Mobility, diffusion and dispersion of single-stranded DNA in sequencing gels

Electrophoresis of single-stranded DNA in denaturing polyacrylamide gels is presently a standard procedure for the sequencing of DNA fragments. A thorough understanding of the factors that determine the resolution of DNA fractionated in polyacrylamide gels is necessary to optimize the performance of DNA sequencers. Significant research on the mobility of double-stranded (ds)DNA molecules in agarose and polyacrylamide gels has been performed, and the phenomenon of band broadening of single-stranded (ss)DNA fragments in DNA sequencing gels has received attention only recently. In this paper, we present a detailed study of mobility, diffusion and dispersion of ssDNA in sequencing gels as a function of molecular size, gel concentration and electric field strength. DNA mobility is shown to be essentially independent of electric field in the range of 0-60 V/cm. The band broadening is greatly enhanced in the presence of an electric field and the dispersion coefficient (DE) can be an order of magnitude higher than the field-free diffusion coefficient. The measured migration parameters approximately follow the predictions of the biased reptation including fluctuations (BRF) theory. However, deviations due to nonidealities of the separation conditions are observed. The measured migration parameters can be used to optimize the performance of separation systems.     

Cyclic capillary electrophoresis

A strategy is described here for increasing both the resolution and the flexibility of capillary electrophoresis performed in a sieving medium of ungelled polymer. This strategy is based on analysis and, sometimes, re-analysis that is done in several stages of constant-field electrophoresis. Enhancement-stages are between the analysis-stages. An enhancement-stage (i) increases the separation between peaks, while (ii) moving DNA molecules in the reverse direction. An enhancement-stage is based on an electrophoretic ratchet generated by a pulsed electrical field that can be zero-integrated. The ratchet-generating pulses are longer than the field pulses that have previously been used to improve the resolution of DNA molecules. No limit has been found to the resolution enhancement achievable. Apparently, diffusion-induced peak broadening is inhibited and, in some cases, may be reversed by the ratchet. The enhancement-stages are critically dependent on the electrical field-dependence of a plot of electrophoretic mobility as a function of DNA length. To generate the pulsed electrical field, a computer-controlled system with a time resolution of 30 microseconds has been developed. Programming is flexible enough to embed other pulses within ratchet-generating pulses. These other pulses can be either the previously used, shorter field-inversion pulses or high-frequency periodic oscillations previously found to sharpen peaks.     

Use of a heterogeneous buffer combination in microchip electrophoresis for high-resolution separation by on-line concentration of DNA samples

We have developed a novel high-resolution separation technique of DNA fragments in a heterogeneous combination of a sample buffer and a separation buffer. The use of a heterogeneous buffer combination is a simple method for on-line concentration of DNA fragments, in which a sample buffer is simply exchanged with one including taurine anions. The mobility of taurine anions, co-ions for DNA, is lower than the that of acetate anions in a separation buffer. The difference in the mobility invokes transient isotachophoresis. The current technique allows DNA fragments to be effectively concentrated and the separation length of microchips to be shorter than that of conventional ones by a factor of three without deterioration in separation resolution and any modification of a chip design. Fragments of 100-bp DNA ladders (100-1000 bp) were separated with high resolution (0.72-10.7) within 60 s with a 10 mm separation length on a polymethyl methacrylate chip. Furthermore, fragments of 10-bp DNA ladders (10-330 bp) were separated with high resolution (0.69-2.00) with a 10 mm separation length within 50 s without band broadening. The current achievements will make it possible to fabricate compact devices for microchip electrophoresis.     

