Automated DNA mutation analysis by single-strand conformation polymorphism using capillary electrophoresis with laser-induced fluorescence detection

Automation is essential for rapid genetic-based mutation analysis in clinical laboratory to screen a large number of DNA samples. We propose in this report an automatic process using Beckman Coulter P/ACE™ capillary electrophoresis (CE) with laser-induced fluorescence (LIF) system to detect a single-point mutation in the codon 12 of human K-ras gene. Polymerase chain reaction (PCR) using a fluorescently labeled reverse primer and a plain forward primer to specifically amplify a selected 50 bp DNA fragment in human K-ras gene. The amplified DNA is placed on the sample tray of the CE system with a pre-programmed step for single-strand conformation polymorphism (SSCP) analysis. Sample injection and denaturation processes are performed online along with separation and real-time data analysis. The concept of automation for rapid DNA mutation analysis using CE-LIF system for SSCP is presented.     

Noncovalent labeling of proteins in capillary electrophoresis with laser-induced fluorescence detection

Interest in the use of capillary electrophoresis (CE) as a tool for protein separations continues to grow. Additionally, laser-induced fluorescence (LIF) detection schemes promise ultrasensitive detection of small quantities of these important biomolecules following their separation. In most cases, LIF detection of proteins necessitates their prior derivatization with a fluorescent label molecule. To minimize the amount of additional sample handling and time associated with such labeling procedures, not to mention the sometimes-stringent pH and temperature controls they require, noncovalent labeling is presented as a viable alternative. This review article considers established methods for noncovalent labeling of proteins for their subsequent analysis by CE-LIF. Label molecules suitable for excitation and emission in the ultraviolet, visible, and near-infrared regions of the spectrum are enumerated for a variety of protein analytes.     

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.     

Direct quantification of gene expression using capillary electrophoresis with laser-induced fluorescence

Quantification of gene expression provides valuable information regarding the response of cells or tissue to stimuli and often is accomplished by monitoring the level of messenger RNA (mRNA) being transcribed for a particular protein. Although numerous methods are commonly used to monitor gene expression, including Northern blotting, real-time polymerase chain reaction, and RNase protection assay, each method has its own drawbacks and limitations. Capillary electrophoresis with laser-induced fluorescence (CE-LIF) can reduce protocol time, eliminate the need for radioactivity, and provide superior sensitivity and dynamic range for quantification of RNA. In addition, CE-LIF can be used to directly determine the amount of an RNA species present, something that is difficult and not normally accomplished using current methods. Gene expression is detected using a fluorescently labeled riboprobe specific for a given RNA species. This direct approach was validated by analyzing levels of 28S RNA and also used to determine the amount of discoidin domain receptor 2 mRNA in cardiac tissue.     

Separation and determination of peptide hormones by capillary electrophoresis with laser-induced fluorescence coupled with transient pseudo-isotachophoresis preconcentration

A new method for the determination of the peptide hormones and their fragments by capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection and transient pseudo-isotachophoresis (pseudo-tITP) preconcentration was established in this study. The LIF detector used an argon ion laser with excitation wavelength at 488 nm and emission wavelength at 535 nm. Fluorescein isothiocyanate (FITC) was used as precolumn derivatization reagent to label cholecystokinin tetrapeptide (CCK-4), neurotensin (NT), neurotensin hexapeptide (NT_8–13), and neurokinin B (NKB). Borate (10 mmol/L, pH 9.0) was selected as derivatization medium to get the high efficiency. When the addition of 70% (v/v) methanol and 1% (m/v) sodium chloride (NaCl) to the sample matrix, and with borate buffer (110 mM, pH 9.5) and 20% (v/v) methanol as running buffer, a preconcentration based on the pseudo-tITP afforded 100-fold improvement in peak heights compared with the traditional hydrodynamic injection (2.3% capillary volume). The detection limits (signal/noise = 3) based on peak height were found to be 0.04, 0.1, 0.2, and 0.08 nmol/L for NT_8–13, NT, NKB, and CCK-4, respectively. The method was validated and applied to qualitative analysis of NT and NT_8–13 in human cerebrospinal fluid sample.     

