A DNA sieving matrix with thermally tunable mesh size

We present a "proof-of-concept" study showing that a blend of thermo-responsive and nonthermo-responsive polymers can be used to create a DNA sieving matrix with a thermally tunable mesh size, or "dynamic porosity". Various blends of two well-studied sieving polymers for CE, including hydroxypropylcellulose (HPC), a thermo-responsive polymer, and hydroxyethylcellulose (HEC), a nonthermo-responsive polymer, were used to separate a double-stranded DNA restriction digest (Phi X174-HaeIII). HPC exhibits a volume-phase transition in aqueous solution which results in a collapse in polymer coil volume at approximately 39 degrees C. Utilizing a blend of HPC and HEC in a ratio of 1:5 by weight, we investigated the effects of changing mesh size on DNA separation, as controlled by temperature. High-resolution DNA separations were obtained with the blended matrix at temperatures ranging from 25 degrees C to 38 degrees C. We evaluated changes in the selectivity of DNA separation with increasing temperature for certain pairs of small and large fragments. A pure HEC (nonthermo-responsive) matrix was used over the same temperature range as a negative control. In the blended matrix, we observe a maximum in selectivity at approximately 31 degrees C for small DNA, while a significant increase in the selectivity of large-DNA separation occurs at approximately 36 degrees C as the polymer mesh "opens". We also demonstrate, through a temperature ramping experiment, that this matrix can be utilized to obtain high-resolution separation of both small and large DNA fragments simultaneously in a single CE run. Blended polymer matrices with "dynamic porosity" have the potential to provide enhanced genomic analysis by capillary array or microchip electrophoresis in microfluidic devices with advanced temperature control.     

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.     

Thermoresponsive N,N-dialkylacrylamide copolymer blends as DNA sieving matrices with a thermally tunable mesh size

In an earlier study we showed that a blend of thermoresponsive and nonthermoresponsive hydroxyalkylcelluloses could be used to create a thermally tunable polymer network for double-stranded (ds) DNA separation. Here, we show the generality of this approach using a family of polymers suited to a wider range of DNA separations: a blended mixture of N,N-dialkylacrylamide copolymers with different thermoresponsive behaviors. A mixture of 47% w/w N,N-diethylacrylamide (DEA)/53% w/w N,N-dimethylacrylamide (DMA) (DEA47; thermoresponsive, transition temperature = 55 degrees C in water) and 30% w/w DEA/70% w/w DMA (DEA30; nonthermoresponsive, transition temperature > 85 degrees C in water) copolymers in the ratio of 1:5 w/w DEA47:DEA30 was used to separate a dsDNA restriction digest (PhiX174-HaeIII). We investigated the effects of changing mesh size on dsDNA separation, as controlled by temperature. We observed good DNA separation performance with the copolymer blend at temperatures ranging from 25 degrees C to 48 degrees C. The separation selectivity was evaluated quantitatively for certain DNA fragment pairs as a function of temperature. The results were compared with those obtained with a control matrix consisting only of the nonthermoresponsive DEA30. Different DNA fragment pairs of various sizes show distinct temperature-dependent selectivities. Over the same temperature range, no significant temperature dependence of selectivity is observed for these DNA fragment pairs in the nonthermoresponsive control matrix. Overall, the results show similar trends in the temperature dependency of separation selectivity to what was previously observed in hydroxyalkylcellulose blends, for the same DNA fragment pairs. Finally, we showed that a ramped temperature scheme enables improved separation in the blended copolymer matrix for both small and large DNA fragments, simultaneously in a single capillary electrophoresis (CE) run.     

