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.     

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.     

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.     

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.     

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.     

Effect of electric field switching on the electrophoretic mobility of single-stranded DNA molecules in polyacrylamide gels

We have examined the effects of pulsed electric fields on the separation of single-stranded DNA molecules in polyacrylamide sequencing gels. Using different electric field pulsing regimens, the mobilities of single-stranded DNA molecules can be retarded or increased as compared to conventional electrophoresis. These results indicated that pulsed field techniques can be applied to gel electrophoresis of small single-stranded DNA molecules.     

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.     

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.     

Orientation of DNA and the agarose gel matrix in pulsed electric fields

Transient electric birefringence has been used as an analytical tool to study the orientation of DNA in agarose gels, and to study the orientation of the matrix alone. The sign of the birefringence of DNA oriented in an agarose gel is negative, as observed in free solution, indicating that the DNA molecules orient parallel to the direction of the electric field. If the median pore diameter of the gel is larger than the contour length of the DNA molecule, the DNA effectively does not see the matrix and the birefringence relaxation time is the same as observed in free solution. However, if the median pore diameter of the gel is smaller than the contour length of the DNA, the DNA molecule becomes stretched as well as oriented. For DNA molecules of moderate size (less than or equal to 4 kb), stretching in the gel causes the birefringence relaxation times to increase to the values expected for fully stretched molecules. Complete stretching is not observed for larger DNA molecules. The orientation and stretching of DNA molecules in the gel matrix indicates that end-on migration, or reptation, is a likely mechanism for DNA electrophoresis in agarose gels. When the electric field is rapidly reversed in polarity, very little change in the orientation of the DNA is observed if the DNA molecules were completely stretched and had reached their equilibrium orientation before the field was reversed in direction. Hence completely stretched, oriented DNA molecules are able to reverse their direction of migration in the electric field with little or no loss of orientation. However, if the DNA molecules were not completely stretched or if the equilibrium orientation had not been reached, substantial disorientation of the DNA molecules is observed at field reversal. The forced rate of disorientation in the reversing field is faster than the field-free rate of disorientation. Complicated patterns of reorientation can be observed after field reversal, depending on the degree of orientation in the original field direction. The effect of pulsed electric fields on the orientation of the agarose gel matrix itself was also investigated.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Temperature dependence of ion mobilities: Experiment and theory

The mobilities of several positive ions plus chloride ion in a variety of polar and nonpolar gases were measured over the temperature range 170–380 K by ICR spectrometry. The experimental results were compared to the predictions of the first-order kinetic theory for the nonpolar neutral systems. For the ion-polar neutral pairs, several theories were used for comparison. In all cases, adequate and reasonable fits were not possible. This result is attributed to theoretical shortcomings. Possible sources of error in the experimental data are discussed in detail. Other mobility measurements (mostly from drift tubes) were also compared with the kinetic theory results. The only systems which could be fit were alkali ion/rare gas neutral pairs. The mobility-temperature curves of other atomic ions and of all polyatomic ions could not be duplicated by the first-order theory.     

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.     

External electric field effects on fluorescence of pyrene butyric acid in a polymer film: concentration dependence and temperature dependence

Fluorescence spectra and electrofluorescence spectra (plots of the electric field-induced change in fluorescence intensity as a function of wavelength) have been measured at different temperatures for pyrene butyric acid (PBA) in a PMMA film at different concentrations. At a low concentration of 0.5 mol % where fluorescence emitted from the locally excited state of PBA (LE fluorescence) is dominant, LE fluorescence spectra show only the Stark shift in the presence of an electric field (F), which results from the difference in molecular polarizability between the ground and emitting states. At a high concentration of 10 mol % where the so-called sandwich-type excimer fluorescence (EX(1)) is dominant, both EX(1) and LE fluorescence are quenched by F. Another fluorescence assigned to a partially overlapped excimer (EX(2)) also exists at room temperature, and this emission is enhanced by F. As the temperature decreases, three fluorescence emissions whose electric field effects are different from each other become clear besides EX(1) and LE fluorescence, indicating that at least five fluorescence components exist at high concentrations at low temperatures. At a medium concentration of 5 mol % where EX(1) is comparable in intensity to the LE fluorescence, the intensity of EX(1) is not affected by F at any temperature, but LE fluorescence and EX(2) are markedly influenced by F at room temperature, and four fluorescence emissions are confirmed at low temperatures.     

The photochemistry of uracil: a reinvestigation

Results from a re-examination of the photochemical reactions undergone by uracil (Ura) are presented. Irradiation of Ura in frozen aqueous solution at -78.5°C produces two diastereomeric (6-4) products, namely the cis and trans isomers of 5-hydroxy-6-4'-(pyrimidin-2'-one)-5,6-dihydrouracil. Upon heating in 0.1m HCl, each of these compounds decomposes to form 6-4'-(pyrimidin-2'-one)uracil. In addition, evidence for production of a hydrate of a trimer of Ura is presented, Irradiation of this compound at 254nm forms Ura and a (6-4) adduct as products. The compounds 5-4'-(pyrimidin-2'-one)uracil and 6-hydroxy-5,6-dihydrouracil were also present after Ura was irradiated in frozen aqueous solution. Cyclobutane dimers (CBDs) are formed when Ura is irradiated in the frozen aqueous state, in fluid aqueous and acetonitrile solution and in the presence of photosensitizers (e.g. acetone). Published information, concerning the identity and relative quantitative importance of the four CBD isomers (cis-syn, cis-anti, trans-syn and trans-anti) formed in these photochemical systems, is incomplete and often in substantial disagreement. Using chemical methods in conjunction with HPLC, the identity and relative amounts of the four dimers have been determined in each of these systems. Consequently, a number of inconsistencies found in the literature concerning dimer product identity and quantitative distribution have been resolved.     

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.     

Well-defined star-shaped calcite crystals formed in agarose gels

Single crystals of calcite exhibiting a morphology of well-defined 8-armed stars, which evolved from original rhombohedral calcite crystals with their 8 points extending radially into eight arms, were produced by crystallization of CaCO3 in agarose gels.     

Atmospheric-pressure ionization studies and field dependence of ion mobilities of isomeric hydrocarbons using a miniature differential mobility spectrometer

The ionization pathways and ion mobility were determined for sets of structural isomeric and stereoisomeric non-polar hydrocarbons (saturated and unsaturated cyclic hydrocarbons and aromatic hydrocarbons) using a novel miniature differential mobility spectrometer with atmospheric-pressure photoionization (APPI) to assess how structural and stereochemical differences influence ion formation and ion mobility. The analytical results obtained using the differential mobility spectrometry (DMS) were compared with the reduced mobility values measured using conventional time-of-flight ion mobility spectrometry (IMS) with the same ionization technique.The majority of differences in DMS ion mobility spectra observed among isomeric cyclic hydrocarbons can be explained by the formation of different product ions. Comparable differences in ion formation were also observed using conventional IMS and by investigations using the coupling of ion mobility spectrometry with mass spectrometry (APPI-IMS-MS) and APPI-MS. Using DMS, isomeric aromatic hydrocarbons can in the majority of cases be distinguished by the different behavior of product ions in the strong asymmetric radio frequency (rf) electric field of the drift channel. The different peak position of product ions depending on the electric field amplitude permits the differentiation between most of the investigated isomeric aromatics with a different constitution; this stands in contrast to conventional IMS in which comparable reduced mobility values were detected for the isomeric aromatic compounds.