The use of capillary electrophoresis for DNA polymorphism analysis

Capillary electrophoresis has advanced enormously over the last 10 yr as a tool for DNA sequencing, driven by the human and other major genome projects and by the need for rapid electrophoresis-based DNA diagnostic tests. The common need of these analyses is a platform providing very high throughput, high-quality data, and low process costs. These demands have led to capillary electrophoresis machines with multiple capillaries providing highly parallel analyses, to new electrophoresis matrices, to highly sensitive spectrofluorometers, and to brighter, spectrally distinct fluorescent dyes with which to label DNA. Capillary devices have also been engineered onto microchip formats, on which both the amount of sample required for analysis and the speed of analysis are increased by an order of magnitude. This review examines the advances made in capillary and chip-based microdevices and in the different DNA-based assays developed for mutation detection and genotype analysis using capillary electrophoresis. The automation of attendant processes such as for DNA sample preparation, PCR, and analyte purification are also reviewed. Together, these technological developments provide the throughput demanded by the large genome-sequencing projects.     

Methylation-dependent fragment separation: direct detection of DNA methylation by capillary electrophoresis of PCR products from bisulfite-converted genomic DNA

Fundamental to understanding the role of cytosine (C) methylation in genomic DNA (gDNA) is the need for robust analysis methods to determine the location and degree of this modification. We report a novel method for methylation detection by denaturing capillary electrophoresis (CE) using standard fragment analysis conditions. Bisulfite treatment of gDNA will selectively deaminate C but not 5-methylcytosine (5mC). Amplicons generated from bisulfite-converted gDNA are analyzed immediately after PCR using a 6-carboxy fluorescein (6-FAM) dye-labeled primer. The amplicons from methylated and unmethylated gDNA separate based solely on base composition due to the presence of multiple C versus thymine (T) differences. By direct detection of PCR amplicons following PCR using primers that anneal independent of methylation status, the overall workflow from gDNA sample input to data analysis is relatively simple. Furthermore, the same PCR product is suitable for additional analyses such as direct sequencing, cloning and sequencing, single-base extension, and post-PCR incorporation of a modified dCTP, the latter of which allows resolution of amplicons with as little as a single C/T difference. We show the utility of this novel CE detection assay by analyzing the hypermethylated region of the fragile-X FMR1 locus.     

Measuring DNA damage using capillary electrophoresis with laser-induced fluorescence detection

Damage to cellular DNA is implicated in the early stages of carcinogenesis and in the cytotoxicity of many anticancer agents, including ionizing radiation. Sensitive techniques are required for measuring cellular levels of DNA damage. We describe in detail a novel immunoassay that makes use of the resolving power of capillary electrophoresis and the sensitivity of laser-induced fluorescence detection. An example is given of the detection of thymine glycol in DNA produced by irradiation of human cells with a clinical dose of 2 Gy. A detection limit of approximately 10(-21) mol allowed us to monitor the repair of the lesion and to suggest that the cellular repair response may be inducible.     

Electrochemical profiles of Herba Saussureae Involucratae by capillary electrophoresis with electrochemical detection

A high-performance capillary electrophoresis with electrochemical detection method has been developed for the determination of the pharmacologically active ingredients, acacetin, rutin, umbelliferone, kaempferol, apigenin, luteolin and quercetin, in Herba Saussureae Involucratae. Under optimum conditions, the seven analytes could be completely separated within 19 min in a 75 cm length capillary at a separation voltage of 16 kV in a 50 mM borax running buffer (pH 9.2). A 300 microm diameter carbon disk electrode, positioned opposite the outlet of the capillary in a wall-jet configuration at a potential of +950 mV (vs a saturated calomel electrode) was used as the working electrode. A good linear relationship was established between peak current and concentration of the analytes over two orders of magnitude with detection limits (signal-to-noise ratio = 3) ranging from 1.2 x 10(-7) to 4.1 x 10(-8) g/mL for all analytes. The proposed method has been successfully applied to the analyses of bio-active components of Herba Saussureae Involucratae samples after a relatively simple extraction procedure. The assay results show that the resultant electrochemical profiles are indicative of the content diversity of each electrochemically active ingredient in the various samples, and may also offer some evidence for phytotaxonomy.     