Nanomaterials and chip-based nanostructures for capillary electrophoretic separations of DNA

Capillary electrophoresis (CE) and microchip capillary electrophoresis (MCE) using polymer solutions are two of the most powerful techniques for the analysis of DNA. Problems, such as the difficulty of filling polymer solution to small separation channels, recovering DNA, and narrow separation size ranges, have put a pressure on developing new techniques for DNA analysis. In this review, we deal with DNA separation using chip-based nanostructures and nanomaterials in CE and MCE. On the basis of the dependence of the mobility of DNA molecules on the size and shape of nanostructures, several unique chip-based devices have been developed for the separation of DNA, particularly for long DNA molecules. Unlike conventional CE and MCE methods, sieving matrices are not required when using nanostructures. Filling extremely low-viscosity nanomaterials in the presence and absence of polymer solutions to small separation channels is an alternative for the separations of DNA from several base pairs (bp) to tens kbp. The advantages and shortages of the use of nanostructured devices and nanomaterials for DNA separation are carefully addressed with respect to speed, resolution, reproducibility, costs, and operation.     

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.     

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.     

The use of light scattering for precise characterization of polymers for DNA sequencing by capillary electrophoresis

The ability of a polymer matrix to separate DNA by capillary electrophoresis (CE) is strongly dependent upon polymer physical properties. In particular, recent results have shown that DNA sequencing performance is very sensitive to both the average molar mass and the average coil radius of the separation matrix polymers, which are affected by both polymer structure and polymer-solvent affinity. Large polymers with high average molar mass provide the best DNA sequencing separations for CE, but are also the most challenging to characterize with accuracy. The methods most commonly used for the characterization of water-soluble polymers with application in microchannel electrophoresis have been gel permeation chromatography (GPC) and intrinsic viscosity measurements, but the limitations and potential inaccuracies of these approaches, particularly for large or novel polymers and copolymers, press the need for a more universally accurate method of polymer molar mass profiling for advanced DNA separation matrices. Here, we show that multi-angle laser light scattering (MALLS) measurements, carried out either alone or in tandem with prior on-line sample fractionation by GPC, can provide accurate molar mass and coil radius information for polymer samples that are useful for DNA sequencing by CE. Wider employment of MALLS for characterization of novel polymers designed as DNA separation matrices for microchannel electrophoresis should enable more rapid optimization of matrix properties and formulation, and assist in the development of novel classes of polymer matrices.     

Polymeric matrices for DNA sequencing by capillary electrophoresis

We review the wide range of polymeric materials that have been employed for DNA sequencing separations by capillary electrophoresis. Intensive research in the area has converged in showing that highly entangled solutions of hydrophilic, high molar mass polymers are required to achieve high DNA separation efficiency and long read length, system attributes that are particularly important for genomic sequencing. The extent of DNA-polymer interactions, as well as the robustness of the entangled polymer network, greatly influence the performance of a given polymer matrix for DNA separation. Further fundamental research in the field of polymer physics and chemistry is needed to elucidate the specific mechanisms by which DNA is separated in dynamic, uncross-linked polymer networks.     

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.     

Pressure-induced transport of DNA confined in narrow capillary channels

Pressure-induced transport of double-stranded DNA (dsDNA) from 10 base pairs (bp) to 1.9 mega base pairs (Mbp) confined in a 750-nm-radius capillary was studied using a hydrodynamic chromatographic technique and four distinct length regions (rod-like, free-coiled, constant mobility, and transition regions) were observed. The transport behavior varied closely with region changes. The rod-like region consisted of DNA shorter than the persistence length (～150 bp) of dsDNA, and these molecules behaved like polymer rods. Free-coiled region consisted of DNA from ～150 bp to ～2 kilo base pairs (kbp), and the effective hydrodynamic radius R(HD) of these DNA scaled to L(0.5) (L is the DNA length in kbp), a characteristic property of freely coiled polymers. Constant mobility region consisted of DNA longer than ～100 kbp, and these DNA had a constant hydrodynamic mobility and could not be resolved. Transition region existed between free-coiled and constant mobility regions. The transport mechanism of DNA in this region was complicated, and a general empirical equation was established to relate the mobility with DNA length. Understanding of the fundamental principles of DNA transport in narrow capillary channels will be of great interest in the development of "lab-on-chip" technologies and nongel DNA separations.     

Separating DNA sequencing fragments without a sieving matrix.

