates and on a PCR product from the retinoblastoma tumor suppressor gene.     

Rapid separation of DNA molecules by agarose gel electrophoresis: use of a new agarose matrix and a survey of running buffer effects.

This report describes the use of a new type of agarose (FastLane agarose) for faster separation of DNA by agarose gel electrophoresis. DNA molecules separated in this agarose exhibited electrophoretic mobilities up to 30% higher than similar separations in standard analytical grade agarose. DNA molecules of all sizes examined showed higher mobilities in FastLane agarose. The mobility increase was predominantly due to the low electroendosmosis of FastLane agarose and was most pronounced in pulsed field gel electrophoresis separations. The magnitude of mobility increase varied depending on the conditions used for electrophoresis.     

Gene expression analysis using single molecule detection

Recent developments of single molecule detection techniques and in particular the introduction of fluorescence correlation spectroscopy (FCS) led to a number of important applications in biological research. We present a unique approach for the gene expression analysis using dual-color cross-correlation. The expression assay is based on gene-specific hybridization of two dye-labeled DNA probes to a selected target gene. The counting of the dual-labeled molecules within the solution allows the quantification of the expressed gene copies in absolute numbers. As detection and analysis by FCS can be performed at the level of single molecules, there is no need for any type of amplification. We describe the gene expression assay and present data demonstrating the capacity of this novel technology. In order to prove the gene specificity, we performed experiments with gene-depleted total cDNA. The biological application was demonstrated by quantifying selected high, medium and low abundant genes in cDNA prepared from HL-60 cells.     

Label-free determination of the number of biomolecules attached to cells by measurement of the cell's electrophoretic mobility in a microchannel

We developed a label-free method for a determination of the number of biomolecules attached to individual cells by measuring the electrophoretic mobility of the cells in a microchannel. The surface of a biological cell, which is dispersed in aqueous solution, is normally electrically charged and the charge quantity at the cell's surface is slightly changed once antibody molecules are attached to the cell, based on which we detect the attachment of antibody molecules to the surface of individual red blood cells by electrophoretic mobility measurement. We also analyzed the number of antibody molecules attached to the cell's surface using a flow cytometer. We found that there is a clear correlation between the number of antibody molecules attached to the individual cells and the electrophoretic mobility of the cells. The present technique may well be utilized not only in the field of cell biology but also in the medical and pharmaceutical industries.     

Double bands in DNA gel electrophoresis caused by bis-intercalating dyes.

Many bis-intercalating dyes used for fluorescence detection of DNA in electrophoresis have been reported to give band-splitting and band-broadening, which results in poor resolution and a decreased detection sensitivity. We have studied the dimeric dye YOYO-1, and to some extent also TOTO-1 and EthD-1, and found that in complex with DNA these dyes give rise to two components with different electrophoretic mobilities. Electrophoresis experiments and spectroscopic measurements on the two components show that they differ in that the DNA molecules have different amounts of dye bound. Our results exclude that the extra bands are caused by intermolecular cross-linking. Incubation of the samples for increasing times before electrophoresis makes the bands move closer and closer to each other as the dye molecules become more homogeneously distributed among the DNA molecules. Finally, the two bands merge into one at an intermediate position. This equilibration process is extremely slow at room temperature (days), and is therefore not a practical method to eliminate band-splitting in routine analysis. However, we find that if the temperature is raised to 50 degrees C, the dye-DNA complexes equilibrate completely in only 2 h.     