Applications of capillary isoelectric focusing with liquid-core waveguide laser-induced fluorescence whole-column imaging detection

Capillary isoelectric focusing (CIEF) with liquid-core waveguide (LCW) laser-induced fluorescence (LIF) whole-column imaging detection (WCID) is a recently developed high-resolution, high-sensitivity, and high-speed analytical tool for protein analysis. Several potential applications of this system were demonstrated in this study. First, this system was employed to separate naturally fluorescent phycobiliproteins. Second, denaturing CIEF was suggested to study the conformational and chemical microheterogeneity and to characterize proteins with identical pI values. Third, a modified noncovalent fluorescent labeling procedure was presented, which allows the simple and effective labeling of proteins, antibodies, and viruses with reduced multiple labeling and preserved activity. Finally, extracellular proteins were suggested as signaling biomarkers for evaluation of cell viability. The separation of cyanobacteria and their extracellular phycoerythrins was demonstrated. The effectiveness of CIEF-LCW-LIF-WCID for the analysis of proteins, antibodies, viruses, and cells has been illustrated.     

Determination of octopamine in human plasma by capillary electrophoresis with optical fiber light-emitting diode-induced fluorescence detection

A new analytical method based on capillary electrophoresis (CE) separation and optical fiber light-emitting diode (LED)-induced fluorescence detection has been developed for the determination of octopamine. Naphthalene-2,3-dicarboxaldehyde (NDA) was used for precolumn derivatization of octopamine. The separation and determination of the derivative was performed using a laboratory-built CE system with an optical fiber LED-induced fluorescence detector. Optimal separation was obtained at 20 kV using a background electrolyte solution consisting of 25 mM sodium borate (pH 9.2). High sensitivity detection was achieved by the optical fiber LED-induced fluorescence detection using a purple LED as the excitation source. The limit of detection (signal/noise=3) for octopamine was 5.0 x 10(-9)M. A calibration curve ranging from 1.0 x 10(-8) to 5.0 x 10(-7)M was shown to be linear. Using this method, the levels of octopamine in human plasma from healthy donors were determined.     

Determination of vigabatrin by capillary electrophoresis with laser-induced fluorescence detection

A new analytical method for vigabatrin based on capillary electrophoretic separation and laser-induced fluorescence detection has been developed. 5-Carboxytetramethylrhodamine succinimidyl ester was used for precolumn derivatization of the non-fluorescent drug. Optimal separation and detection were obtained with an electrophoretic buffer of 50 mM sodium borate (pH9.5) containing 10 mM sodium dodecyl sulfate and a green He-Ne laser (excitation at 543.5 nm, emission at 589 nm). The concentration limit of detection in aqueous solution was 24 nM. Combined with a simple cleanup procedure, this method can be applied to the determination of vigabatrin in human plasma. A calibration curve ranging from 1.5 to 200 microM shown to be linear. Both the within-day and day-to-day reproducibilities and accuracies were less then 14.3% and 4.9% respectively. The limit of detection of vigabatrin in plasma was about 0.13 microM     

Measurement of chloramphenicol by capillary zone electrophoresis following end-column amperometric detection at a carbon fiber micro-disk array electrode