The possibility of separating appropriately labeled DNA fragments using free-flow capillary electrophoresis was predicted a few years ago based on simple theoretical arguments. Free-flow separation of double-stranded DNA (dsDNA) fragments in the 100-1000 base range was later demonstrated using a streptavidin label. In this article, we now report that end-labeled free-flow electrophoresis (ELFSE) can also be used to sequence single-stranded DNA (ssDNA). The first 100 bases of a DNA sequencing reaction were read without any sieving matrix when fractionated streptavidin was added to the 5'-end of the ssDNA fragments. These separations required only 18 min and did not require coated capillaries. An analysis of the results indicates that sample injection, analyte-wall interactions and thermal diffusion are the limiting factors at this time. Extrapolating from our data, we predict that several hundred bases could be sequenced in less than 30 min with the proper conditions. ELFSE thus offers an attractive potential alternative to polymer solutions for DNA sequencing in capillaries and microchips.     

DNA analysis on electrophoretic microchips: effect of operational variables

Applicability of modern microfabrication technology to electrophoresis microchips initiated a rapidly moving interdisciplinary field in analytical chemistry. Electric field mediated separations in microfabricated devices (electrophoresis microchips) are significantly faster than conventional gel electrophoresis, usually completed in seconds to minutes. Electrophoretic separation of DNA molecules on microfabricated devices proved to have the potential to improve the throughput of analysis by orders of magnitude. The flexibility of electrophoresis microchips allows the use of a plethora of separation matrices and conditions. In this paper, we report on electric field mediated separation of fluorescent intercalator-labeled dsDNA fragments in polyvinylpyrrolidone matrix-filled microchannel structures. The separations were detected in real time by a confocal, single-point laser-induced fluorescence/photomultiplier setup. Effects of the sieving matrix concentration (Ferguson plot), migration characteristics (reptation plot), separation temperature (Arrhenius plot), as well as applied electric field strength and intercalator concentration on the separation of DNA fragments are thoroughly discussed.     

A design of nanosized PEGylated-latex mixed polymer solution for microchip electrophoresis

We report here advanced microchip electrophoresis using a nanoparticle doped polymer solution that enables greater separation of DNA. The proposed system is simple and effective without any new apparatus or complicated procedures. Various amounts and sizes (80 nm, 110 nm, and 193 nm) of polymer nanoparticle solutions (PEGylated-latex) were mixed with a conventional polymer solution for microchip electrophoresis. When a 0.49 wt% hydroxyl propyl methyl cellulose (HPMC) buffer solution was mixed with a 2.25 wt% 80 nm-PEGylated-latex a higher separation efficiency and a higher mobility of a wider molecular range of dsDNA (10 bp to 2 kbp) was achieved under low viscosity conditions (<5.5 cP) than in conventional 0.7% HPMC. The separation performance was dependent on the particle size and concentration. Furthermore, the effectiveness of the larger PEGylated-latex (193 nm) was not as high as the smaller one (80 to 110 nm). The observed separation improvement by polymer solution with latex-nanoparticles seems to derive from the balance between wider polymer mesh size and the structural obstacles of particles in the buffer.     

Nanoparticle-filled capillary electrophoresis for the separation of long DNA molecules in the presence of hydrodynamic and electrokinetic forces

We report the analysis of long DNA molecules by nanoparticle-filled capillary electrophoresis (NFCE) under the influences of hydrodynamic and electrokinetic forces. The gold nanoparticle (GNP)/polymer composites (GNPPs) prepared from GNPs and poly(ethylene oxide) were filled in a capillary to act as separation matrices for DNA separation. The separations of lambda-DNA (0.12-23.1 kbp) and high-molecular-weight DNA markers (8.27-48.5 kbp) by NFCE, under an electric field of -140 V/cm and a hydrodynamic flow velocity of 554 microm/s, were accomplished within 5 min. To further investigate the separation mechanism, the migration of lambda-DNA was monitored in real time using a charge-coupled device (CCD) imaging system. The GNPPs provide greater retardation than do conventional polymer media when they are encountered during the electrophoretic process. The presence of interactions between the GNPPs and the DNA molecules is further supported by the fluorescence quenching of prelabeled lambda-DNA, which occurs through an energy transfer mechanism. Based on the results presented in this study, we suggest that the electric field, hydrodynamic flow, and GNPP concentration are the three main determinants of DNA separation in NFCE.     

