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.     

DNA analysis on microfabricated electrophoretic devices with bubble cells

Microfluidic devices with bubble cells have been fabricated on poly(methyl methacrylate) (PMMA) plates and have been employed for the analysis of DNA using polyethylene oxide (PEO) solutions. First, the separation channel was fabricated using a wire-imprinting method. Then, wires with greater sizes or a razor blade glued in a polycarbonate plate was used to fabricate bubble cells, with sizes of 190-650 microm. The improvements in resolution and sensitivity have been achieved for large DNA (> 603 base pair, bp) using such devices, which depend on the geometry of the bubble cell. The main contributor for optimal resolution is mainly due to DNA migration at lower electric field strengths inside the bubble cell. On the other hand, slight losses of resolution for small DNA fragments have been found mainly due to diffusion, supported by the loss of resolution when separating two small solutes. With a bubble cell of 75 microm (width) x 500 microm (depth), the sensitivity improvement up to 17-fold has been achieved for the 271 bp fragment in the separation of PhiX-174/HaeIII DNA restriction fragments. We have also found that a microfluidic device with a bubble cell of 360 microm x 360 microm is appropriate for DNA analysis. Such a device has been used for separating DNA ranging from 8 to 2176 bp and polymerase chain reaction (PCR) products amplified after 30 cycles, with rapidity and improvements in the sensitivity as well as resolution.     

Separation of dsDNA in the presence of electroosmotic flow under discontinuous conditions

Separations of phiX-174/HaeIII DNA restriction fragments have been performed in the presence of electroosmotic flow (EOF) using five different polymer solutions, including linear polyacrylamide (LPA), poly(ethylene oxide) (PEO), hydroxypropylcellulose (HPC), hydroxyethylcellulose (HEC), and agarose. During the separation, polymer solutions entered the capillary by EOF. When using LPA solutions, bulk EOF is small due to adsorption on the capillary wall. On the other hand, separation is faster and better for the large DNA fragments (> 872 base pairs, bp) using derivative celluloses and PEO solutions. Several approaches to optimum resolution and speed by controlling EOF and/or altering electrophoretic mobility of DNA have been developed, including (i) stepwise changes of ethidium bromide (0.5-5 microg/mL), (ii) voltage programming (125-375 V/cm), (iii) use of mixed polymer solutions, and (iv) use of high concentrations of Tris-borate (TB) buffers. The DNA fragments ranging from 434 to 653 bp that were not separated using 2% PEO (8,000,000) under isocratic conditions have been completely resolved by either stepwise changes of ethidium bromide or voltage programming. Compared to PEO solutions, mixed polymer solutions prepared from PEO and HEC provide higher resolving power. Using a capillary filled with 600 mM TB buffers, pH 10.0, high-speed (< 15 min) separation of DNA (pBR 322/HaeIII digest, pBR 328/ Bg/l digest and pBR 328/Hinfl digest) has been achieved in 1.5% PEO.     

Separation of double-stranded DNA fragments by capillary electrophoresis: Impacts of poly(ethylene oxide), gold nanoparticles, ethidium bromide, and pH

The separation of DNA by capillary electrophoresis using poly(ethylene oxide) (PEO) containing gold nanoparticles (GNPs) is presented. The impacts of PEO, GNPs, ethidium bromide (EtBr), and pH on the separation of double-stranded DNA have been carefully explored. Using a capillary dynamically coated with 5.0% poly(vinylpyrrolidone) and filled with 0.2% PEO containing 0.3 x GNPs (the viscosity less than 15 cP), we have demonstrated the separation of DNA markers V and VI within 5 min at pH 8.0 and 9.0. In terms of resolution and reproducibility, GNPs have a greater impact on the separation of DNA at pH 9.0. Resolution improvements for large DNA fragments (> 300 base pairs, bp) are greater than those for small ones in the presence of GNPs. It is important to point out that reproducibility is excellent (relative standard deviations for the migration times less than 0.5%) and thus no further dynamic coating is required in at least 20 consecutive runs in the presence of GNPs. Using 0.2% PEO (pH 9.0) containing 0.3 x GNPs, the separation of DNA fragments ranging in size from 21 to 23,130 bp was accomplished in 7 min. The results presented in this study show the advantage of PEO containing GNPs for DNA separation, including rapidity, high resolving power, excellent reproducibility, and ease of filling capillaries.     

