Evaluation of different nucleic acid stains for sensitive double-stranded DNA analysis with capillary electrophoretic separation

This paper outlines the first use of SYTOX Orange, SYTO 82 and SYTO 25 nucleic acid stains for on-column staining of double-stranded DNA (dsDNA) fragments separated by capillary electrophoresis (CE). Low-viscosity, replaceable poly(vinylpyrrolidone) (PVP) polymer solution was used as the sieving matrix on an uncoated fused-silica capillary. The effects of PVP concentration, electric field strength, and incorporated nucleic acid stain concentrations on separation efficiency were examined for a wide range of DNA fragment sizes. Our study was focused on using nucleic acid stains efficiently excitable at a wavelength of 532 nm. Among the five tested nucleic acid stains, SYTOX Orange stain was shown to have the best sensitivity for dsDNA detection by CE. About a 500-fold lower detection limit was obtained compared to commonly used ethidium bromide and propidium iodide. SYTOX Orange stain also provided a wide linear dynamic range for direct DNA quantitation with on-line CE detection. Use of SYTOX Orange stain can greatly improve the measurement of DNA fragments by CE, which will enable an expanded set of applications in genomics and diagnostics.     

On-chip potential gradient detection with a portable capillary electrophoresis system

A portable chip-CE system with potential gradient detection (PGD) was developed and applied to the determinations of alkali metals and alkaloids. The separation efficiency appeared to be satisfactory and nonaqueous capillary electrophoresis (NACE) proved to be applicable to PGD or conductivity detection. The power supplies, separation and detection were built on a device of 3 kg in weight. A branch channel near the end of the separation channel was designed to perform PGD and make the application of relatively high field strength possible. The study is the first report on the application of PGD on the microchip platform. The design of the chip-CE system shows several advantages, such as simplicity, miniaturization and wide applicability.     

Rapid separation and laser-induced fluorescence detection of mutated DNA by capillary electrophoresis in a self-coating, low-viscosity polymer matrix

The detection of point and other simple mutations in DNA is important for cancer research and diagnosis and other biological studies. Capillary electrophoresis has been successfully used for separating DNA fragments. However, a low-viscosity polymer sieving buffer for DNA separation with on-line coating has never been reported. In this paper, a new method using capillary electrophoresis with on-line coating and laser-induced fluorescence detection (CE-LIF) for screening for point or simple DNA mutations has been demonstrated. The method uses an on-line dynamic coating technique that increases capillary lifetime and analysis reproducibility, and employs a low-viscosity polymer solution, which allows the user to rinse the capillary rapidly and refill with polymer solution easily. Experiments proved that the additives in the separation buffer for on-line capillary coating do not affect the separation efficiency of the running buffer, and do not interfere with the formation of hydrogen-bonded network between boric acid, mannitol and hydroxypropylmethylcellulose polymers. The stability of the dynamically coated capillary was quantitatively studied; the capillary lifetime was increased 6- to 7-fold compared with that of permanently coated CE columns. Standard DNA fragments containing mutations, with sizes of 209, 219, and 338 bps, were successfully separated and detected with this system, after the mutated DNA fragments were cleaved by CEL-I endonuclease. The technique is very sensitive for the size-separation of low-range, middle-range, and high-range DNA fragments. Results were compared with the HPLC methods developed by Transgenomic, Inc. and were in good agreement. The method should be applicable to mutation detection for all relevant biological and clinical studies. The factors influencing separations and the stability of dynamic capillary coatings are also discussed in the paper.     

Identifying the orientation of DNA fragment in recombinant plasmid by capillary electrophoresis with a non-gel sieving solution.

It was demonstrated that a capillary electrophoresis (CE) method with a non-gel sieving solution has been developed to identify the orientation of DNA fragments in recombinant plasmids in molecular biology. The influences of the concentration of sieving polymer HEC, the applied electric field strength and sampling on CE separation were analyzed concerning the optimization of separation. YO-PRO-1 was used as a DNA intercalating reagent to facilitate fluorescence detection. Under the chosen conditions (buffer, 1 x TBE containing 1 microM YO-PRO-1 and 1.2% HEC; applied electric field strength, 200 V/cm; electrokinetic sampling: time, 5 s; voltage, -6 kV), three DNA markers (phi 174/HaeIII, pBR322/HaeIII and lambda DNA/HindIII) were tested for further evaluating the relationship between the DNA size and the mobility. The established CE method conjugated with the enzymatic approach was successfully applied to identifying the DNA orientation of recombinant plasmid in transgene operations of a newly cloned gene from Arabidopsis Thaliana.     

