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.     

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.     

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.     

The electric field dependence of DNA mobilities in agarose gels: a reinvestigation

The electric field dependence of the electrophoretic mobility of linear DNA fragments in agarose gels was reinvestigated in order to correct the observed mobilities for the different temperatures actually present in the gel during electrophoresis in different electric field gradients. When corrected to a common temperature, the electrophoretic mobilities of DNA fragments less than or equal to 1 kilobase pairs (kbp) in size were independent of electric field strength at all field strengths from 0.6 to 4.6 V/cm if the gels contained less than or equal to 1.4% agarose. The mobilities of larger DNA fragments increased approximately linearly with electric field strength. If the agarose concentration was higher than 2%, the mobilities of all DNA fragments increased with increasing electric field strength. The electric field dependence of the mobility was larger in gels cast and run in Tris-borate buffer (TBE) than in gels cast and run in Tris-acetate buffer (TAE), and was more pronounced in gels without ethidium bromide incorporated in the matrix. Ferguson plots were constructed for the various DNA fragments, both with and without extrapolating the temperature-corrected mobilities to zero electric field strength. Linear Ferguson plots were obtained for all fragments less than or equal to 12 kbp in size in agarose gels less than or equal to 1.4% in concentration if the mobilities were first extrapolated to zero electric field strength. Concave upward curvature of the Ferguson plots was observed for DNA fragments greater than or equal to 2 kbp in size at finite electric field strengths. Convex downward curvature of the Ferguson plots was observed for DNA fragments greater than or equal to 1 kbp in size in agarose gels greater than or equal to 2% in concentration. The mobilities of the various DNA fragments, extrapolated to zero agarose concentration and zero electric field strength, decreased with increasing DNA molecular weight; extrapolating to zero molecular weight gave an "intrinsic" DNA mobility of 2.7 x 10(-4) cm2/Vs at 20 degrees C. The pore sizes of LE agarose gels cast and run in TAE and TBE buffers were estimated from the mobility of the DNA fragments.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Enhancing the sensitivity of DNA detection and recovery from agarose gels

By inserting nitrocellulose strips into agarose gels alongside the electrophoresed lanes and passing an electric current perpendicularly in the direction of the strips, highly efficient transfer of DNA bands onto the membrane in the form of concentrated dots is achieved. DNA detection limits by this technique are enhanced, at least three times as visualized by ethidium bromide fluorescence and at least twice more by radiolabeling.     

Effect of linear polymer additives on the electroosmotic characteristics of agarose gels in ultrathin-layer electrophoresis.

Electroosmotic properties of agarose gels with low, medium, high and super high electroendosmosis (EEO) were evaluated based on the apparent electric field mediated mobility of a neutral, fluorescent marker under constant field strength using ultrathin-layer separation configuration. Electroosmotic flow mobility values were measured in different gel concentrations and also in the absence and the presence of various linear polymer additives. Under ultrathin-layer separation conditions, a slight decrease in electroosmotic flow mobility was observed with increasing agarose gel concentration of 1 to 3% for all agarose gels investigated. When linear polymer additives, such as linear polyacrylamide, hydroxyethyl cellulose or polyethylene oxide were added to 1% low electroendosmosis agarose gel, significant reduction of the electroosmotic flow properties were observed with increasing additive concentration. Effect of the intrinsic electroosmotic properties of the various electroendosmosis agaroses on the apparent mobilities and separation performance of double-stranded DNA fragments during automated ultrathin-layer agarose gel electrophoresis was also investigated.     

Preparative two-dimensional gel electrophoresis with agarose gels in the first dimension for high molecular mass proteins

A two-dimensional gel electrophoresis (2-DE) method that uses an agarose isoelectric focusing (IEF) gel in the first dimension (agarose 2-DE) was compared with an immobilized pH gradient 2-DE method (IPG-Dalt). The former method was shown to produce significant improvements in the 2-D electrophoretic separation of high molecular mass proteins larger than 150 kDa, up to 500 kDa, and to have a higher loading capacity, as much as 1.5 mg proteins in total for micropreparative runs. The extraction medium found best in this study for agarose 2-DE of mammal tissues was 6 M urea, 1 M thiourea, 0.5% 2-mercaptoethanol, protease inhibitor cocktail (Complete Mini EDTA-free), 1% Triton X-100 and 3% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Trichloroacetic acid (TCA) treatment of the agarose gel after IEF is to be carefully weighed beforehand, because some high molecular mass proteins were less likely to enter the second-dimensional polyacrylamide gel after TCA fixation, and proteins such as mouse skeletal muscle actin gave pseudospots in the agarose 2-DE patterns without TCA fixation. As a good compromise we suggest fixation of proteins in the agarose gel with TCA for one hour or less. The first-dimensional agarose IEF gel containing Pharmalyte as a carrier ampholyte was 180 mm in length and 2.5-4.8 mm in diameter. The gel diameter was shown to determine the loading capacity of the agarose 2-DE, and 1.5 mg liver proteins in total were successfully separated by the use of a 4.8 mm diameter agarose gel.     

