Separation performance of single-stranded DNA electrophoresis in photopolymerized cross-linked polyacrylamide gels

Considerable effort has been directed toward optimizing performance and maximizing throughput in ssDNA electrophoresis because it is a critical analytical step in a variety of genomic assays. Ultimately, it would be desirable to quantitatively determine the achievable level of separation resolution directly from measurements of fundamental physical properties associated with the gel matrix rather than by the trial and error process often employed. Unfortunately, this predictive capability is currently lacking, due in large part to the need for a more detailed understanding of the fundamental parameters governing separation performance (mobility, diffusion, and dispersion). We seek to address this issue by systematically characterizing electrophoretic mobility, diffusion, and dispersion behavior of ssDNA fragments in the 70-1,000 base range in a photopolymerized cross-linked polyacrylamide matrix using a slab gel DNA sequencer. Data are collected for gel concentrations of 6, 9, and 12%T at electric fields ranging from 15 to 40 V/cm, and resolution predictions are compared with corresponding experimentally measured values. The data exhibit a transition from behavior consistent with the Ogston model for small fragments to behavior in agreement with the biased reptation model at larger fragment sizes. Mobility data are also used to estimate the mean gel pore size and compare the predictions of several models.     

DNA electrophoresis in agarose gels: a simple relation describing the length dependence of mobility

Electrophoretic mobilities of DNA molecules ranging in length from 100 to 10 000 base pairs (bp) were measured in gels of eleven concentrations of agarose from 0.5 to 1.5%. Excellent fits of the dependence of mobility on DNA length were obtained with the relationship [equation: see text] showing an e(-L/gamma) crossover, where L is the length of a DNA fragment and gamma is a crossover length ranging from 8000 to 12000 bp. The other parameters in the fit are mu(s) the mobility of short DNA with unit charge in the limit as length is extrapolated to zero, and muI, the mobility of long DNA as length is extrapolated to infinity. This exponential relationship should be a useful interpolation function for determining DNA lengths over a wide range. The simplicity of this relationship may be of more fundamental significance and suggests that some common feature dominates the electrophoresis of double stranded DNA fragments in agarose gels, regardless of length.     

Diffusion coefficient of DNA molecules during free solution electrophoresis

The free-draining properties of DNA normally make it impossible to separate nucleic acids by free-flow electrophoresis. However, little is known, either theoretically or experimentally, about the diffusion coefficient of DNA molecules during free-flow electrophoresis. In fact, many authors simply assume that the Nernst-Einstein relation between the mobility and the diffusion coefficient still holds under such conditions. In this paper, we present an experimental study of the diffusion coefficient of both ssDNA and dsDNA molecules during free-flow electrophoresis. Our results unequivocally show that a simplistic use of Nernst-Einstein's relation fails, and that the electric field actually has no effect on the thermal diffusion process. Finally, we compare the dependence of the diffusion coefficient upon DNA molecular size to results obtained previously by other groups and to Zimm's theory.     

DNA electrophoresis in agarose gels: effects of field and gel concentration on the exponential dependence of reciprocal mobility on DNA length

Electrophoretic mobilities of DNA molecules ranging in length from 200 to 48 502 base pairs (bp) were measured in agarose gels with concentrations T = 0.5% to 1.3% at electric fields from E = 0.71 to 5.0 V/cm. This broad data set determines a range of conditions over which the new interpolation equation nu(L) = (beta+alpha(1+exp(-L/gamma))(-1) can be used to relate mobility to length with high accuracy. Mobility data were fit with chi(2) > 0.999 for all gel concentrations and fields ranging from 2.5 to 5 V/cm, and for lower fields at low gel concentrations. Analyses using so-called reptation plots (Rousseau, J., Drouin, G., Slater, G. W., Phys. Rev. Lett. 1997, 79, 1945-1948) indicate that this simple exponential relation is obeyed well when there is a smooth transition from the Ogston sieving regime to the reptation regime with increasing DNA length. Deviations from this equation occur when DNA migration is hindered, apparently by entropic-trapping, which is favored at low fields and high gel concentrations in the ranges examined.     