Optical absorption and valence band photoemission from uncapped CdTe nanocrystals

CdTe nanocrystals have been successfully fabricated by a mechanical alloying process. X-ray diffraction (XRD) patterns demonstrate that a single-phase CdTe compound with a zinc blende structure has been formed after ball milling elemental Cd and Te mixture powders for 27 h. The large broadening effect for the width of the {111} diffraction peak of uncapped CdTe nanocrystals on smaller size was observed in slowly scanned XRD patterns. The X-ray photoelectron spectrum was used to study the surface of the uncapped CdTe nanocrystals within both core level and valence band regions. The presence of tellurium oxide film on the surface of the uncapped CdTe nanocrystals has been detected in the X-ray photoelectron spectrum of the Te 3d core level, which was comparable to the observed amorphous oxide thin layer on the surface of uncapped CdTe nanocrystals in a high resolution transmission electron microscopy (HRTEM) image. The energy of the valence band maximum for uncapped CdTe powders blue shifts to the higher energy side with smaller particle sizes. In UV-visible optical absorption spectra of the suspension solution containing uncapped CdTe nanocrystals, the absorption peaks were locating within the ultraviolet region, which shifted toward the higher energy side with prolonged ball milling time. Both blue shifts of valence band maximum energy and absorption peaks with decreasing particle size provide a unique pathway to reveal the quantum confinement effect of uncapped CdTe nanocrystals.     

Dispersion of discrete sample signals within aerosol spray chambers: preliminary investigations

A barrel-type spray chamber with a baffle system and various other structures were used for characterization of band-broadening phenomena occurring within aerosol spray chambers, with an ICP-AES detector used in this case. Absolute values of aerosol particle size distributions from nebulizer/spray chamber systems were measured and found to be affected by the relative positions of impact surfaces with respect to the nebulizer. Smaller particles, but less total mass was observed as the baffle was moved closer to the nebulizer tip. However, based on measurements herein, it does not appear that particle sizes in any case will be small enough within the spray chamber for diffusion to influence band broadening for the bulk of the aerosol mass, as particularly important for mass sensitive detectors such as ICP-AES, although diffusion is likely to be important to particle number sensitive detectors.

Dispersion within the turbulent gas-phase regions of spray chambers appears to be relatively small. Geometries which introduce stagnant gas flow regions appear to have larger effects on band-broadening. The smoke tests utilized in these studies were found to be useful aids for visualization of spray chamber flow phenomena that influence dispersion of discrete signals.

Recirculation phenomena can also play a role in band broadening. Recirculation of aerosol re-entrained into the nebulizer jet appears to have an effect on dispersion, as represented by the full width at half maximum values. On the other hand, recirculation renebulization leads to substantial peak tailing.     

On-chip fraction collection for multiple selected ssDNA fragments using isolated extraction channels

High efficiency and high-purity fraction collection is highly sought in analysis of fragments-of-interest from selective polymerase chain reaction (PCR) products generated by High Coverage Gene Expression Profiling (HiCEP) methods. Here we demonstrate a new electrophoretic chip device enabling automatic high-efficient fractionation of multiple ssDNA target fragments during a run of separation. We used thoroughly isolated extraction channels for each selected target to reduce the risk of cross-contamination between targets due to cross-talk of extraction channels. Fragments of 35, 108 and 138 b, were successfully isolated, then the recovery was PCR-amplified and assessed by capillary electrophoresis (CE) analysis. Total impurity level of the targets due to unwanted fragments of 0.7%, 2% and 6% respectively, was estimated. Difficulties in collecting multiple target factions are due to band diffusion and DNA adsorption to the walls for the fragments in the separation channel, which is generated by transferring the DNA target fraction from the extraction section to the target reservoir. Therefore, we have carefully measured band broadening and analyzed its influence on the separation resolution due to the delay.     

Contribution of axial dispersion to band spreading in perfusion chromatography

A new interpretation of the band spreading data in perfusion chromatography is proposed by investigating the relative importance of axial dispersion in perfusive beds. Elution chromatography of proteins (bovine serum albumin and lysozyme) under non-retained conditions on two kinds of reversed-phase perfusive supports (POROS R1/H and POROS R2/H), which have different pore structures, were carried out to obtain the axial dispersion data. The Knox equation and some empirical correlations for dispersion coefficients in porous media were applied to correlate the experimental data. The influences of particle properties, solute molecular sizes and flow velocity on the dispersion coefficient were elucidated. Axial dispersion was recognised to be the main contributor to peak broadening in perfusion chromatography. The dependence of the height equivalent to a theoretical plate on flow-rate was found to be the result of the velocity dependence of the axial dispersion. The dispersion coefficient in a perfusive column can be well represented both by a power-law relationship and a correlation derived based on stochastic theory. Pursuant to these, it was found that pore size distribution of the perfusive particles and solute molecular size are important parameters, which influenced the dispersion results significantly.     