Multiplexed DNA sizing by capillary electrophoresis using entangled polymer solutions and diode array detection

We developed a method for the analysis of multiplexed double-stranded DNA (dsDNA) samples complexed to various intercalating dyes using entangled polymer solution. A commercial single-column capillary electrophoresis (CE) instrument with diode array detection was used for multiplexed detection of DNA samples by addition of intercalating fluorescent molecules. A Phi X174HinfI and a pGEM DNA ladder (1 mg/mL) were used for the electrophoretic separation of dsDNA fragments ranging in size from 24 to 726 and 36 to 2645 bp, respectively. The results suggested that simultaneous electrophoretic separation of different DNA ladders multiplexed with different dyes could be performed in the same capillary yielding fast DNA sizing separations. CE analysis, which is often overpowered by slab gel in sample throughput, could now overcome this disadvantage by allowing multiplexed sample analysis in a fraction of the time needed for slab gel analysis. The separation efficiency of stained DNA molecules with both dyes were dramatically improved with buffers containing a large cation such as tetrapentylammonium ion (Npe(4) (+)) as the only cation in the buffer.     

A simple microfluidic system for efficient capillary electrophoretic separation and sensitive fluorimetric detection of DNA fragments using light-emitting diode and liquid-core waveguide techniques

A miniaturized CE system has been developed for fast DNA separations with sensitive fluorimetric detection using a rectangle type light-emitting diode (LED). High sensitivity was achieved by combining liquid-core waveguide (LCW) and lock-in amplification techniques. A Teflon AF-coated silica capillary on a compact 6x3 cm baseplate served as both the separation channel for CE separation and as an LCW for light transmission of fluorescence emission to the detector. An electronically modulated LED illuminated transversely through a 0.2 mm aperture, the detection point on the LCW capillary without focusing, and fluorescence light was transmitted to the capillary outlet. To simplify the optics and enhance collection of light from the capillary outlet, an outlet reservoir was designed, with a light transmission window, positioned directly in front of a photomultiplier tube (PMT), separated only by a high pass filter. Automated sample introduction was achieved using a sequential injection system through a split-flow interface that allowed effective release of gas bubbles. In the separation of a phiX174 HaeIII DNA digest sample, using ethidium bromide as labeling dye, all 11 fragments of the sample were effectively resolved in 400 s, with an S/N ratio comparable to that of a CE system with more sophisticated LIF.     

Genetic mutation analysis by CE with LIF detection using inverse-flow derivatization of DNA fragments

Inverse-flow derivatization is a novel approach to obtain fluorescent DNA derivatives in DNA analysis based on CE with LIF detection. In the present work, we want to explore the feasibility of the application of this method into the mutation detection based on constant denaturant capillary electrophoresis (CDCE) and SSCP analysis. The DNA fragments were first amplified by PCR using a pair of common primers without fluorescent label, and then the mutations were determined by CDCE or SSCP analysis based on CE-LIF with inverse-flow derivatization of DNA fragments. The experimental conditions were investigated systematically, and different labeling modes including inverse-flow derivatization, on-column derivatization and fluorescent labeled primer technique were compared. The inverse-flow derivatization was successfully used in the detection of C677T mutation in the methylenetetrahydrofolate reductase gene by CDCE or SSCP analysis. Our preliminary results demonstrate that inverse-flow derivatization is very simple, inexpensive and sensitive and well suitable for the genetic analysis in clinical diagnosis.     

Capillary electropherograms for restriction fragment length polymorphism of Helicobacter pylori

Rapid identification of Helicobacter pylori strains is of importance for diagnosis and then treatment of duodenal and gastric ulcers. We developed a CE approach for the analysis of RFLP of the PCR products of urease (UreAB) gene and flagellin A (FlaA) gene fragments. Prior to CE analysis, the 2.4-kbp UreAB and 1.5-kbp FlaA PCR products were digested with the restriction enzymes HaeIII and HhaI, respectively. The DNA fragments were then separated by CE in conjunction with laser-induced fluorescence detection using poly(ethylene oxide) in the presence of electroosmotic flow. The DNA fragments range in sizes 259-1831 bp and 12-827 bp for UreAB and FlaA restriction fragments, respectively. Of 27 samples, the CE approach provided five and ten different RFLP patterns of the HaeIII and HhaI digests. The RFLP of PCR products of the two genes allow great sensitivity of identification of H. pylori strains. When compared with slab gel electrophoresis, the present CE approach provides advantages of rapidity (within 6 min per run), simplicity, and automation. The preliminary results have shown great practicality of the CE approach for screening H. pylori strains.     

