On-column concentration and separation of double-stranded DNA by gradient capillary electrophoresis

We describe the separation of dsDNA by capillary electrophoresis in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO). Using 1.0% PEO, the separation of DNA fragments with sizes ranging from 51 bp to 23 kbp has been achieved in less than 12 min, which is better than conventional methods (in the absence of EOF) in terms of speed and resolution. In order to concentrate and separate the DNA sample, gradient changes in the concentrations of PEO and ethidium bromide (EtBr) have been conducted. Different concentrations of PEO solutions are injected to the polyethylene tubes by pressure, where they enter the capillary by EOF. Because the large DNA fragments migrate faster towards the cathode end under counterflow conditions, the introduction sequence is from low to high concentrations of PEO solutions after sample injection. Using the gradient CE approach, the separations of the DNA sample injected at 30 cm height for times up to 120 s have been demonstrated. The linearity between injection time and peak height shows that the DNA fragments stacked during migration from the sample zone to PEO. We found that stacking efficiency is greater when the analysis was performed by simultaneously changing the PEO and EtBr concentration, compared to individual changes in PEO concentration.     

Mixed triblock copolymers used as DNA separation medium in capillary electrophoresis

A polymer solution, formed by mixing two polyoxybutylene-polyoxyethylene-polyoxybutylene (BEB) triblock copolymers (B10E270B10 and B6E46B6), was tested as a new separation medium for double-stranded DNA separation in capillary electrophoresis. The mixture of B10E270B10 and B6E46B6 has a viscosity-adjustable property and a dynamic coating ability, which makes the medium very easy to handle. The performance of the mixture on the DNA separation is greatly affected by the mass ratio of the two constituents. There is a minimum amount of concentration for B10E270B10, below which the medium will lose its performance. The addition of B6E46B6 increases both the selectivity and the separation efficiency. The optimal concentration, with 3% (w/v) B10E270B10 and 5% (w/v) B6E46B6, is determined with the consideration of both speed and resolution. A resolution of 1.3 was achieved on the separation of 123/124 base pairs in the pBR322/HaeIII digest within 20 min by using a 10 cm column of 75 microm I.D., demonstrating the potential use of mixtures of amphiphilic block copolymers as an effective DNA separation medium.     

Interaction between netropsin and double-stranded DNA in capillary zone electrophoresis and affinity capillary electrophoresis

Poor sensitivity and low phase ratio are the main drawbacks of open tubular capillary electrochromatography (OTCEC). The poor sensitivity results from the use of narrow bore size capillary, whereas the low phase ratio, which limits the separation capability, is caused by the limited surface area of conventional capillary. Two strategies may be useful to overcome these disadvantages. First, an extended light path (ELP) capillary, which has a bubble cell at the detection point, is used to improve the sensitivity. Secondly, an etched capillary of a 1,000-fold increased surface area is used to enhance the phase ratio. In this work, use of an ELP capillary and an etched capillary in OTCEC was evaluated with a chiral stationary phase of avidin prepared with the physical adsorption method. With a 20 microm I.D. ELP capillary with a 150 microm bubble cell, the peak height was enhanced by 4-10-fold and the corrected peak area was increased by 12-fold relative to a 20 microm I.D. conventional capillary. However, the peak efficiency and resolution decreased noticeably. The phase ratio on the etched capillary was slightly enhanced, by a factor of 1.64 relative to an unetched capillary. Consequently, the separation capability was slightly improved. The increase in the phase ratio was much lower than that expected from the increase in surface area, the reason for which is probably the reduced density of surface silanol group and the generation of nitrogen-containing groups due to the etching process.     

Spatial open-network formed by mixed triblock copolymers as a new medium for double-stranded DNA separation by capillary electrophoresis

A mixture of two polyoxybutylene-polyoxyethylene-polyoxybutylene (BEB) triblock copolymers (B6E46B6 and B10E271B10, respectively) was used as a new separation medium for separating double-stranded DNA (dsDNA) fragments by capillary electrophoresis (CE). The two block copolymer mixtures were designed to form mixed flower-like micelles in dilute solution and a homogeneous gel-like open-network with hydrophobic clusters as cross-linking points at higher polymer concentrations. Being a polyoxyalkylene block copolymer gel, the separation medium has some special advantages, including the temperature-dependent sol-gel transition that makes sample injection easy, and the self-coating of the inner capillary wall that makes experimental procedures simple and reproducible. Furthermore, it can shorten the elution time and further improve the separation resolution, especially for small dsDNA fragments, when compared with EPE-type separation media, e.g., F127 (E99P69E99, with P being polyoxypropylene) block copolymer gels formed by the closed packing of spherical micelles. Single base pair resolution can be achieved by using the new separation medium for dsDNA fragments up to over 100 base pairs.     