Capillary zone electrophoresis was employed for the measurement of chloramphenicol using end-column amperometric detection with a carbon fiber micro-disk array electrode, at a constant potential of −1.00 V vs. saturated calomel electrode. The effect of oxygen in the buffer has been investigated. It is found that when the area of the carbon fiber electrode is smaller than 1.1 mm², the interference of oxygen can be overcome. In this procedure deoxygenation is not necessary. The effect of pH, the concentration of the buffer and the high separation voltage across the capillary on the migration time, electrophoretic peak current and separation efficiency has been studied. The optimum conditions of separation and detection are 8.4×10⁻⁴ mol/l HOAc–3.2×10⁻³ mol/l NaOAc for the buffer solution, 20 kV for the separation voltage, 5 kV and 5 s for the injection voltage and the injection time, respectively. The calibration plot was found to be linear in the range 5×10⁻⁶ to 1×10⁻³ mol/l and the limit of detection is 9.1×10⁻⁷ mol/l or 1.4 fmol (S/N=2). The relative standard deviation is 1.1% for the migration time and 2.3% for the electrophoretic peak current. The method was applied to the determination of chloramphenicol in human serum.     

Determination of uric acid in human urine and serum by capillary electrophoresis with chemiluminescence detection

A simple and sensitive method based on capillary electrophoresis (CE) with chemiluminescence (CL) detection has been developed for the determination of uric acid (UA). The sensitive detection was based on the enhancement effect of UA on the CL reaction between luminol and potassium ferricyanide (K₃[Fe(CN)₆]) in alkaline solution. A laboratory-built reaction flow cell and a photon counter were deployed for the CL detection. Experimental conditions for CL detection were studied in detail to achieve a maximum assay sensitivity. Optimal conditions were found to be 1.0 × 10⁻⁴ M luminol added to the CE running buffer and 1.0 × 10⁻⁴ M K₃[Fe(CN)₆] in 0.2 M NaOH solution introduced postcolumn. The proposed CE-CL assay showed good repeatability (relative standard deviation [RSD] = 3.5%, n = 11) and a detection limit of 3.5 × 10⁻⁷ M UA (signal/noise ratio [S/N] = 3). A linear calibration curve ranging from 6.0 × 10⁻⁷ to 3.0 × 10⁻⁵ M UA was obtained. The method was evaluated by quantifying UA in human urine and serum samples with satisfactory assay results.     

Steroid analysis in single cells by capillary electrophoresis with collinear laser-induced fluorescence detection.

Capillary electrophoresis with collinear laser-induced fluorescence detection was used for the analysis of steroids in single R2C cells. Progesterone secretion was monitored from cultured cells and subsequently detected in single cells. Mass detection limit of 10(-18) mol for dansylated steroids was achieved with the 325-nm line of a helium-cadmium laser. Dansylhydrazine proved to be an effective fluorescent tag for derivatization of steroids outside and inside the biological cell. Fluorescence microscopy indicates that a dimethyl sulfoxide-containing physiological buffer was sufficient to incorporate the tag inside the cell for subsequent steroid derivatization.     

Determination of LSD in blood by capillary electrophoresis with laser-induced fluorescence detection

Capillary electrophoresis (CE) with HeCd laser-induced fluorescence (LIF) detection and its application in forensic toxicology is demonstrated by the determination of

Full-size image

-lysergic acid diethylamide (LSD) in blood. Following precipitation of proteins, washing of the evaporated supernatant and extraction, the residue was reconstituted in methanol and injected electrokinetically (10 s, 10 kV). The total analysis time for quantification of LSD was 8 min using a citrate–methanol buffer, pH 4.0. With this buffer system it is possible to separate LSD, nor-LSD, iso-LSD and iso-nor-LSD. Using a specific sample preparation, electrokinetic injection, extended light path (bubble cell) capillaries and especially LIF detection (λ_ex 325 nm, λ_em 435 nm), a limit of detection of 0.1–0.2 ng LSD per ml blood could be obtained. The limit of quantitation was about 0.4–0.5 ng/ml. The quantitative evaluation for LSD was carried out using methylergometrine as internal standard. The precision expressed as coefficient of variation (C.V.) and accuracy of the method were <20% and 86–110%, respectively. The application of the method to human blood samples from two forensic cases and a comparison with radioimmunoassay demonstrated that the results were consistent.     