Electrophoretic motion of a charged porous sphere within micro- and nanochannels

Electrophoretic motion of a charged porous sphere within micro- and nanochannels is investigated theoretically. The Brinkman model and the full non-linear Poisson-Boltzmann equation are adopted to model the system, with the charged porous sphere resembling polyelectrolytes like proteins and DNA. General electrokinetic equations are employed and solved with a pseudo-spectral method. Key parameters of electrokinetic interest are examined for their respective effect as well as overall impact on the particle motion. We found, among other things, that the confinement effect of the channel can be so drastic that 75% reduction of particle mobility is observed in some situations for a poorly permeable particle. However, only 15% for the corresponding highly permeable particle due to the allowance of fluid penetration which alleviates the retarding shear stress significantly. In particular, an intriguing phenomenon is observed for the highly permeable particle: the narrower the channel is, the faster the particle moves! This was experimentally observed as well in the literature on DNA electrophoresis within nanostructures. The reason behind it is thoroughly explained here. Moreover, charged channels can exert electroosmosis flow so dominant that sometimes it may even reverse the direction of the particle motion. Comparison with experimental data available in the literature for some polyelectrolytes is excellent, indicating the reliability of this analysis. The results of this study provide fundamental knowledge necessary to interpret experimental data correctly in various microfluidic and nanofluidic operations involving bio-macromolecules, such as in biosensors and Lab-on-a-chip devices.     

Sieving properties of end group‐halogenated Pluronic polymer matrix in DNA separation under nondenaturing CE analysis

CE‐SSCP analysis is a well‐established DNA separation method that is based on variations in mobility caused by sequence‐induced differences in the conformation of single‐stranded DNA. The resolution of CE‐SSCP analysis was improved by using a Pluronic polymer matrix, and it has been successfully applied in various genetic analyses. Because the Pluronic polymer forms a micellar cubic structure in the capillary, it provides a stable internal structure for high‐resolution CE‐SSCP analysis. We hypothesized that formation of micellar cubic structure is influenced by the end hydroxyl group of the Pluronic polymer, which affords structural stability through hydrogen bonding. To test this hypothesis, the hydroxyl group was halogenated to eliminate the hydrogen bonding without disturbing the polarity of polymer matrix. CE‐SSCP resolution of two DNA fragments with a single base difference was significantly worse in the halogenated polymer matrices due to band broadening. The viscoelastic properties of control (which has hydroxyl group), chlorinated, and brominated F108 solution upon heating were also investigated by rheological experiments, and we found that gelation was significantly associated with resolution. In this series of experiments, the effect of the hydroxyl group in Pluronic polymer matrix on separation resolution of CE‐SSCP analysis was demonstrated.     

On the optimization of the shell thickness of superficially porous particles

The thickness of the porous shells of superficially porous particles influences the separation power of columns packed with these packing materials. Models of the mass transfer kinetics across porous adsorbents permit the prediction of the HETP curves of columns packed with particles having shells of different thicknesses, for molecules of different sizes. Decreasing the thickness of the porous layer potentially results in lower values of the “C-term” of the HETP curve and of the minimum of these curves. The Poppe plots calculated under isocratic and gradient conditions show that the separation power of columns packed with superficially porous particles increases significantly with decreasing thickness of the porous layer but this increase is more important for larger than for smaller molecules. The resolution between pairs of compounds increases at constant values of their retention factors when the strength of the eluent must be reduced to compensate for the decrease of their retention that is caused by the reduction of the surface area of the stationary phase. Thus, the separation power of columns packed with superficially porous particles increases with decreasing shell thickness. In contrast, if analysts do not compensate for the retention decrease, the resolution between small molecular weight compounds becomes worse with thin than with thick superficially porous particles. Finally, the importance of using instruments providing low extra-column band broadening contributions is stressed.     