Methodologies for monitoring nanoparticle formation by self-assembly of DNA with poly(l-lysine)

DNA self-assembly with polycations produces nanoparticles suitable for gene delivery, although there is no standard methodology to measure particle formation and stability. Here we have compared three commonly used assays, namely, light scattering, inhibition of ethidium bromide fluorescence, and modified electrophoretic mobility of DNA. Analysis by light scattering and loss of ethidium bromide fluorescence both showed poly(l-lysine) (pLL)/DNA nanoparticles form over the lysine/phosphate ratio range 0.6-1.0, although retardation of DNA electrophoretic mobility commenced at lower lysine/phosphate ratios. This probably indicates that the first two assays monitor DNA collapse into particles, while the electrophoresis assay measures neutralization of the charge on DNA. Gel analysis of the complexes showed disproportionation during nanoparticle formation, probably reflecting cooperative binding of the polycation. The assays were used to examine stability of complexes to dilution in water and physiological salts. Whereas all pLL/DNA nanoparticles were stable to dilution in water, the presence of physiological salts provoked selective disruption of complexes based on low-molecular-weight pLL. Polyelectrolyte complexes for targeted application in vivo should therefore be based on high-molecular-weight polycations, or should be stabilized to prevent their dissociation under physiological salt conditions.     

Electrophoresis and movements of fluorescence pattern after photobleaching of large DNA fragments in agarose gels

By combining electrophoresis with movements of fluorescence pattern after photobleaching (MOFPAP), which is abbreviated as EMOFPAP, we are able to measure electrophoretic mobilities of large DNA fragments in an agarose gel within a fairly short time scale (about 10 min or even down to 1 min). The new method represents a significant improvement in experiment time when compared with the time (typically on the order of hours) required to determine the average electrophoretic mobility of large DNA fragments in agarose gels by means of either conventional gel electrophoresis or pulsed-field gel electrophoresis. In this article, we present the EMOFPAP experimental setup and consider optical conditions, including beam profile geometry and fluorescence pattern formation. A realistic formula that can explain the parameters governing the EMOFPAP method using our present optical setup has been derived. A comparison of results between experimental and computer simulation data is made, and an optimization of the EMOFPAP method is proposed.     

Capillary electrophoresis monitors changes in the electrophoretic behavior of mitochondrial preparations

The presence of electrical charges on the surface of an organelle is the source of the organelle's electrophoretic mobility. Recently, we reported that capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) can be used to determine the electrophoretic mobility of individual mitochondria. Here, we describe the use of CE-LIF to monitor changes in the electrophoretic mobility distributions of: (i). mitochondria isolated from cultured NS-1 mouse hybridoma cells disrupted by nitrogen cavitation or mechanical homogenization; (ii). mitochondria isolated from rat liver and purified by gradient centrifugation before and after being frozen in liquid nitrogen; and (iii). mitochondria chemically transformed into mitoplasts. These results indicate that the organelle electrophoretic mobility observed by researchers is affected by preparation procedures and that CE-LIF is a complementary technique for monitoring the quality of mitochondrial preparations.     

Capillary electrophoresis of DNA in uncrosslinked polymer solutions: evidence for a new mechanism of DNA separation

The electrophoretic separation of DNA molecules is usually performed in thin slabs of agarose or polyacrylamide gel. However, DNA separations can be achieved more rapidly and efficiently within a microbore fused silica capillary filled with an uncrosslinked polymer solution. An early assumption was that the mechanism of DNA separation in polymer solution(SINGLEBOND)capillary electrophoresis (PS(SINGLEBOND)CE) is the same as that postulated to occur in slab gel electrophoresis, i.e., that entangled polymer chains form a network of "pores" through which the DNA migrates. However, we have demonstrated that large DNA restriction fragments (2.0(SINGLEBOND)23.1 kbp) can be separated by CE in extremely dilute polymer solutions, which contain as little as 6 parts per million [0.0006% (w/w)] of uncrosslinked hydroxyethyl cellulose (HEC) polymers. In such extremely dilute HEC solutions, far below the measured polymer entanglement threshold concentration, pore-based models of DNA electrophoresis do not apply. We propose a transient entanglement coupling mechanism for the electrophoretic separation of DNA in uncrosslinked polymer solutions, which is based on physical polymer/DNA interactions. (c) 1996 John Wiley & Sons, Inc.     