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.     

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.     

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.     

Sparsely cross-linked "nanogels" for microchannel DNA sequencing

We have developed sparsely cross-linked "nanogels", sub-colloidal polymer structures composed of covalently linked, linear polyacrylamide chains, as novel DNA sequencing matrices for capillary electrophoresis. The presence of covalent cross-links affords nanogel matrices with enhanced network stability relative to standard, linear polyacrylamide (LPA), improving the separation of large DNA fragments. Nanogels were synthesized via inverse emulsion (water-in-oil) copolymerization of acrylamide and N,N-methylenebisacrylamide (Bis). In order to retain the fluidity necessary in a replaceable polymer matrix for capillary array electrophoresis (CAE), a low percentage of the Bis cross-linker (< 10(-4) mol%) was used. Nanogels were characterized by multiangle laser light scattering and rheometry, and were tested for DNA sequencing by CAE with four-color laser-induced fluorescence (LIF) detection. The properties and performance of nanogel matrices were compared to those of a commercially available LPA network, which was matched for both weight-average molar mass (Mw) and extent of interchain entanglements (c/c*). Nanogels presented in this work have an average radius of gyration of 226 nm and a weight-average molar mass of 8.8 x 10(6) g/mol. At concentrations above the overlap threshold, nanogels form a clear, viscous solution, similar to the LPA matrix (Mw approximately 8.9 x 10(6) g/mol). The two matrices have similar flow and viscosity characteristics. However, because of the physical network stability provided by the internally cross-linked structure of the nanogels, a substantially longer read length ( approximately 63 bases, a 10.4% improvement) is obtained with the nanogel matrix at 98.5% accuracy of base-calling. The nanogel network provides higher-selectivity separation of ssDNA sequencing fragments longer than 375 bases. Moreover, nanogel matrices require 30% less polymer per unit volume than LPA. This is the first report of a sequencing matrix that provides better performance than LPA, in a side-by-side comparison of polymer matrices matched for Mw and extent of interchain entanglements.     

高效毛细管电泳用于以聚环氧乙烷为筛分介质分离DNA的机理研究

用毛细管电泳以聚环氧乙烷(PEO)为筛分介质对pUC19DNA/Msp Ⅰ(HpaⅡ)Marker中的12条DNA片段进行了分离,并尝试用Ogston模型、爬行模型以及线性模型对分离机理进行研究,最终发现26～147bp的小片段,在低电场强度时能很好地符合Ogston模型理论,而190～501bp中等长度的DNA片段电泳迁移率与其尺寸间存在很好的负相关的线性关系,为此,提出一种新的线性模型来进行解释.此外,还探讨了PEO的浓度和电场强度对分离的影响.其结论可更好地从理论上指导对中小片段DNA的分离,对肿瘤基因突变点的分析和PCR扩增产物的分离分析具有重要的意义.     

An inexpensive microslab gel DNA electrophoresis system with real-time fluorescence detection

In this paper, we describe the construction of a simple yet powerful gel electrophoresis apparatus that can be used to perform size-selective separations of DNA fragments in virtually any laboratory. This system employs a microslab gel format with a novel gel casting technique that eliminates the need for delicate combs to define sample loading wells. The compact size of the microslab gel format allows rapid separations to be performed at low voltages using submicroliter sample volumes. Real time fluorescence detection of the migrating DNA fragments is accomplished using an inexpensive digital microscope that directly connects to any PC with a USB interface. The microscope is readily adaptable for this application by replacing its white light source with a blue light-emitting diode (LED) and adding an appropriate emission filter. Both polyacrylamide and agarose gels can be used as separation matrices. Separation performance was characterized using standard dsDNA ladders, and correct sizing of a 191 bp PCR product was achieved in 15 min. The low cost and simplicity of this system makes it ideally suited for use in a variety of laboratory and educational settings.