Single-molecule measurements of trapped and migrating circular DNA during electrophoresis in agarose gels

The effect of agarose gel concentration and field strength on the electrophoretic trapping of open (relaxed) circular DNA was investigated using microscopic measurements of individual molecules stained with a fluorescent dye. Three open circles with sizes of 52.5, 115, and 220 kbp were trapped by the electric field (6 V/cm) and found to be predominately fixed and stretched at a single point in the gel. The length of the stretched circles did not significantly change with agarose concentration of the gels (mass fractions of 0.0025, 0.01, and 0.02). The relaxation kinetics of the trapped circles was also measured in the gels. The relaxation of the large open circles was found to be a slow process, taking several seconds. The velocity and average length of the 52.5 kbp open circles and 48.5 kbp linear DNA were measured during electrophoresis in the agarose gels. The velocity increased when the agarose concentrations were lowered, but the average length of the open-circle DNA (during electrophoresis) did not significantly change with agarose gel concentrations. The circles move through the gels by cycles of stretching and relaxation during electrophoresis. Linear dichroism was also used to investigate the trapping and alignment of the 52.5 kbp open circles. The results in this study provide information that can be used to improve electrophoretic separations of circular DNA, an important form of genetic material and commonly used to clone DNA.     

Separation of large DNA molecules by pulsed-field gel electrophoresis A review of the basic phenomenology

Pulsed-field gel electrophoresis is a method of separating large DNA molecules. The distinctive feature of this method is that the direction of the electric field is changed periodically. During the five years since Schwartz and Cantor introduced this technique, there has been dramatic progress in pulsed-field instrumentation and in associated electrophoretic methods. Progress has been driven by practical experience with little guidance from theory. In this review, the basic phenomenology of pulsed-field gel electrophoresis is summarized and some speculations are advanced about possible molecular mechanisms.     

A new approach for separating low-molecular-weight RNA molecules by staircase electrophoresis in non-sequencing gels

Low-molecular-weight (LMW) RNA profiles, which include ribosomal and transfer RNA molecules with similar small sizes, are molecular signatures of microorganisms with a great potential in microbial identification. The greatest resolution of these profiles was achieved by staircase electrophoresis in sequencing gels. Nevertheless, this technique is difficult to use because it takes 7 h, the gels have large sizes and it is necessary to heat the system and to recycle the buffer to maintain the denaturing conditions and avoid smile effects. Most available sequencing slabs have no internal temperature control or homogenizing devices, which by contrast are present in some newly designed non-sequencing slabs. Nevertheless, these slabs present two important problems for separating LMW RNA molecules, the size of gels is only 20 cm (instead of 40 cm) and the maximum voltage that can be reached is only 840 V (instead 2400 V). Staircase electrophoresis follows a model in which the external polarization is incrementally modified with a constant time step value. In the present work, we experimentally confirmed that by reducing the time step and increasing the total number of steps a suitable resolution is achieved. Under these conditions, despite the smaller size of the gels and the lower values of the electric field, the intensity reaches higher values than in sequencing gels and the LMW RNA profiles are correctly separated in 5 h. The resolution of these profiles obtained in non-sequencing gels is similar to that obtained in sequencing ones facilitating the analysis of large populations of microorganisms in any laboratory.     

Formamide modified polyacrylamide gels for DNA sequencing by capillary gel electrophoresis.

Compressions are occasionally found during the separation of DNA sequencing fragments, particularly in G/C-rich regions and in gels operated at room temperature. Addition of at least 10% formamide to urea/polyacrylamide sequencing gels improves the denaturing capacity of the gel, minimizing compressions. Addition of 20% or more formamide decreases the separation rate, theoretical plate count, and resolution for normally migrating fragments. An optimum concentration of 10% formamide improves resolution of compressed regions without degrading the other characteristics of the gel. Operation of gels at room temperature simplifies the engineering associated with automated sequencers based on capillary gel electrophoresis.     

Design of separation length and electric field strength for high-speed DNA electrophoresis

Gel-based DNA separation on microchip will play an important role in future genomic analysis due to its potential for high-efficiency and high-speed. Optimal design of microchip and separation condition is essential to take full advantage of high-speed separation on microchip. Separation length L and electric field strength E, which are crucial for design of microchip system, are focused on in this paper. Simultaneous optimization of L and E was carried out to achieve the most rapid separation. It was shown that the condition of L and E and the shortest separation time is closely related to the shape of resolution Rs surface in a three-dimensional space with axes E, L, and Rs. This surface was investigated, taking sample injection, detector, diffusion, and Joule heating into account. Thermal gradient broadening due to Joule heating helps to produce camber or ridge shape of Rs surface, which is essential for the shortest separation length and separation time. Sample plug length and detection volume should be more carefully controlled in microchip. The property of diffusion coefficient was shown to play a key role in determining Rs surface.     