UV- and visible-excited fluorescence of nucleic acids separated by capillary electrophoresis

UV- and visible-excited fluorescence detection strategies were compared for nucleic acids separated by capillary electrophoresis (CE). A dual-polymer sieving matrix consisting of hydroxypropylmethylcellulose and poly(vinylpyrrolidone) was used to separate DNA fragments from a 100-base pair ladder and RNA from individual cells. Two nucleic acid dyes, SYBR Gold and SYBR Green I, were evaluated for their performance at both UV (275 nm) and visible (488 nm) excitation wavelengths. While SYBR Gold-bound RNA from single cells yielded a substantially reduced UV-excited signal compared to that with visible excitation (as expected), the sensitivity of SYBR Gold-bound double-stranded DNA was comparable for UV and Vis excitation wavelengths. This study reveals the first demonstration of using SYBR Gold dyes for DNA detection following separation with CE and also the first example of SYBR-based detection of RNA sampled and separated from individual cells.     

Study on detection of mutation DNA fragment in gastric cancer by restriction endonuclease fingerprinting with capillary electrophoresis

The DNA fragment detection focusing technique has further enhanced the sensitivity and information of DNA targets. The DNA fragment detection method was established by capillary electrophoresis with laser‐induced fluorescence detection and restriction endonuclease chromatographic fingerprinting (CE‐LIF‐REF) in our experiment. The silica capillary column was coated with short linear polyarclarylamide (SLPA) using nongel sieving technology. The excision product of various restricted enzymes of DNA fragments was obtained by REF with the molecular biology software Primer Premier 5. The PBR322/BsuRI DNA marker was used to establish the optimization method. The markers were focused electrophoretically and detected by CE‐LIF. The results demonstrate that the CE‐LIF‐REF with SLPA can improve separation, sensitivity and speed of analysis. This technique may be applied to analysis of the excision product of various restricted enzymes of prokaryotic plasmid (pIRES2), eukaryote plasmid (pcDNA3.1) and the PCR product of codon 248 region of gastric cancer tissue. The results suggest that this method could very sensitively separate the excision products of various restricted enzymes at a much better resolution than the traditional agarose electrophoresis. Copyright © 2011 John Wiley & Sons, Ltd.     

Inverse-flow derivatization for capillary electrophoresis of DNA fragments with laser-induced fluorescence detection

A novel method is presented to detect DNA fragments separated by capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection using inverse-flow derivatization. In electrophoresis, the intercalating dye, thiazol orange was only added to the separation buffer at the positive polarity. The negatively charged DNA fragments migrated from the negative polarity to the positive polarity, while the positively charged dye migrated in the opposite direction. When DNA fragments met with dye ions, the DNA–dye complexes were formed. The complexes continued migrating to the positive end, due to their net negative charges. When the complexes passed through the detection window, the fluorescent signals were generated. Importantly, DNA fragments migrated as their native state before DNA–dye complexes were formed. This procedure was used to detect double stranded DNA (dsDNA) and single stranded DNA (ssDNA) fragments, and polymerase chain reaction (PCR) products. The excellent resolution and good reproducibility of DNA fragments were achieved in non-gel sieving medium. This procedure may be useful in genetic mutation/polymorphism detections.     