Reduced viscosity polymer matrices for microchip electrophoresis of double-stranded DNA

On a polymethylmethacrylate (PMMA) microchip, double-stranded DNA fragments with a wide size range from 50 bp to 20 kbp were separated by two polymer solutions. One was a hydroxypropylmethylcellulose-4000 (HPMC-4000) solution of 1.3% (w/v) to separate fragments below 590 bp, and another was a mixed four molecular weight poly(ethylene oxide) solution at a total concentration of 0.1% to separate fragments above 520 bp. The widths at half height (wh) of the fragments had a good relationship with their migration times (tR) in both polymer solutions. Such a relationship was suitable for obtaining the wh values of unresolved peaks, calculating the resolution of two adjacent fragments, and optimizing microchip separation matrices. Based on the relativity, a low viscosity medium containing 2% HPMC-50 and 8% glucose was optimized for high-performance separation of a phiX174 HaeIII restriction fragment digest.     

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.     

Ultrafast, efficient separations of large-sized dsDNA in a blended polymer matrix by microfluidic chip electrophoresis: a design of experiments approach

Double-stranded (ds) DNA fragments over a wide size range were successfully separated in blended polymer matrices by microfluidic chip electrophoresis. Novel blended polymer matrices composed of two types of polymers with three different molar masses were developed to provide improved separations of large dsDNA without negatively impacting the separation of small dsDNA. Hydroxyethyl celluloses with average molar masses of ～27 kDa and ～1 MDa were blended with a second class of polymer, high-molar mass (～7 MDa) linear polyacrylamide. Fast and highly efficient separations of commercially available DNA ladders were achieved on a borosilicate glass microchip. A distinct separation of a 1-kb DNA extension ladder (200-40,000 bp) was completed in 2 min. An orthogonal design of experiments was used to optimize experimental parameters for DNA separations over a wide size range. We find that the two dominant factors are the applied electric field strength and the inclusion of a high concentration of low-molar mass polymer in the matrix solution. These two factors exerted different effects on the separations of small dsDNA fragments below 1 kbp, medium dsDNA fragments between 1 and 10 kbp, and large dsDNA fragments above 10 kbp.     

Comparison of sieving matrices for on-the-fly fluorescence lifetime detection of dye-labeled DNA fragments

Commercially available, replaceable sieving matrices and their solvent modulated forms were evaluated for use in on-the-fly fluorescence lifetime detection of dye-labeled DNA fragments in capillary electrophoresis. The fragments were labeled with dyes that can be excited by the 488 nm line of an argon ion laser and have lifetimes in the range of 0.8 ns to 3.8 ns. The sieving matrices and buffer systems included poly(vinylpyrrolidone) (PVP), poly(ethyleneoxide) (PEO), hydroxyethylcellulose (HEC), Tris-borate-EDTA (TBE) and Tris-TAPS-EDTA buffers modified with DMSO and formamide. Selection of the optimal sieving matrix is based on the separation efficiency and the enhancement of lifetime resolution of DNA fragments. Best results for both electrophoretic resolution and lifetime detection were obtained using a poly(ethyleneoxide)/TBE gel buffer in the presence of 10% formamide.     

Comparison of sieving matrices for on-the-fly fluorescence lifetime detection of dye-labeled DNA fragments

Commercially available, replaceable sieving matrices and their solvent modulated forms were evaluated for use in on-the-fly fluorescence lifetime detection of dye-labeled DNA fragments in capillary electrophoresis. The fragments were labeled with dyes that can be excited by the 488 nm line of an argon ion laser and have lifetimes in the range of 0.8 ns to 3.8 ns. The sieving matrices and buffer systems included poly(vinylpyrrolidone) (PVP), poly(ethyleneoxide) (PEO), hydroxyethylcellulose (HEC), Tris-borate-EDTA (TBE) and Tris-TAPS-EDTA buffers modified with DMSO and formamide. Selection of the optimal sieving matrix is based on the separation efficiency and the enhancement of lifetime resolution of DNA fragments. Best results for both electrophoretic resolution and lifetime detection were obtained using a poly(ethyleneoxide)/TBE gel buffer in the presence of 10% formamide. Received: 25 August 2000 / Revised: 7 November 2000 / Accepted: 14 November 2000     

Stepwise capillary electrophoretic separation of DNA fragments using poly(ethylene oxide) solutions in the presence of electroosmotic flow.

