Effect of pulsed and reversing electric fields on the orientation of linear and supercoiled DNA molecules in agarose gels

When linear or supercoiled DNA molecules are imbedded in agarose gels and subjected to electric fields, they become oriented in the gel matrix and give rise to an electric birefringence signal. The sign of the birefringence is negative, indicating that the DNA molecules are oriented parallel to the electric field lines. If the DNA molecules are larger than about 1.5 kilobase pairs, a delay is observed before the birefringence signal appears. This time lag, which is roughly independent of DNA molecular weight, decreases with increasing electric field strength. The field-free decay of the birefringence is much slower for the DNA molecules imbedded in agarose gels than observed in free solution, indicating that orientation in the gel is accompanied by stretching. Both linear and supercoiled molecules become stretched, although the apparent change in conformation is much less pronounced for supercoiled molecules. When the electric field is rapidly reversed in polarity, very little change in the birefringence signal is observed for linear or supercoiled DNAs if the equilibrium orientation (i.e., birefringence) had been reached before field reversal. Apparently, completely stretched, oriented DNA molecules are able to reverse their direction of migration with little or no loss of orientation. If the steady-state birefringence had not been reached before the field reversal, complicated orientation patterns are observed after field reversal. Very large, partially stretched DNA molecules exhibit a rapid decrease in orientation at field reversal. The rate of decrease of the birefringence signal in the reversing field is faster than the field-free decay of the birefringence and is approximately equal to the rate of orientation in the field (after the lag period).(ABSTRACT TRUNCATED AT 250 WORDS)     

Orientation of the agarose gel matrix in pulsed electric fields.

The technique of transient electric birefringence was used to investigate the effect of pulsed electric fields on the orientation of the agarose gel matrix. Orientation of the gel was observed at all electric field strengths. Very slow, time-dependent effects were observed when pulses of 10-100 V/cm were applied to 1% gels for 0.5-2 seconds, indicating that domains of the matrix were being oriented by the electric field. The sign of the birefringence reversed when the direction of the applied electric field was reversed, indicating that the domains tend to orient in the perpendicular direction after field reversal. Theories of gel electrophoresis will need to incorporate the orientation of the matrix in order to provide a complete explanation of electrophoresis in agarose gels.     

Transient electric birefringence of agarose gels. I. Unidirectional electric fields

The orientation of agarose gels in pulsed electric fields has been studied by the technique of transient electric birefringence. The unidirectional electric fields ranged from 2 to 20 V/cm in amplitude and 1 to 100 s in duration, values within the range typically used for pulsed field gel electrophoresis (PFGE). Agarose gels varying in concentration from 0.3 to 2.0% agarose were studied. The sign of the birefringence varied randomly from one gel to another, as described previously [J. Stellwagen & N. C. Stellwagen (1989), Nucleic Acids Research, Vol. 17, 1537–1548]. The sign and amplitude of the birefringence also varied randomly at different locations within each gel, indicating that agarose gels contain multiple subdomains that orient independently in the electric field. Three or four relaxation times of alternating sign were observed during the decay of the birefringence. The various relaxation times, which range from 1 to ～ 120 s, can be attributed to hierarchies of aggregates that orient in different directions in the applied electric field. The orienting domains range up to ～ 22 μm in size, depending on the pulsing conditions. The absolute amplitude of the birefringence of the agarose gels increased approximately as the square of the electric field strength. The measured Ker constants are ～ 5 orders of magnitude larger than those observed when short, high-voltage pulses are applied to agarose gels. The increase in the Kerr constants in the low-voltage regime parallels the increase in the relaxation times in low-voltage electric fields. Birefringence saturation saturation curves in both the low- and high-voltage regimes can be fitted by theoretical curves for permanent dipole orientation. The apparent permanent dipole moment increase approximately as the 1.6 power of fiber length, consistent with the presence of overlapping agarose helices in the large fiber bundles orienting in low-voltage electric fields, the optical factor is approximately independent of fiber length. Therefore, the marked increase in the Kerr constants observed in the low-voltage regime is due to the large increase in the electrical orientation factor, which is due in turn to the increased length of the fiber bundles and domains orienting in low-voltage electric fields. Since the size of the fiber bundles and domains approximates the size of the DNA molecules being separated by PFGE, the orientation of the agarose matrix in the applied electric field may facilitate the migration of large DNA molecules during PFGE. © 1994 John Wiley & Sons, Inc.     