Portable capillary electrophoresis system with potential gradient detection for separation of DNA fragments

A portable capillary electrophoresis (CE) system with a novel potential gradient detection (PGD) was utilized to separate DNA fragments. For the first time it was demonstrated that separation of DNA fragments in polymer solution could be detected by a portable CE system integrated with PGD, with a limit of detection (LOD) comparable to that of the CE-ultraviolet (UV) method. Effects of buffer solution, sieving medium, and applied voltage were also investigated. The portable CE-PGD system shows several potential advantages, such as simplicity, cost effectiveness, and miniaturization.     

Designing polymer matrix for microchip-based double-stranded DNA capillary electrophoresis

An optimization strategy for ternary solvent-strength gradient elution RP chromatography is described in which a two-dimensional model of gradient time (2 levels) against ternary proportions of organic modifiers (4 levels) was constructed. From the resolution surface the optimum ratio of organic modifiers could be selected. Excellent retention time and acceptable peak width and resolution simulations were obtained. The separation could be further optimized from the same input data by using a standard one-dimensional model in order to optimize for gradient slope, duration and shape. Excellent retention time and acceptable peak width and resolution simulations were obtained (< 1, 2 and 6% error, respectively).     

Capillary electrophoresis of double-stranded DNA in an untreated capillary.

Using the zwitterionic buffer N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) in the presence of a high-molecular-mass hydroxypropylmethylcellulose (HPMC) as a sieving polymer and ethidium bromide double-stranded DNA (dsDNA) was separated in an untreated capillary. The HEPES buffer shielded the DNA against the capillary wall interaction and decreased the electroosmotic flow enabling a good separation of the DNA similar to that obtained in a commercially coated capillary. In addition to the low cost of the untreated capillary it can be washed after each run. Furthermore, stacking with hydrodynamic injection filling about half of the capillary volume is demonstrated.     

Characterization of single-stranded DNA separation by capillary gel electrophoresis

We have investigated the effect of polymer gel reconditioning, the shape of the capillary, the applied electric field, and the capillary length for single-stranded DNA. The polyethylene oxide gel had deformed under the high electric field causing the degradation of the separation power. By the reintroduction of the fresh polyethylene oxide gel for the next run, one-base resolution was recovered. It turned out that the tip of the capillary at the injection side needed to be clean and symmetric for much improved resolution. Changing DNA motion by the pulsed electric field resulted in the separation of DNA far more than 500 bases.     

Improved separation of double-stranded DNA fragments by capillary electrophoresis using poly(ethylene oxide) solution containing colloids

The analysis of double-stranded (ds) DNA fragments by capillary electrophoresis (CE) using poly(ethylene oxide) (PEO) solution containing gold nanoparticles (GNPs) is presented, focusing on evaluating size dependence of the GNPs and PEO on resolution and speed. To prevent the interaction of the capillary wall with DNA, the capillary was dynamically coated with polyvinylpyrrolidone. Using different PEO solutions containing GNPs ranging in diameter from 3.5 to 56 nm, we have achieved reproducible, rapid, and high-resolution DNA separations. The results indicate that the sizes of PEO and GNPs as well as the concentration of PEO affect resolution. The separation of DNA ranging in size from 8 to 2176 base pairs (bp) was accomplished in 5 min using 0.2% PEO (8 MDa) containing 56 nm GNPs. We have also demonstrated the separations of the DNA fragments ranging from 5 to 40 kbp using 0.05% PEO (2 MDa) containing 13 nm GNPs or 0.05% PEO (4 MDa) containing 32 nm GNPs. With very low viscosity (< 15 cP), automatic replacement of the sieving matrices is easy, indicating a great potential for high-throughput DNA analysis using capillary array electrophoresis systems.     

Principles of DNA separation with capillary electrophoresis

During the last decade, capillary electrophoresis (CE) of DNA has undergone rapid development. This improvement was especially important for DNA sequencing, where CE has now become a standard method facilitating to decipher several genomes within a very short time. Here, we give a review of the fundamentals of DNA separation in CE and the major factors influencing the performance.     

Prediction of DNA separation by capillary electrophoresis with polymer additives

The paper is focused on the powerful prediction ability of the quantitative DNA sieving model in DNA separations by capillary electrophoresis, which was proposed by us previously. First, the DNA resolution can be predicted by the theory. The model predicts that the most difficult and easiest separation will be 184bp/192bp and 234bp/267bp respectively, which is consist with experimental results. Furthermore, the average relative differences of predicted and experimental resolution values (R(S)) for ssDNA 184b/192b or dsDNA 184bp/192bp were all smaller than 2.8% if the diffuse parameter D considered was 8×10(-5) cm(2)/s. Secondly, the optimum polymer concentrations for DNA separation were also calculated by the model, and the results show that polymer concentration should be as high as possible in DNA separation. Thirdly, the sieving ability of polymer will be predicted by the model. Polymer with smaller k, a polymer parameter calculated by the model, is prior to use as DNA sieving media.     