Resolution of a paradox in the electrophoresis of DNA in agarose gels

A paradox was observed in a previous study of the electrophoresis of linear DNA fragments in agarose gels (D. L. Holmes and N. C. Stellwagen, Electrophoresis 1990, 11, 5-15). The pore size of the agarose matrix was more accurately determined if the root-mean-square radius of gyration was used to measure DNA macromolecular size. However, the Ogston equations were obeyed and other gel parameters such as the apparent fiber radius and fiber volume appeared to be better described if the geometric mean radius was used to measure DNA size. This paradox can be resolved if relative mobilities (with respect to the smallest DNA molecule in the data set) are used to construct the Ferguson plots, instead of absolute mobilities. Using relative mobilities and the root-mean-square radius of gyration, the Ogston equations are obeyed and the pore size of the matrix is consistent with values determined by other methods.     

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 the electrokinetic properties of reversible electrophoresis gels for the separation and preparation of DNA

The effect of adding linear polymers to a novel reversible electrophoretic was measured. Reversible gels are formed using the polyanionic carbohydrate polymer, gellan gum. Gellan gum forms strong stable gels in the presence of divalent cations or diamines. The gels are reversible (return to solution) by changing the ionic environment or pH. Gellan gum is an anionic polymer, and the electrophoresis gels have considerable electroosmotic flow (EOF) toward the negative electrode. We measured the EOF in gellan gum electrophoresis gels as a function of gel concentration, buffer composition, and linear polymer additive. The linear polymers used in this study were polyethylene oxide and hydroxyethyl cellulose. Both polymers reduced EOF in the gels, in a manner dependent on molecular weight. Polymers with high molecular weight were more effective at reducing EOF. The addition of polymers increased the resolution of low molecular weight DNA. Native gellan gum resolved DNA from approx 50,000 to 1000 bp. Addition of the polymers resolved DNA down to approx 50 bp, in some instances. The influence of the polymers on circular plasmid DNA was also investigated. Addition of high molecular weight polyethylene oxide reduced the electrophoretic mobility of the nicked circular form compared to the supercoiled form.     

Using in situ rheology to characterize the microstructure in photopolymerized polyacrylamide gels for DNA electrophoresis

Photopolymerized cross-linked polyacrylamide hydrogels are attractive sieving matrix formulations for DNA electrophoresis owing to their rapid polymerization times and the potential to locally tailor the gel pore structure through spatial variation of illumination intensity. This capability is especially important in microfluidic systems, where photopolymerization allows gel matrices to be precisely positioned within complex microchannel networks. Separation performance is also directly related to the nanoscale gel pore structure, which is in turn strongly influenced by polymerization kinetics. Unfortunately, detailed studies of the interplay among polymerization kinetics, mechanical properties, and structural morphology are lacking in photopolymerized hydrogel systems. In this paper, we address this issue by performing a series of in situ dynamic small-amplitude oscillatory shear measurements during photopolymerization of cross-linked polyacrylamide electrophoresis gels to investigate the relationship between rheology and parameters associated with the gelation environment including UV intensity, monomer and cross-linker composition, and reaction temperature. In general, we find that the storage modulus G' increases with increasing initial monomer concentration, cross-linker concentration, and polymerization temperature. The steady-state value of G', however, exhibits a more complex dependence on UV intensity that varies with gel concentration. A simple model based on rubber elasticity theory is used to obtain estimates of the average gel pore size that are in surprisingly good agreement with corresponding data obtained from analysis of DNA electrophoretic mobility in gels cast under identical polymerization conditions.     

On-site manipulation of single chromosomal DNA molecules by using optically driven microstructures

We report a novel method for manipulation of single giant DNA molecules under a video microscope. Using optically driven microstructures, we manipulated chromosomal DNA of length in the order of millimetres, extended by electroosmotic flow without DNA breakage in aqueous solution: we picked up DNA, using microfabricated hooks and wound it around microfabricated bobbins.     