Single-base resolution in the separation of DNA markers V and VI was achieved in the presence of electroosmotic flow (EOF), using poly(ethylene oxide) (PEO) solutions containing ethidium bromide (EtB) under isocratic conditions. Furthermore, a new approach called stepwise capillary electrophoresis (SCE) has been developed for DNA analysis, including stepwise changes in PEO concentration, EtB concentration as well as both PEO and EtB concentrations, wherein the EOF was used to introduce different PEO solutions into the capillary during the separation. DNA fragments smaller than 80 bp were both detected under isocratic conditions using 20 micrograms/ml EtB, and SCE using 1 and 20 micrograms/ml EtB, but not under isocratic conditions using 1 microgram/ml EtB. Resolution and speed of the DNA separation in SCE were different from those obtained from isocratic means, indicating that DNA underwent different concentrations of PEO and EtB in SCE. For example, DNA fragments with 458 and 504 base pairs (bp) were partially resolved in SCE, but not under isocratic conditions. The results further suggest that it is worth developing gradient techniques for widening the separation range and enhancing resolution in DNA analysis.     

Separation of long DNA fragments by inversion field capillary electrophoresis

This study reports improved pulsed field capillary electrophoresis (PFCE) for separation of large DNA ladders. Important analytical conditions, including gel polymer concentration, ratio of forward to backward pulse duration, and separation potential, were investigated for their effects on the separation performance of DNA ranging in size from 0.1 to 10.0 kilo base pairs (kbp). Results show that DNA fragments from 0.1 to 8.0 kbp can be resolved with high resolution, simultaneously, in a short time. The ratio of forward to backward pulse duration affects the separation performance for DNA fragments greater than 1.5 kbp, and 3 or 4 is the optimum value of the ratio for separation of DNA up to 10 kbp. Furthermore, the separations that were obtained with 74–19,329 bp λ-DNA restriction fragments clearly demonstrate a dramatic improvement in the separation time and resolution over the conventionally used square-wave PFCE. The inversion field capillary electrophoresis reported here may help enable future DNA analysis studies to be performed quickly and effectively.     

Modification of poly(methyl methacrylate) microchannels for highly efficient and reproducible electrophoretic separations of double-stranded DNA

This paper deals with dynamic coating of the microchannels fabricated on poly(methyl methacrylate) (PMMA) chips and DNA separation by microchip electrophoresis (MCE). After testing a number of polymers, including 2-hydroxyethyl cellulose, hydroxypropylmethyl cellulose, different sizes of poly(ethylene oxide) (PEO), and poly(vinyl pyrrolidone) (PVP), we found that coating of the PMMA microchannels with PEO(Mr = 6.0 x 10(5) g/mol) on the first layer is essential to minimize the interaction of DNA with PMMA surface. To achieve high efficiency, multilayer coating of PMMA chips with PEO, PVP, and PEO containing gold nanoparticles [PEO(GNP)] is important. A 2-(PEO-PVP)-PEO(GNP) PMMA chip, which was repeatedly coated with 1.0% PEO and 5.0% PVP twice, and then coated with 0.75% PEO(GNP) each for 30 min, provided a high efficiency (up to 1.7 x 10(6) plates/m) for the separation of DNA markers V (pBR 322/HaeIII digest) and VI (pBR 328/BgiI digest and pBR 328/HinfI digest) when using 0.75% PEO(GNP). With such a high efficiency, we demonstrated the separation of hsp65 gene fragments of Mycobacterium HaeIII digests by MCE within 90 s. The advantages of this approach to DNA analysis include ease of filling the microchannel with 0.75% PEO(GNP), rapidity, and reproducibility.     