Preconcentration and separation of double-stranded DNA fragments by electrophoresis in plastic microfluidic devices

We have evaluated double-stranded DNA separations in microfluidic devices which were designed to couple a sample preconcentration step based on isotachophoresis (ITP) with a zone electrophoretic (ZE) separation step as a method to increase the concentration limit of detection in microfluidic devices. Developed at ACLARA BioSciences, these LabCard trade mark devices are plastic 32 channel chips, designed with a long sample injection channel segment to increase the sample loading. These chips were designed to allow stacking of the sample into a narrow band using discontinuous ITP buffers, and subsequent separation in the ZE mode in sieving polymer solutions. Compared to chip ZE, the sensitivity was increased by 40-fold and we showed baseline resolution of all fragments in the PhiX174/HaeIII DNA digest. The total analysis time was 3 min/sample, or less than 100 min per LabCard device. The resolution for multiplexed PCR samples was the same as obtained in chip ZE. The limit of detection was 9 fg/microL of DNA in 0.1xpolymerase chain reaction (PCR) buffers using confocal fluorescence detection following 488 nm laser excitation with thiazole orange as the fluorescent intercalating dye.     

Polymer solutions and entropic-based systems for double-stranded DNA capillary electrophoresis and microchip electrophoresis

We give an overview of recent development of low-viscosity polymer solutions and entropic trapping networks for double-stranded DNA (dsDNA) separations by conventional capillary electrophoresis and microchip electrophoresis. Theoretical models for describing separation mechanisms, commonly used noncross-linked polymer solutions, thermoresponsive (viscosity-adjustable) polymer solutions, and novel entropic trapping networks are included. The thermoresponsive polymer solutions can be loaded at one temperature into microchannels at lower viscosities, and used in separation at another temperature at entanglement threshold concentrations and higher viscosities. The entropic-based separations use only arrays of regular obstacles acting as size-separations and do not need viscous polymer solutions. These progresses have potential in integration to automated capillary and microfluidic chip systems, enabling better reusability of separation microchannels, much shorter DNA separation times, and higher reproducibility due to less matrix degradation.     

Mathematical model for DNA separation by capillary electrophoresis in entangled polymer solutions

A mathematical model of DNA separation by capillary electrophoresis in entangled polymer solution is presented. The mechanism is modeled as a DNA molecule moving through transient pores formed in polymer solutions and colliding with blobs of polymer molecules encountered during migration. By taking account of the average retardation time (t(c)) of DNA-blob collision and calculating the total collision number (N(c)), a quantitative mathematical equation was reported, leading to predictions for the DNA mobility as a function of the experimental conditions like the size of DNA, the polymer concentration and the electric field strength. For DNA fragments in frequent size range, the initial experimental data agree well with the model. The DNA shape function (f(E)) was suggested and then discussed by the experimental data. The relationship between f(E) and electric field strength E was empirically estimated. Then, the average retardation time t(c) was obtained as about (2 approximately 3)x10(-6)s in linear polyacrylamide (LPA) and hydroxyethylcellulose (HEC) solution.     

Analysis of double-stranded DNA by microchip capillary electrophoresis using polymer solutions containing gold nanoparticles

The impact of gold nanoparticles (GNPs) on the microchip electrophoretic separation of double-stranded (ds) DNA using poly(ethylene oxide) (PEO) is described. Coating of the 75-microm separation channel on a poly(methyl methacrylate) (PMMA) plate in sequence with poly(vinyl pyrrolidone), PEO, and 13-nm GNPs is effective to improve reproducibility and resolution. In this study, we have also found that adding 13-nm GNPs to 1.5% PEO is extremely important to achieve high resolution and reproducibility for DNA separation. In terms of the stability of the GNPs, 100 mM glycine-citrate buffer at pH 9.2 is a good buffer system for preparing 1.5% PEO. The separation of DNA markers V and VI ranging in size from 8 to 2176 base pairs has been demonstrated using the three-layer-coated PMMA microdevice filled with 1.5% PEO containing the GNPs. Using these conditions, the analysis of the polymerase chain reaction products of UGT1A7 was complete in 7 min, with the relative standard deviation values of the peak heights and migration times less than 2.3% and 2.0%, respectively. In conjunction with stepwise changes of the concentrations of ethidium bromide (0.5 and 5 microg/ml), this method allows improved resolution and sensitivity for DNA markers V and VI.     