Incorporation of guanosine gels into sieving matrices for length- and sequence-based separation of DNA in capillary electrophoresis

Sieving gels are used in capillary gel electrophoresis to resolve DNA strands of different lengths. For complex samples, however, such as those encountered in metagenomic analysis of microbial communities or biofilms, length-based separation may mask the true genetic diversity of the community since different organisms may contribute same-length DNA with different sequences. There is a need, therefore, for DNA separations based on both the length and sequence. Previous work has demonstrated the ability of guanosine gels (G-gels) to separate four single-stranded DNA 76-mers that differ by only a few A/G base substitutions. The goal of the present work is to determine whether G-gels could be combined with commercial sieving gels in order to simultaneously separate DNA based on both length and sequence. The results are given for the four 76-mers and for a standard dsDNA ladder. Commercial sieving gels were used alone and in combination with G-gels. For the 76-mers, the combined medium was less efficient than the G-gel alone but was able to achieve partial resolution. The combined medium was at least as effective as the sieving gel alone at resolving the denatured DNA ladder and showed indications of sequence-based resolution as well, as supported by MALDI-MS. The results show that the combined sieving gel/G-gel medium retains the selectivity of the individual media, providing a promising approach to simultaneous length- and sequence-based DNA separation for metagenomic analysis of complex systems.     

Detection of DNA curvature by transverse pore gradient gel electrophoresis on PhastSystem gels.

It has been known since 1990 that DNA curvature can be recognized on transverse pore gradient gels by an intersection of "Ferguson curves" with those of DNA size standards. The miniaturized PhastSystem polyacrylamide gels allow one to detect DNA curvature effortlessly and fast and at great economy of sample relative to alternative methods of electrophoresis. Using the transverse gradient gel electrophoresis method, it was found that the 660 bp length subfragment of the matrix attachment region (MAR) sequence of the chicken lysosyme gene migrates as a fragment of 800-900 bp length. When subjected to digestion with the restriction enzyme HaeIII, the fragment gives rise to two species of 248 and 412 bp length, respectively. The Ferguson curves of both species intersect with those of DNA size standards, indicating that both exhibit curvature. Only the curvature of the 412 bp fragment conforms to prediction. Ethidium bromide abolishes the effect of curvature on the fragment, reducing its apparent size from 900 to 660, the value obtained by agarose gel electrophoresis.     

Sieving of double-stranded DNA during agarose gel electrophoresis

Agarose gel electrophoresis is used to fractionate linear, double-stranded DNA by its length. Sieving of the gel is the cause of this fractionation and has been investigated by developing theoretical models and by quantifying sieving observed during electrophoresis. Here are reviewed the following aspects of the fractionation of linear, double-stranded DNA by agarose gel electrophoresis: (1) the basic observations that qualitatively characterize these fractionations, (2) evidence for the deformation of DNA's random coil, (3) quantitative analysis of the relationship of observed electrophoretic mobility to the DNA's length, (4) theoretical models that have been developed to explain data presented in Sections 1-3, (5) observations not yet quantitatively explained by models, and (6) some aspects of the use of a variable electrical field (pulsed-field gel electrophoresis) to improve separations.     

Improving the length-fractionation of DNA during capillary electrophoresis

The present study develops a path-lengthening strategy for capillary electrophoresis of short double-stranded DNA molecules, in an aqueous solution of neutral polymer (hydroxypropylmethylcellulose). Tests of the dependence of fractionations on pulse times reveal the operation of at least one mechanism in addition to increase in effective path length. Electrophoresis is performed in the following two-stage cycles (cyclic electrophoresis): The first analysis-stage of each cycle is a constant field (forward) capillary electrophoresis. This analysis-stage reveals the length distribution of the shortest DNA molecules not previously analyzed. The second, enhancement-stage of each cycle is zero-integrated field electrophoresis (ZIFE). The enhancement-stage improves the DNA length-fractionation for the next DNA molecules to be analyzed. A slight reverse migration occurs in the enhancement-stage. Increase in both peak separation and peak sharpness contribute to improvement in the length-fractionation of DNA molecules.