Fabrication and performance of a microfluidic traveling-wave electrophoresis system

A microfluidic traveling-wave electrophoresis (TWE) system is reported that uses a locally defined traveling electric field wave within a microfluidic channel to achieve band transport and separation. Low voltages, over a range of -0.5 to +0.5 V, are used to avoid electrolysis and other detrimental redox reactions while the short distance between electrodes, ～25 μm, provides high electric fields of ～200 V cm(-1). It is expected that the low voltage requirements will simplify the future development of smaller portable devices. The TWE device uses four interdigitated electrode arrays: one interdigitated electrode array pair is on the top of the microchannel and the other interdigitated electrode array pair is on the microchannel bottom. The top and bottom substrates are joined by a PDMS spacer that has a nominal height of 15 μm. A pinched injection scheme is used to define a narrow sample band within an injection cross either electrokinetically or hydrodynamically. Separation of two dyes, fluorescein and FLCA, with baseline resolution is achieved in less than 3 min and separation of two proteins, insulin and casein is demonstrated. Investigation of band broadening with fluorescein reveals that sample band widths equivalent to the diffusion limit can be achieved within the microfluidic channel, yielding highly efficient separations. This low level of band broadening can be achieved with capillary electrophoresis, but is not routinely observed in microchannel electrophoresis. Sample enrichment can be achieved very easily with TWE using a device with converging electric field waves controlled by two sets of independently controlled interdigitated electrodes arrays positioned serially along the microchannel. Sample enrichment of 40-fold is achieved without heterogeneous buffer/solvent systems, sorptive, or permselective materials. While there is much room for improvement in device fabrication, and many capabilities are yet to be demonstrated, it is anticipated that the capabilities and performance demonstrated herein will enable new lab-on-a-chip processes and systems.     

Why ultra high performance liquid chromatography produces more tailing peaks than high performance liquid chromatography, why it does not matter and how it can be addressed

The purpose of this study is to demonstrate, with experiments and with computer simulations based on a firm chromatographic theory, that the wide spread perception of that the United States Pharmacopeia tailing factor must be lower than 2 (T(f)<2) is questionable when using the latest generation of LC equipment. It is shown that highly efficient LC separations like those obtained with sub-2 μm porous and 2.7 μm superficially porous particles (UHPLC) produce significantly higher T(f)-values than the corresponding separation based on 3 μm porous particles (HPLC) when the same amount of sample is injected. Still UHPLC separations provide a better resolution to adjacent peaks. Expressions have been derived that describe how the T(f)-value changes with particle size or number of theoretical plates. Expressions have also been derived that can be used to scale the injection volume based on particle size or number of theoretical plates to maintain the T(f)-value when translating a HPLC separation to the corresponding UHPLC separation. An aspect that has been ignored in previous publications. Finally, data obtained from columns with different age/condition indicate that T(f)-values should be complemented by a peak width measure to provide a more objective quality measure.     

Preparation of porous polylactide microspheres by emulsion‐solvent evaporation based on solution induced phase separation

Porous polylactide (PLA) microspheres were fabricated by an emulsion‐solvent evaporation method based on solution induced phase separation. Scanning electron microscopy (SEM) observations confirmed the porous structure of the microspheres with good connectivity. The pore size was in the range of decade micrometers. Besides large cavities as similarly existed on non‐porous microspheres, small pores were found on surfaces of the porous microspheres. The apparent density of the porous microspheres was much smaller than that of non‐porous microspheres. Fabrication conditions such as stirring rate, good solvent/non‐solvent ratio, PLA concentration and dispersant (polyvinyl alcohol, PVA) concentration had an important influence on both the particle size and size distribution and the pore size within the microspheres. A larger pore size was achieved at a slower stirring rate, lower good solvent/non‐solvent ratio or lower PLA concentration due to longer coalescence time. Copyright © 2005 John Wiley & Sons, Ltd.     