A new quasi-interpenetrating network formed by poly(N-acryloyl-tris-(hydroxymethyl)aminomethane and polyvinylpyrrolidone: separation matrix for double-stranded DNA and single-stranded DNA fragments by capillary electrophoresis with UV detection

The preparation of a new separation matrix, quasi-interpenetrating networks (quasi-IPNs) formed by poly(N-acryloyl-Tris) (poly(tris-A)) and PVP, and its application for dsDNA and ssDNA fragments separation by CE with UV detection, are presented. This new quasi-IPN exhibited high sieving performance, good dynamic coating ability, and low viscosity. Single-base resolutions of dsDNA fragments (Rs = 0.92 for 123/124 bp) and ssDNA fragments (Rs = 0.65 for 123/124 base, Rs = 0.48 for 309/310 base) were achieved by using the quasi-IPN of poly(tris-A)/PVP (2% + 2%) solution in a 31 cm effective length linear polyacrylamide (LPA)-coated column. Single-base separation of dsDNA fragments (Rs = 0.92 for 123/124 bp) was also obtained within 28 min in a 46.7 cm effective length bare column at higher 160 V/cm electric field strength by using the same quasi-IPN solution. The RSD of the migration time measured for each DNA fragments was less than 1.5% in the bare column for nine continuous runs. The effects of temperature and electric field strength on the DNA separation were also investigated.     

In-line coupling SPE and CE for DNA preconcentration and separation

An in-line SPE method coupled to CE was developed for the analysis of DNA. The amino silica monolith was prepared in situ by polymerization of tetraethoxysilane and N-(β-aminoethyl)-γ-aminopropyltriethoxysilane in ethanol aqueous solution at the inlet end of a 100?μm id fused-silica capillary, and the remaining part of the capillary was used as separation channel. The procedure for this in-line SPE-CE method was constructed on the basis of investigation on operational conditions such as the introduction mode of sieving matrix, the composition of elution solvent and the elution time. Twenty millimolar ammonium hydroxide was demonstrated to be effective for DNA desorption from the monolith, and linear poly(N-isopropylacrylamide) was used as the separation matrix. The proposed method could achieve limits of detection of 0.065-0.123?ng/mL for six DNA fragments ranging 100-2000?bp. Compared with conventional CE, preconcentration factors of over 100 times were obtained. The applicability of the in-line SPE-CE method was further demonstrated by analyzing plasmid DNA from Escherichia coli crude lysate.     

Capillary electrophoresis with dual laser detection in separation of amplified fragment length polymorphism fragments

We are presenting the application of CE technique with dual‐channel LIF detection for the simultaneous separation of DNA fragments labeled with two different fluorescence dyes. The optimal conditions of the analysis were determined for the separation of amplified fragment length polymorphism (AFLP) fragments labeled with 5′‐6‐carboxyfluorescein (6‐FAM) and the DNA size standard labeled with sulfoindocyanine succinimidyl ester (Cy‐5). CE equipped with both argon ion and diode lasers is a good alternative for sequencers and might be applied in analyses of PCR products generated by various fingerprinting methods.     

Single strand conformation polymorphism analysis of ras oncogene by capillary electrophoresis with laser-induced fluorescence detector

Single strand conformation polymorphism (SSCP) analysis of the N-ras oncogene was achieved by capillary electrophoresis with a laser-induced fluorescence detector (CE-LIF) using methylcellulose as a molecular sieving agent. The PCR-amplified N-ras oncogene, which is known to have a point mutation at codon 61 in the neuroblastoma, was investigated by CE-LIF combined with SSCP (SSCP-CE-LIF). A mixture of wild- and mutant-type single strand DNA fragments (103 bp) of the N-ras oncogene was separated by buffer solution containing 1.0% methylcellulose and 0.2 microM fluorescent dye (YO-PRO-1) at 25 degrees C. The SSCP-CE-LIF technique gave good resolution for wild- and mutant-type single strand DNA fragments with separation completed within 7 min. SSCP analysis using a CE system with a LIF detector was successfully applied to the detection of the one point mutation on the N-ras oncogene.     

In situ determination of nerve agents in various matrices by portable capillary electropherograph with contactless conductivity detection

Rapid, efficient and robust methods for sampling and extracting genuine nerve agents sarin, soman and VX were developed for analyzing these compounds on various solid matrices, such as concrete, tile, soil and vegetation. A portable capillary electrophoretic (CE) system with contactless conductometric detection was used for the in situ analysis of the extracted samples. A 7.5 mM MES/HIS-based separation electrolyte accomplished the analysis of target analytes in less than 5 min. The overall duration of the process including instrument start-up, sample extraction and analysis was less than 10 min, which is the fastest screening of nerve agents achieved with liquid phase separation methods to date. The procedure can easily be performed by a person in a protective suit and is therefore suitable for real-life applications. The CE results were validated by an independent GC-MS method and a satisfactory correlation was obtained. The use of a proper sampling strategy with two internal standards and "smart" data-processing software can overcome the low reproducibility of CE. This has a significant impact on the potential acceptance of portable CE instrumentation for the detection and analysis of genuine chemical warfare agents (CWA).     