Transient electric birefringence of agarose gels. II. Reversing electric fields and comparison with other polymer gels

The transient electric birefringence of low electroendosmosis (LE) agarose gels oriented by pulsed unidirectional electric fields was described in detail in Part I [J. Stellwagen and N. C. Stellwagen (1994), Biopolymers, Vol. 34, p. 187]. Here, the birefringence of LE agarose gels in rapidly reversing electric fields, similar in amplitude and duration to those used for field inversion gel electrophoresis, is reported. Symmetric reversing electric fields cause the sign of the birefringence of LE agarose gels, and hence the direction of orientation of the agarose fibers, to Oscillate in phase with the applied electric field. Because of long-lasting memory effects, the alternating sign of the birefringence appears to be due to metastable changes in gel structure induced by the electric field. If the reversing field pulses are equal in amplitude but different in duration, the orientation behavior depends critically on the applied voltage. If E < 7 V/cm, the amplitude of the birefringence gradually decreases with increasing pulse number and becomes unmeasurably small. However, if E > 7 V/cm, the amplitude of the birefringence increase more than 10-fold after ～ 20 pulses have been applied to the gel, suggesting that a cooperative change in gel structure has occurred. Because there is no concomitant change birefringence must be due to an increase in the number of agarose fibers and /or fiber bundles orienting in the lectric field, which in turn indicates a cooperatice breakdown of the noncovalent “junction zones” that corss-link the fibers in to the fgel matrix. The sign of the birefringence of LE agarose gels is always positive after extensive junction zone breakdown, indicating that the agarose fibers and fiber bundles preferentially orient parallel to the lectric field when they are freed from the constraints of the gel matrix. Three other gel-forming polymers, high electroendosmosis (HEEO) agarose (a more highly changed agarose), β-carrageenan (a stereoisomer of agarose), and polyacrylamide (a chemically corss-linked polymer) were alos studied in unidirectional and rapidly reversing electric fields. The birefringence of HEEO agarose backbone chain. The β-carrageenan gels exhibit variable orientation behavior in reversing electric fields, suggesting that its internal gel structure is not as tightly interconnected as that of agaroise gels. Both HEEO agarose and β-carrageenan gels exhibit a large increase in the amplitude of the birefringence with increasing pulse number when asymmetric reversing pulses > 7 V/cm are applied to the gels, suggesting that junction zone breakdown in a common feature of polysaccharide gels. Chemically cross-linked polyacrylamide gels exhibit very small birefringence signals, indicating that very little orientation occurs in pulsed lectric fields. The sign of the birefringence is independent of the polarity of the lectric field, as expected from the Kerr law, and normal orientation behavior is observed in reversing electric fields. Hence, the anomalous change in sign of the birefringence observed for agarose gels in reversing electric fields must be due to the metastable junction zones in the agarose gel matrix, which allow gel fiber rearrangements to occur. © 1994 John Wiley & Sons, Inc.     

Pulse time and agarose concentration affect the electrophoretic mobility of cccDNA during PFGE and FIGE [corrected].

Circular DNAs have been shown to migrate in an unusual manner during field inversion gel electrophoresis (FIGE) and orthogonal field alternating gel electrophoresis (OFAGE). We studied the effect of varying pulse time and agarose concentration on the electrophoretic mobility of supercoiled (ccc) DNAs ranging from 2 kbp to 16 kbp during FIGE and contoured homogeneous electric fields (CHEF). Both supercoiled and linear molecules display a minimum mobility as a function of pulse time in a CHEF apparatus. Linear and cccDNAs of the same size are differently affected by pulse time. Pulse-time dependence was observed for cccDNAs in both systems. Pulse-time dependence in FIGE is very small at a 1.0% agarose concentration, but is pronounced in 0.8% or 1.2% gels.     