Nonmonotonous variation of DNA angular separation during asymmetric pulsed field electrophoresis

Asymmetric pulsed field electrophoresis within crystalline arrays is used to generate angular separation of DNA molecules. Four regimes of the frequency response are observed, a low frequency rise in angular separation, a plateau, a subsequent decline, and a second plateau at higher frequencies. It is shown that the frequency response for different sized DNA is governed by the relation between pulse time and the reorientation time of DNA molecules. The decline in angular separation at higher frequencies has not previously been analyzed. Real‐time videos of single DNA molecules migrating under high frequency‐pulsed electric field show the molecules no longer follow the head to tail switching, ratchet mechanism seen at lower frequencies. Once the pulse period is shorter than the reorientation time, the migration mechanism changes significantly. The molecule reptates along the average direction of the two electric fields, which reduces the angular separation. A freely jointed chain model of DNA is developed where the porous structure is represented with a hexagonal array of obstacles. The model qualitatively predicts the variation of DNA angular separation with respect to frequency.     

Absorption and fluorescence spectroscopy of growing ZnO quantum dots: size and band gap correlation and evidence of mobile trap states

ZnO nanoparticles constitute a convenient model system for fundamental studies with many possible technical applications in, for example, sensors and the field of catalysis and optoelectronics. A large set of ZnO quantum dots in the size range 2.5-7 nm have been synthesized and analyzed in detail. Time resolved in situ UV-vis absorption measurements were used to monitor the growth of these particles in solution by correlating the optical band gap to particle size given from X-ray diffraction (XRD) measurements. The particles formed were isotropic in shape, but small initial deviations gave indications of a transition from thermodynamic to kinetically controlled growth for particles around 4 nm in diameter. On the basis of this, the behavior and mechanisms for the particle growth are discussed. The fluorescence dependence on particle size was investigated by combining fluorescence and UV-vis measurements on growing particles. This revealed that the positions of the fluorescence trap states are mobile toward the conduction- and valence band. A broadening of the trap states was also found, and a surface dependent mechanism of the trap state shift and broadening is proposed.     

Study of the influence of the aspect ratio on efficiency, flow resistance and retention factors of packed capillary columns in pressure- and electrically-driven liquid chromatography

The influence of the aspect ratio, rho (rho = column diameter/particle diameter), on column parameters such as efficiency, retention factors and flow resistance was studied in both high-performance liquid chromatography and capillary electrochromatography with packed capillary columns. In order to compare the true efficiencies of different columns, a procedure to account for external band broadening was applied. High efficiencies (reduced plate height h approximately 2) were obtained with capillary columns with internal diameters of 150-, 100-, and 75-microm, packed with 10-microm particles. In contrast to previous reports in the literature, no significant improvements in efficiency or flow resistance were observed when the aspect ratio of such columns was decreased. Our observations suggest that the wall effect in these types of columns is not significant. When the aspect ratio was decreased by increasing the particle size, a decrease in reduced plate height was observed. However, the results of flow resistance measurements showed that the latter effect should be attributed to differences in packing and particle batch quality rather than to differences in the aspect ratio.     

Ion-pair reversed-phase high-performance liquid chromatography analysis of oligonucleotides: retention prediction

An ion-pair reversed-phase HPLC method was evaluated for the separation of synthetic oligonucleotides. Mass transfer in the stationary phase was found to be a major factor contributing to peak broadening on porous C18 stationary phases. A small sorbent particle size (2.5 microm), elevated temperature and a relatively slow flow-rate were utilized to enhance mass transfer. A short 50 mm column allows for an efficient separation up to 30mer oligonucleotides. The separation strategy consists of a shallow linear gradient of organic modifier, optimal initial gradient strength, and the use of an ion-pairing buffer. The triethylammonium acetate ion-pairing mobile phases have been traditionally used for oligonucleotide separations with good result. However, the oligonucleotide retention is affected by its nucleotide composition. We developed a mathematical model for the prediction of oligonucleotide retention from sequence and length. We used the model successfully to select the optimal initial gradient strength for fast HPLC purification of synthetic oligonucleotides. We also utilized ion-pairing mobile phases comprised of triethylamine (TEA) buffered by hexafluoroisopropanol (HFIP). The TEA-HFIP aqueous buffers are useful for a highly efficient and less sequence-dependent separation of heterooligonucleotides.     