Scattering properties of agarose gels

Static light scattering and small angle neutron scattering measurements are reported for agarose hydrogels prepared under various conditions of concentration and temperature. For the wide range of transfer wave vector explored, these measurements show that the gels do not display fractal behaviour. Their structure is better described by a stretched exponential form, in which the value of the exponent is n = 0.2. As found by other authors, a maximum in the scattering intensity is observed in the light scattering spectra. The position of the maximum, q_max, depends on the concentration and on the thermal history of the sample. The inverse length 1/q_max is in good agreement with published measurements of the pore size D in this system. Preliminary measurements by small angle scattering indicate that the sol-gel transition is not of spinodal type.     

Capillary electrophoresis of DNA in agarose solutions at 40 degrees C.

DNA fragments ranging from 72 to 1353 bp in length (phi X174 RF DNA/HaeIII) were separated by capillary electrophoresis in 0.3-2.0% solutions of agarose (Sea-Plaque GTG) at 40 degrees C. Liquified agarose above its gelling temperature is easily filled and refilled into capillaries. Its background absorbance at 260 nm was sufficiently low to allow for DNA detection at an estimated DNA load of 13 ng/10 components. Sample injection proceeded at 1 kV for 16 s. The internal capillary diameter was 150 mu, the migration path 27 cm. Migration times varied from 5 to 14 min at 185 V/cm. Potentially, the applicability of capillary electrophoresis in agarose solutions can be expected to extend to the entire size range of DNA, in view of the recent demonstration of kb-sized circular DNA separations in agarose solutions, and those of Mb-sized DNA-agarose complexes in linear polyacrylamide solutions.     

Oxygen-17 relaxation in aqueous agarose gels

Nuclear magnetic relaxation of oxygen-17 in H₂¹⁷O enriched agarose gels shows that existing explanations of water behaviour are oversimplified. Satisfactory models must include at least three proton phases, two of which involve water molecules.     

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.     

Adaptive weighted least squares method for the estimation of DNA fragment lengths from agarose gels

The size of DNA fragments is most frequently estimated from their electrophoretic mobilities. Agarose gels are used to estimate the size of DNA fragments ranging from a few hundred nucleotides to more than 20 kbp. The common practice when estimating the unknown fragment sizes is to plot the log of the size of molecular weight standards against their mobility and read the values of unknowns from this graph. However, due to perturbations in the gel, such plots often show pronounced curvature which may introduce significant subjectivity into the interpolation process. We present a new method "adaptive weighted least squares (AWLS)" based on the significance test to choose the order of polynomial. We compare this with the method introduced by Schaffer based on the modification of the Southern method. The results obtained by AWLS are significantly better than the method introduced by Schaffer. Different lanes are tested for consistency.     

Direct chemiluminescent immunodetection of proteins in agarose gels

Chemiluminescent immunodetection of proteins separated by polyacrylamide gel electrophoresis is generally performed only after Western blotting. Agarose gels are adequately permeable to allow immunoprobing directly in the gel. Chemiluminescent substrates had not been applied for direct immunoprobing of agarose gels. In a comparison with direct immunostaining of fibrinogen derivatives with horse radish peroxidase (HRP)-conjugated primary antibody using 3,3'-diaminobenzidene (DAB) yielding a sensitivity in the low nanogram range, a luminol-based chemiluminescent detection extended sensitivity to the mid-picogram range with seemingly no interference from either regular or glyoxyl agarose gels. The high sensitivity of chemiluminescence extends utility of direct immunoprobing of either agarose or glyoxyl agarose composite gels for detection and measurement of both high and low molecular weight proteins/peptides which are not easily detected/measured by Western blotting. However, due to the thickness of the gels, direct immunoprobing can be quite laborious. To eliminate that drawback, we describe a simplified approach, converting the thick gels to thin ones prior to probing, that makes direct immunoprobing as easy as Western blotting.     