Factors influencing the size of PEO complexes with a tyrosine-rich polypeptide

Aqueous complexes of PEY1, a 36 100 Da random synthetic peptide of tyrosine (50 mol %) and glutamic acid (50 mol %), with poly(ethylene oxide) (PEO) were studied as functions of PEO molecular weight, mixing ratio of PEO and PEY1, and the presence of calcium ions. Without calcium ions, the complexes were water soluble, with each complex consisting of a single PEO chain with many bound PEY1 chains. In the presence of 1 mM calcium ions, PEY1 formed colloidal aggregates with intermediate molecular weight PEO (10(5) to 10(6) Da). By contrast, very high molecular weight PEO (8 x 10(6) Da) with calcium ions formed large hydrogels when mixed with PEY1. It is proposed that PEY1 molecules completely bind to low molecular PEO and thus are deactivated from causing the coupling of multiple PEO chains, whereas deactivation of PEY1 on very high molecular weight PEO clusters is a slower process, giving an opportunity for cluster-cluster aggregation.     

Single-molecule analysis enables free solution hydrodynamic separation using yoctomole levels of DNA

Single-molecule free solution hydrodynamic separation (SML-FSHS) cohesively integrates cylindrical illumination confocal spectroscopy with free solution hydrodynamic separation. This technique enables single-molecule analysis of size separated DNA with 100% mass detection efficiency, high sizing resolution and wide dynamic range, surpassing the performance of single molecule capillary electrophoresis. Furthermore, SML-FSHS required only a bare fused silica microcapillary and simple pressure control rather than complex high voltage power supplies, sieving matrices, and wall coatings. The wide dynamic range and high sizing resolution of SML-FSHS was demonstrated by separating both large DNA (23 vs 27 kbp) and small DNA (100 vs 200 bp) under identical conditions. Separations were successfully performed with near zero sample consumption using as little as 5 pL of sample and 240 yoctomoles (～150 molecules) of DNA. Quantitative accuracy was predominantly limited by molecular shot noise. Furthermore, the ability of this method to analyze of single molecule nanosensors was investigated. SML-FSHS was used to examine the thermodynamic equilibrium between stochastically open molecular beacon and target-bound molecular beacon in the detection of E. coli 16s rRNA targets.     

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.     

Effect of ionic strength, pH and polymer concentration on the separation of DNA fragments in the presence of electroosmotic flow

DNA separations in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO) solutions have been demonstrated. During the separations, PEO entered capillaries filled with Tris-borate (TB) free buffers by EOF and acted as sieving matrices. We have found that ionic strength and pH of polymer and free solutions affect the bulk EOF and resolution differently from that in capillary zone electrophoresis. The EOF coefficient increases with increasing ionic strength of the free TB buffers as a result of decreases in the adsorption of PEO molecules. In contrast, the bulk EOF decreases with increasing the ionic strength of polymer solutions using capillaries filled with high concentrations of free TB buffers. Although resolution values are high due to larger differential migration times between any two DNA fragments in a small bulk EOF using 10 mM TB buffers, use of a capillary filled with at least 100 mM TB free buffers is suggested for high-speed separations. On the side of PEO solutions, 1.5% PEO solutions prepared in 100 to 200 mM TB buffers are more proper in terms of resolution and speed. The separation of DNA markers V and VI was accomplished less than 29 min in 1.5% PEO solutions prepared in 100 mM TB buffers, pH 7.0 at 500 V/cm using a capillary filled with 10 mM free TB buffers, pH 7.0.     

A novel pathogen detection system based on high‐resolution CE‐SSCP using a triblock copolymer matrix

Although CE‐SSCP analysis combined with 16S ribosomal RNA gene‐specific PCR has enormous potential as a simple and versatile pathogen detection technique, low resolution of CE‐SSCP causes the limited application. Among the experimental conditions affecting the resolution, the polymer matrix is considered to be most critical to improve the resolution of CE‐SSCP analysis. However, due to the peak broadening caused by the interaction between hydrophobic moiety of polymer matrices and DNA, conventional polymer matrices are not ideal for CE‐SSCP analysis. A poly(ethyleneoxide)‐poly(propyleneoxide)‐poly(ethyleneoxide) (PEO‐PPO‐PEO) triblock copolymer, with dynamic coating ability and a propensity to form micelles to minimize exposure of hydrophobic PPO block to DNA, can be an alternative matrix. In this study, we examined the resolution of CE‐SSCP analysis using the PEO‐PPO‐PEO triblock copolymer as the polymer matrix and four same‐sized DNA fragments of similar sequence content. Among 48 commercially available PEO‐PPO‐PEO triblock copolymers, three were selected due to their transparency in the operable range of viscosity and PEO₁₃₇PPO₄₃PEO₁₃₇ exhibited the most effective separation. Significant improvement in resolution allowed discrimination of the similar sequences, thus greatly facilitated CE‐SSCP analysis compared to the conventional polymer matrix. The results indicate that PEO‐PPO‐PEO triblock copolymer may serve as an ideal matrix for high‐resolution CE‐SSCP analysis.     