Effect of organic solvent, electrolyte salt and a loading of cellulose tris (3,5-dichlorophenyl-carbamate) on silica gel on enantioseparation characteristics in capillary electrochromatography

The effect of the amount of the chiral selector, cellulose tris(3,5-dichlorophenylcarbamate) (CDCPC) on the separation characteristics of enantiomers of some charged and neutral analytes was studied in capillary electrochromatography (CEC). For better understanding of the effect of the loading of the chiral selector on the particles and pore size of the packing material, laser-beam particle size analyzer and scanning electron microscopy (SEM) were used. As shown in this study, CDCPC can be used for CEC enantioseparations of a wide range of chiral charged and neutral analytes with high efficiency. The loading of the polysaccharide derivative on the surface of silica materials even in high amounts does not markedly affect the particle size and porous structure of the packing material. The separation characteristics are strongly affected by the loading of CDCPC onto silica gel in both CEC and capillary liquid chromatography (CLC).     

Peak broadening from an electrothermal vaporization sample introduction source into an inductively coupled plasma

Signal broadening using electrothermal vaporization with inductively coupled mass spectrometry (ETV-ICPMS) occurs at a rate much faster than would be predicted by simple longitudinal diffusion. A Monte Carlo simulation that focused on particle motion within the transport tubing was created to elucidate the causes of this dispersion within ETV-ICPMS. Several parameters, including the diffusion coefficient, tube diameter, transport tube length, and flow rate were varied to discern their role in signal broadening. Using typical instrumental parameters, the parabolic flow profile generated by laminar flow of the carrier gas was shown to be the primary cause of dispersion. Manipulating the aforementioned variables to lessen the effects of laminar flow led to a decrease in dispersion. Conversely, increasing the role of laminar flow promoted broadening. The broadening processes should be applicable to any transient introduction system where material must be transported to a detection system. Due to the difference in the rate of broadening expected for particles of different sizes, the simulation was used to calculate the average size of particles generated in the ETV using different mass amounts of sample. No change in particle size (∼1 nm) was seen for mass amounts ranging from 10–10 000 pg, which suggests that the particle number is increased with increasing sample mass rather than the average particle size. Using this method of determining particle size, it might be possible to further evaluate the mechanisms of physical ‘carrier’ action.     

Divergent dispersion behavior of ssDNA fragments during microchip electrophoresis in pDMA and LPA entangled polymer networks

Resolution of DNA fragments separated by electrophoresis in polymer solutions ("matrices") is determined by both the spacing between peaks and the width of the peaks. Prior research on the development of high-performance separation matrices has been focused primarily on optimizing DNA mobility and matrix selectivity, and gave less attention to peak broadening. Quantitative data are rare for peak broadening in systems in which high electric field strengths are used (>150 V/cm), which is surprising since capillary and microchip-based systems commonly run at these field strengths. Here, we report results for a study of band broadening behavior for ssDNA fragments on a glass microfluidic chip, for electric field strengths up to 320 V/cm. We compare dispersion coefficients obtained in a poly(N,N-dimethylacrylamide) (pDMA) separation matrix that was developed for chip-based DNA sequencing with a commercially available linear polyacrylamide (LPA) matrix commonly used in capillaries. Much larger DNA dispersion coefficients were measured in the LPA matrix as compared to the pDMA matrix, and the dependence of dispersion coefficient on DNA size and electric field strength were found to differ quite starkly in the two matrices. These observations lead us to propose that DNA migration mechanisms differ substantially in our custom pDMA matrix compared to the commercially available LPA matrix. We discuss the implications of these results in terms of developing optimal matrices for specific separation (microchip or capillary) platforms.