Separation of double-stranded DNA fragments by capillary electrophoresis in interpenetrating networks of polyacrylamide and polyvinylpyrrolidone

Mixtures of two polymers with totally different chemical structures, polyacrylamide and polyvinylpyrrolidone (PVP) have been successfully used for double-stranded DNA separation. By polymerization of acrylamide in a matrix of PVP solution, the incompatibility of these two polymers was suppressed. Laser light scattering (LLS) studies showed that highly entangled interpenetrating networks were formed in the solution. Further systematic investigation showed that double-stranded DNA separation was very good in these interpenetrating networks. With a concentration combination of as low as 2% w/v PVP (weight-average molecular mass Mr = 1 x 10(6) g/mol) + 1% w/v polyacrylamide (Mr = 4 x 10(5) g/mol), the 22 fragments in pBR322/HaeIII DNA, including the doublet of 123/124 bp, have been successfully separated within 6.5 min. Under the same separation conditions, similar resolution could only be achieved by using polyacrylamide (Mr = 4 x 10(5) g/mol) with concentrations higher than 6% w/v and could not be achieved by using only PVP (Mr = 1 x 10(6) g/mol) with a concentration as high as 15% w/v. It is noted that the interpenetrating network formed by 2% PVP and 1% polyacrylamide has a very low viscosity and can dynamically coat the inner wall of a fused-silica capillary. The separation reached an efficiency of more than 10(7) theoretical plate numbers/m and a reproducibility of less than 1% relative standard deviation of migration time in a total of seven runs. The interpenetrating network could stabilize polymer chain entanglements. Consequently, the separation speed was increased while retaining resolution.     

Separation of double-stranded DNA fragments in plastic capillary electrophoresis chips by using E99P69E99 as separation medium.

The separation of double-stranded DNA (dsDNA) fragments in polymethylmethacrylate (PMMA) capillary electrophoresis (CE) chips by using E99P69E99 as a separation medium has been demonstrated. The PMMA CE chips were simply manufactured by micromachining and adhesive tape sealing. To make the separation channel compatible with the separation medium, a dynamic nonionic surfactant coating procedure was developed, which made the plastic separation channel sufficiently hydrophilic to allow the separation medium to fill the channel by capillary action. Subsequent separation of DNA fragments was successful with a separation efficiency of the order of 10(4) theoretical plates over an effective separation distance of 1.5 cm. By using an applied electric field strength of 200 V/cm, the separation of low DNA mass ladder was completed within 5 min. The simple coating procedure, together with the self-assembled viscosity-adjustable separation medium, should be useful to meet some of the essential requirements for developing single-use disposable CE chips. Coating the channels with polymer blends of PMMA and the separation medium also showed promise.     

Chiral separation of cathinone derivatives used as recreational drugs by cyclodextrin-modified capillary electrophoresis

In recent years, cathinone derivatives have entered the global drug market and caused serious social problems in many European countries. Modification of the basic structure of cathinone leads to a multitude of derivatives, including the most popular representative mephedrone. All those substances contain a stereogenic center and therefore two isoforms exist. As it is the case with many chiral active pharmaceutical ingredients, even the pharmacological effect of the enantiomers of those psychoactive compounds may differ. During this research, an easy-to-prepare chiral capillary zone electrophoresis method for the enantioseparation of a set of 19 cathinone derivatives was developed. Testing different types of cyclodextrin (CD), including native-β-CD, carboxymethyl-β-CD, 2-hydroxypropyl-β-CD, sulfated-β-CD, and native γ-CD, best results were obtained with the negatively charged sulfated-β-CD. The effect of the CD concentration, the temperature, and the addition of ACN to the BGE on the enantioseparation is shown by three model compounds. Under optimal conditions, using 20 mg/mL sulfated-β-CD in 50 mM ammonium acetate buffer pH?= 4.5 containing 10% v/v ACN at a cassette temperature of 40°C and with an applied voltage of 20 kV, all derivatives except methedrone were resolved in their enantiomers within 20 min.     

Copolymer solutions as separation media for DNA capillary electrophoresis

Capillary electrophoresis techniques offer high plate numbers and are highly suited for the efficient separations of a wide variety of chemical components in diverse matrices. Because of the small capillary and detection cell dimensions, together with the minute volumes of samples to be injected, sensitive detection schemes based on different physicochemical principles are being developed. One logical approach to increased sensitivity in capillary electrophoresis detection has been the development of chemiluminescence-based detectors. The development of on-line ultrasensitive chemiluminescence detection (referred to the concentration detection limit of nM order of magnitude or mass detection limit of amol order of magnitude) in capillary electrophoresis system is reviewed. The applications and limitations of the current detection methodology are briefly considered and future prospects for the development are discussed.