Cloning of AFLP markers detected by fluorescence capillary electrophoresis

The available methods to isolate specific amplified fragment length polymorphism (AFLP) markers can be used only if markers are detected by radioactive labeling, silver staining, or ethidium bromide staining; these methods are useless if modern and automated genetic analyzers are used to detect AFLP markers by fluorescent labeling. We have developed a method that allows for isolation and cloning of specific AFLP markers obtained with a laser-induced fluorescence capillary electrophoresis system. This procedure has been tested on 5Arabidopsis thaliana polymorphic AFLP markers, and the nucleotide sequences obtained from these cloned markers were identified and located in theArabidopsis genome.     

Analysis of single nucleotide incorporation reactions by capillary electrophoresis

Single nucleotide incorporation assays have been used to probe the kinetic parameters of many DNA and RNA polymerases. Traditionally, oligonucleotide primers are 5'-(32)P labeled using T4 kinase and annealed to a complementary template with a 5' overhang. To quantify the reaction kinetics, the products of the primer extension reactions are usually separated using denaturing polyacrylamide gel electrophoresis and quantified using a phosphorimager or other method to measure radioactivity. We have developed a method using a 5' fluorescently labeled oligonucleotide to examine the kinetics of single nucleotide incorporation catalyzed by recombinant human mitochondrial polymerase gamma (Pol gamma) holoenzyme. Using laser-induced fluorescence detection in the P/ACE MDQ instrument, primers 5' labeled with fluorescent probes such as 6-carboxyfluorescein can be rapidly separated and quantified. However, we also show that only select probes can be used, presumably due to unfavorable interactions between Pol gamma and certain 5' labels.     

Self-dissociative primers for nucleic acid amplification and detection based on DNA quadruplexes with intrinsic fluorescence

We have developed a new method, quadruplex priming amplification, to greatly simplify nucleic acid amplification and real-time quantification assays. The method relies on specifically designed guanine-rich primers, which after polymerase elongation are capable of spontaneous dissociation from target sites and forming DNA quadruplex. The quadruplex is characterized by significantly more favorable thermodynamics than the corresponding DNA duplexes. As a result, target sequences are accessible for the next round of priming and DNA amplification proceeds under isothermal conditions with improved product yield. In addition, the quadruplex formation is accompanied by an increase in intrinsic fluorescence of the primers, allowing simple and accurate detection of product DNA.     

Rapid analysis of furosemide in human urine by capillary electrophoresis with laser-induced fluorescence and electrospray ionization-ion trap mass spectrometric detection

Furosemide, a drug that promotes urine excretion, is used in the pharmacotherapy of various diseases and is considered as a doping agent in sports. Using alkaline electrolytes, analysis of furosemide by dodecyl sulfate based micellar electrokinetic capillary chromatography (MECC) and capillary zone electrophoresis (CZE) with laser-induced fluorescence detection (LIF, analyte excitation with the 325 nm line of a HeCd laser) is described. Data produced by injection of plain or diluted patient urines are confirmed with those obtained via analysis of urinary solid-phase extracts. CZE-LIF and MECC-LIF are thereby shown to permit unambiguous recognition of furosemide in urines collected after ingestion of therapeutic doses of this drug. This is in contrast to solute detection via UV absorbance for which the extraction of furosemide is required. MECC based electropherograms are somewhat more complex compared to those obtained by CZE-LIF, this suggesting that the latter approach is more suitable for rapid screening of urines with direct sample injection and LIF detection. Alternatively, capillary electrophoresis with negative electrospray ionization-ion-trap tandem mass spectrometry (CE-MS2) is shown to permit the direct confirmation of furosemide in human urine. This approach is based upon the monitoring of the m/z 329.3-->4m/z 285.2 precursor-product ion transition. CZE-LIF and CE-MS2 with injection of plain or diluted urine represent simple, rapid and attractive urinary screening and confirmation assays for furosemide in patient urines.