Electrophoretic migration behavior of DNA fragments in polymer solution

A newly developed polymer coil shrinking theory is described and compared with the existing entangled solution theory to explain electrophoretic migration behaviour of DNA in hydroxypropylmethylcellulose (HPMC) polymer solution in buffer containing 100 mM tris(hydroxymethyl)aminomethane 100 mM boric acid, 2 mM ethylenediaminetetraacetic acid at pH 8.3. The polymer coil shrinking theory gave a better model to explain the results obtained. The polymer coil shrinking concentration, Cs, was found to be 0.305% and the uniform entangled concentration, C+, 0.806%. The existence of three regions (the dilute, semidilute, and concentrated solution) at different polymer concentrations enables a better understanding of the system to guide the selection of the best conditions to separate DNA fragments. For separating large fragments (700/ 800 bp), dilute solutions (HPMC < 0.3%) should be used to achieve a short migration time (10 min). For small fragments (200/300 bp), concentrated solutions are preferred to obtain constant resolution and uniform separation. The best resolution is 0.6% HPMC due to a combined interaction of the polymer coils and the entangled structure. The possibility of DNA separation in semidilute solution is often neglected and the present results indicate that this region has a promising potential for analytical separation of DNA fragments.     

Analysis of double-stranded DNA by capillary electrophoresis using poly(ethylene oxide) in the presence of hexadecyltrimethylammonium bromide

The impact of hexadecyltrimethylammonium bromide (CTAB) on the separation of ds-DNA by capillary electrophoresis in conjunction with laser-induced fluorescence (CE-LIF) detection using poly(ethylene oxide) (PEO) solution is described. The use of CTAB for improved separation reproducibility and efficiency of DNA has not been demonstrated although it is widely used for controlling the magnitude and direction of electroosmotic flow in CE. With increasing CTAB concentration, the interactions of DNA with ethidium bromide (EtBr) and with the capillary wall decrease. For the separation of DNA fragments with the sizes ranging from several base pairs (bp) to 2,176 bp, a polymer solution consisting of 0.75% poly(ethylene oxide), 100 mM TB buffer (pH 8.0), 25 microg/mL EtBr, and 0.36 microg/mL CTAB is proper. Using the PEO solution, we separated a mixture of DNA markers V (pBR 322/HaeIII digest) and VI (pBR 328/BglI digest and pBR 328/HinfI digest) within 8 min at -375 V/cm, with the limit of detection of 2.0 ng/mL based on the peak height for the 18-bp DNA fragment. The method is highly efficient (>10(6)plate/m), repeatable (RSD of the migration times <1.5%), and sensitive. In addition, it is convenient to fill a capillary (75 microm in diameter) with such a low-viscosity PEO solution by syringe pushing.     

聚环氧乙烷无胶筛分毛细管电泳分离宽分子量范围DNA片段

在无胶筛分毛细管电泳中,以聚环氧乙烷为筛分介质,用硅烷化处理的毛细管柱（31.2 cm×75 μm有效长度21.0 cm）分离DL5000 DNA Marker（DNA长度为100~5000 bp）,研究筛分介质浓度、缓冲液pH、分离电压和溴化乙锭浓度对分离双链DNA片段的影响,优化出分离100~5000 bp DNA片段的最佳条件。毛细管电泳的最佳条件为PEO浓度0.5%、缓冲液pH值8.0、电压12 kV、溴化乙锭浓度3.0 μg/mL。此条件下,对山梨醇脱氢酶基因（SDH）和乙烯受体基因（ETR1）的聚合酶链式反应（PCR）扩增产物同时检测,分离、鉴定效果良好。