Counterion-dye staining method for DNA in agarose gels using crystal violet and methyl orange

Sensitive and safe methods for visualization of DNA in agarose gels are described. 0.001% crystal violet dissolved in distilled water was used for DNA staining on agarose gels and it could detect as little as 16 ng of DNA (3 kb, pGem-7Zf/EcoRI) without destaining procedure. The detection limit is four times lower than that of ethidium bromide. To improve the sensitivity, we studied a counterion-dye staining method using methyl orange as a counterion-dye which contributes to reduce excessive background staining by crystal violet. Dye concentration, pH of staining solution, mixing molar ratio of two dyes, and staining times were optimized for the counterion-dye staining. By the staining with a mixed solution of 0.0025% crystal violet and 0.0005% methyl orange in distilled water, 8 ng of the 3 kb DNA in an agarose gel was detected within 30 min.     

Discontinuous buffer system for polyacrylamide and agarose gel electrophoresis of DNA fragments

DNA fragments up to 9 kb in size were stacked and separated by polyacrylamide gel electrophoresis, and those up to 50 kb in size by agarose gel electrophoresis, using a discontinuous buffer system. Polyacrylamide gels at pH 8.9, 2 degrees C, 0.01 M ionic strength, yielded sharp bands with DNA loads of 8 micrograms/cm2 of gel of a mixture of 19 DNA fragments in the size range of 72-23130 bp, while agarose gels at pH 8.5, 25 degrees C, provided well-resolved, unperturbed bands at 0.04 M ionic strength with DNA loads of 1 microgram/cm2 of the same mixture. Note that the ionic strength of the agarose gels is comparable to the conventionally used 0.5 x TBE (Tris-borate-EDTA) buffer, while that successfully applied to polyacrylamide is seven-fold less than the ionic strength of conventionally used 1 x TBE buffer, with a substantially shorter duration of electrophoresis as a result. The application of a discontinuous buffer system to the gel electrophoresis of DNA results in (i) Band identification by Rf, the migration distance relative to a sharply defined "buffer front" (moving boundary). This is sufficiently labor saving, compared to determining absolute mobilities, so as to render practical the expression of bands as numbers, with benefits for data storage, statistical manipulations and physico-chemical exploitation of mobility data. The use of Rf's also circumvents loss of precision in mobility measurement resulting from progressive band spreading of dye bands used as a front. (ii) A uniformly and highly concentrated starting zone, beneficial to resolution, is obtained, without the losses by which separate concentration steps are usually burdened.(ABSTRACT TRUNCATED AT 250 WORDS)     

Controlling electrophoretic trapping of circular DNA by addition of starch preparations to agarose gels

Starch preparations were added to agarose gels to enhance the electrophoretic trapping of circular plasmid DNA. The critical voltages required to trap the open circular (OC) and the supercoiled (SC) forms of a 13.1-kbp plasmid were measured in gels composed of agarose and added starch preparations. Modified starch preparations reduced the critical voltage required to trap the OC form of the plasmid to approximately one-third of the control value (in 1% agarose gels). Amylose (a fraction of starch with a low amount of branching) also reduced the critical voltage to trap the OC form in a similar manner. The critical voltage to trap the SC form of the plasmid was not significantly reduced by the starch preparations. The capacity to trap OC DNA was increased by the addition of higher amounts of the starch preparations added to the gels. Field inversion gel electrophoresis was used to characterize the length of the traps in the gels. The starch preparations and amylose increased the trap lengths approximately twofold. The increased trap length correlated with the decreased critical voltage required to trap the OC form of the 13.1-kbp plasmid. Certain commercial equipment, instruments, or materials are identified in this article to specify adequately the experimental procedures. Such identification does not imply recommendation by National Institute of Standards and Technology, nor does it imply that the materials or equipment are necessarily the best available for the purpose.     

Ternary complexes in gels from agarose and from chemically‐modified agarose

Thermodynamic data and mechanical measurements are shown for gels prepared in aqueous binary solvents (water/DMSO, water/DMF, water/methyl formamide and water/formamide). When electrostatic interactions, as opposed to hydrogen bonding, can be established with the cosolvent (DMSO, DMF, methyl formamide) we come to the conclusion that ternary complexes are formed (agarose/water/cosolvent). In the case of chemically‐modified agarose (OH groups replaced by OCH₃ groups) we suggest that these cosolvents are directly involved in the formation of the gel.