Electrochemical Detection for Capillary Electrophoresis Microchips: A Review

Electrochemistry detection offers considerable promise for capillary‐electrophoresis (CE) microchips, with features that include remarkable sensitivity, portability, independence of optical path length or sample turbidity, low cost and power requirements, and high compatibility with modern micromachining technologies. This article highlights key strategies in controlled‐potential electrochemical detectors for CE microchip systems, along with recent advances and directions. Subjects covered include the design of the electrochemical detection system, its requirements and operational principles, common electrode materials, isolation from the separation voltage, derivatization reactions, typical applications, and future prospects. It is expected that electrochemical detection will become a powerful tool for CE microchip systems and will lead to the creation of truly portable (and possibly disposable) devices.     

Comparison of the separation of large DNA fragments in the presence and absence of electroosmotic flow at high pH

This paper describes the analysis of large DNA fragments at pH > 10.0 by capillary electrophoresis (CE) in the presence of electroosmotic flow (EOF) using hydroxyethylcellulose (HEC) solution. HEC solution in the anodic reservoir enters the capillaries filled with high-pH buffer by EOF after sample injection. With respect to resolution, sensitivity, and speed, separation conducted under discontinuous conditions (different pH values of HEC solutions and buffer filling the capillary) is appropriate. Using HEC solution at concentrations higher than its entanglement threshold ensures a good separation of large DNA fragments in the presence of EOF at high pH. In addition to pH and HEC, the electrolyte species, dimethylamine, methylamine, and piperidine, play different roles in determining the resolution. The separation of DNA fragments ranging in size from 5 to 40 kilo base pairs was completed in 6 min using 1.5% HEC prepared in 20 mM methylamine-borate, pH 12.0, and the capillary filled with 40 mM dimethylamine-borate, pH 10.0. In comparison, this method allows faster separations of large DNA fragments compared with that conducted in the absence of EOF using dilute HEC solutions.     

Analysis of DNA restriction fragments and polymerase chain reaction products by capillary electrophoresis

Capillary electrophoresis (CE) is a new, high-resolution tool for the analysis of DNA restriction fragments and DNA amplified by the polymerase chain reaction (PCR). By combining many of the principles of traditional slab gel methods in a capillary format, it is possible to perform molecular size determinations of human and plant PCR amplification products and DNA restriction fragments. DNA restriction fragments and PCR products were analyzed by dynamic sieving electrophoresis (DSE) and capillary gel electrophoresis (CGE). As part of this study, sample preparation procedures, injection modes, and the use of molecular mass markers were evaluated. Optimum separations were performed using the uPage-3 (3% T, 3% C) CGE columns with UV detection at 260 nm. Membrane dialysis and ultrafiltration/centrifugation proved to be nearly equivalent methods of sample preparation. Reproducibility studies demonstrated that blunt-ended, non-phosphorylated markers (specifically allele generated markers) provide the most accurate calibration for PCR product analysis. This study demonstrates that CE offers a high-speed, high-resolution analytical method for accurately determining molecular size and/or allelic type as compared with traditional methodologies.     

Characterization of acridone dyes for use in four-decay detection in DNA sequencing

Four acridone dyes and dye-labeled primers were characterized for use in four-decay DNA sequencing. In the four-decay scheme, fluorescence lifetime replaces spectral ("color") selectivity for distinguishing between four base-specific labels in a single-lane capillary electrophoresis (CE) separation of the DNA fragments. Prior to the introduction of the acridone dyes, a major obstacle to four-decay detection was the lack of four suitable dyes with resolvable lifetimes. The four acridone dyes, whether free in solution or tethered to DNA primer, exhibit significant differences among their lifetimes and are well-suited to use together in four-decay sequencing. The lifetimes of the four dye-labeled DNA primers that were sequentially injected and detected on-the-fly in a 2% POP6 sequencing gel were 4, 6, 11 and 14 ns. A 405 nm violet laser diode provides optimal excitation of the four dyes.