Capillary electrophoresis for the study of affinity interactions

Molecular recognition may be characterized both qualitatively and quantitatively by electrophoretic methods if complexed molecules differ in electrophoretic mobility from unbound ones. The use of capillary zone electrophoresis (CE) for the characterization of affinity interactions is advantageous because of the high resolution, reproducibility and wide applicability of the technique and because of the mild conditions, i.e., physiological buffers without additions of organics or detergents, that are often sufficient for highly efficient separations. CE gives the ability to characterize binding between small amounts of unlabelled reactants in solution, has few requirements for special characteristics of the interacting molecules and is also applicable to the study of interactions of individual components in mixtures, as detection of binding and analytical separation are achieved in one step. This is unique compared with other techniques for the study of non-covalent interactions. The advantages and disadvantages of using CE to demonstrate molecular interactions, to screen for specific ligand binding in complex mixtures and to calculate binding constants will be discussed.     

Fragment screening using capillary electrophoresis (CEfrag) for hit identification of heat shock protein 90 ATPase inhibitors

CEfrag is a new fragment screening technology based on affinity capillary electrophoresis (ACE). Here we report on the development of a mobility shift competition assay using full-length human heat shock protein 90α (Hsp90α), radicicol as the competitor probe ligand, and successful screening of the Selcia fragment library. The CEfrag assay was able to detect weaker affinity (IC(50) >500 μM) fragments than were detected by a fluorescence polarization competition assay using FITC-labeled geldanamycin. The binding site of selected fragments was determined by co-crystallization with recombinant Hsp90α N-terminal domain and X-ray analysis. The results of this study confirm that CEfrag is a sensitive microscale technique enabling detection of fragments binding to the biological target in near-physiological solution.     

On-line melting of double-stranded DNA for analysis of single-stranded DNA using capillary electrophoresis

Capillary electrophoresis (CE) is a convenient, fast and non-radioactive method with possibilities for automatization. To analyse single-stranded DNA molecules in a more automated way, we developed a heating device to melt double-stranded DNA fragments in the capillary during electrophoresis. In this study we used this device to obtain single-stranded DNA, necessary for the detection of point mutations in DNA using the single-strand conformation polymorphism technique. Results show that double-stranded DNA molecules can be melted on-line into single-stranded DNA molecules, although not for 100%. In an attempt to find universal electrophoretic conditions for the analysis of single-stranded DNA, we investigated the influence of several parameters on the yield of single-stranded DNA molecules and on the resolution of the single-stranded DNA peaks. We demonstrate that this heating device is a technical adjustment of CE which contributes to more automated analyses of DNA fragments.     

Single nucleotide polymorphism detection by combinatorial fluorescence energy transfer tags and biotinylated dideoxynucleotides

Combinatorial fluorescence energy transfer (CFET) tags, constructed by exploiting energy transfer and combinatorial synthesis, allow multiple biological targets to be analyzed simultaneously. We here describe a multiplex single nucleotide polymorphism (SNP) assay based on single base extension (SBE) using CFET tags and biotinylated dideoxynucleotides (biotin-ddNTPs). A library of CFET-labeled oligonucleotide primers was mixed with biotin-ddNTPs, DNA polymerase and the DNA templates containing the SNPs in a single tube. The nucleotide at the 3′-end of each CFET-labeled oligonucleotide primer was complementary to a particular SNP in the template. Only the CFET-labeled primer that is fully complementary to the DNA template was extended by DNA polymerase with a biotin-ddNTP. We isolated the DNA extension fragments that carry a biotin at the 3′-end by capture with streptavidin-coated magnetic beads, while the unextended primers were eliminated. The biotinylated fluorescent DNA fragments were subsequently analyzed in a multicolor fluorescence electrophoresis system. The distinct fluorescence signature and electrophoretic mobility of each DNA extension product in the electropherogram coded the SNPs without the use of a sizing standard. We simultaneously distinguished six nucleotide variations in synthetic DNA templates and a PCR product from the retinoblastoma tumor suppressor gene. The use of CFET-labeled primers and biotin-ddNTPs coupled with the specificity of DNA polymerase in SBE offered a multiplex method for detecting SNPs.