Agarose Gel Electrophoresis for the Separation of DNA Fragments

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb¹. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits². During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel''s molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight³. The leading model for DNA movement through an agarose gel is "biased reptation", whereby the leading edge moves forward and pulls the rest of the molecule along⁴. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation⁵; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: 1. Understand the mechanism by which DNA fragments are separated within a gel matrix 2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix 3. Identify an agarose solution of appropriate concentration for their needs 4. Prepare an agarose gel for electrophoresis of DNA samples 5. Set up the gel electrophoresis apparatus and power supply 6. Select an appropriate voltage for the separation of DNA fragments 7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 8. Determine the sizes of separated DNA fragments       

DNA gel electrophoresis: effect of field intensity and agarose concentration on band inversion

We study the effect of electric field intensity and agarose gel concentration on the anomalous electrophoretic mobility recently predicted by the biased reptation model and experimentally observed for linear DNA fragments electrophoresed in continuous electric fields. We show that high fields and low agarose concentrations eliminate the physical mechanism responsible for anomalous DNA mobility and band inversion, in good agreement with theory, thus restoring the monotonic mobility-size relationship necessary for unambiguous interpretation of the results of DNA gel electrophoresis.     

Fluorographic detection of [3H]thymidine-labeled deoxyribonucleic acid using agarose gels infused with 2,5-diphenyloxazole prior to electrophoresis

A fluorographic procedure for the detection of [3H]thymidine-labeled deoxyribonucleic acids electrophoresed in agarose gels was developed. 2,5-Diphenyloxazole (PPO) was added to the agarose solution before pouring of the gel for electrophoresis. This procedure did not interfere with the electrophoretic mobility of the DNA molecules. The radioactive detection efficiency was found to be improved over an existing procedure whereby the agarose gel was infused with PPO after electrophoresis with the aid of acetic acid.     

Separation of the DNA molecules beyond conventional size limits by gel electrophoresis with sodium dodecyl sulfate.

Electrophoretic mobility of DNA through polyacrylamide as well as agarose gels is greatly increased by sodium dodecyl sulfate (SDS). DNA molecules well beyond the conventionally separable size limits are separated readily and rapidly by gel electrophoresis with SDS in a conventional static electric field. Furthermore in optimal concentration gels DNA molecules of similar molecular sizes are separated better from one another in the presence of SDS than without it. Evidence is presented that SDS may act at least in part by altering conformation of DNA. This simple and readily available means for high resolution separation of hitherto impossible sizes of DNA molecules in polyacrylamide and agarose gels in an ordinary static electric field should find general use in molecular genetic analyses. Structural analyses of DNA-protein complexes are also facilitated by virtue of the simultaneous separation of the DNA and protein components on the same gel lane.     

Unidirectional pulsed-field electrophoresis of single- and double-stranded DNA in agarose gels: analytical expressions relating mobility and molecular length and their application in the measurement of strand breaks

Unidirectional pulsed-field electrophoresis improves the separation of single-stranded DNA molecules longer than 20 kilobases (kb) in alkaline agarose gels compared to static-field electrophoresis. The greatest improvement in separation is for molecules longer than 100 kb. The improved resolution of long molecules with unidirectional pulsed-field electrophoresis makes possible the measurement of lower frequencies of single-strand breaks. The analytical function that relates the length and mobility of single-stranded DNA electrophoresed with a static field also applies to unidirectional pulsed field separations. Thus, the computer programs used to measure single-strand breaks are applicable to both undirectional pulsed- and static-field separations. Unidirectional pulsed-field electrophoresis also improves the separation of double-stranded DNA in neutral agarose gels. The function relating molecular length and mobility for double-stranded DNA separated by unidirectional pulsed-field electrophoresis is a superset of the function for single-stranded DNA. The coefficients of this function can be determined by iterative procedures.     

Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis

A new type of gel electrophoresis separates DNA molecules up to 2000 kb with resolutions exceeding the logarithmic molecular weight dependence of conventional electrophoresis. The technique uses 1.5% agarose, 10 to 20 micrograms of DNA per well, and low ionic strength buffers. It employs alternately pulsed, perpendicularly oriented electrical fields, at least one of which is inhomogeneous. The duration of the applied electrical pulses is varied from 1 sec to 90 sec to achieve optimal separations for DNAs with sizes from 30 to 2000 kb. This pulsed field gradient gel electrophoresis fractionates intact S. cerevisiae chromosomal DNA, producing a molecular karyotype that greatly facilitates the assignment of genes to yeast chromosomes. Each yeast chromosome consists of a single piece of DNA; the chromosome sizes are consistent with the genetic linkage map. We also describe a general method for preparing spheroplasts, and cell lysates, without significant chromosomal DNA breakage.     

Electrophoresis and orientation of F-actin in agarose gels.

F-Actin was electrophoresed on agarose gels. In the presence of 2 mM MgCl2 and above pH 8.5 F-actin entered 1% agarose; when the electric field was 2.1 V/cm and the pH was 8.8, F-actin migrated through a gel as a single band at a rate of 2.5 mm/h. Labeling of actin with fluorophores did not affect its rate of migration, but an increase in ionic strength slowed it down. After the electrophoresis actin was able to bind phalloidin and heavy meromyosin (HMM) and it activated Mg2+-dependent ATPase activity of HMM. The mobility of F-actin increased with the rise in pH. Acto-S-1 complex was also able to migrate in agarose at basic pH, but at a lower rate than F-actin alone. The orientation of fluorescein labeled F-actin and of fluorescein labeled S-1 which formed rigor bonds with F-actin was measured during the electrophoresis by the fluorescence detected linear dichroism method. The former showed little orientation, probably because the dye was mobile on the surface of actin, but we were able to measure the orientation of the absorption dipole of the dye bound to S-1 which was attached to F-actin, and found that it assumed an orientation largely parallel to the direction of the electric field. These results show that actin can migrate in agarose gels in the F form and that it is oriented during the electrophoresis.     

Effects of supercoiling in electrophoretic trapping of circular DNA in polyacrylamide gels.

Electrophoretic velocity and orientation have been used to study the electric-field-induced trapping of supercoiled and relaxed circular DNA (2926 and 5386 bp) in polyacrylamide gels (5% T, 3.3% C) at 7.5-22.5 V/cm, using as controls linear molecules of either the same contour length or the same radius of gyration. The circle-specific trapping is reversible. From the duration of the reverse pulse needed to detrap the molecules, the average trap depth is estimated to be 90 A, which is consistent with the molecular charge and the field strengths needed to keep molecules trapped. Trapped circles exhibit a strong field alignment compared to the linear form, and there is a good correlation between the enhanced field alignment for the circles and the onset of trapping in both constant and pulsed fields. The circles do not exhibit the orientation overshoot response to a field pulse seen with linear DNA, and the rate of orientation growth scales as E(-2+/-0.1) with the field, as opposed to E(-1.1+/-0.1) for the linear form. These results show that the linear form migrates by cyclic reptation, whereas the circles most likely are trapped by impalement on gel fibers. This proposal is supported by very similar velocity and orientation behavior of circular DNA in agarose gels, where impalement has been deemed more likely because of stiffer gel fibers. The trapping efficiency is sensitive to DNA topology, as expected for impalement. In polyacrylamide the supercoiled form (superhelical density sigma = -0.05) has a two- to fourfold lower probability of trapping than the corresponding relaxed species, whereas in agarose gels the supercoiled form is not trapped at all. These results are consistent with existing data on the average holes in the plectonemic supercoiled structures and the fiber thicknesses in the two gel types. On the basis of the topology effect, it is argued that impalement during pulsed-field electrophoresis in polyacrylamide gels may be useful for the separation of more intricate DNA structures such as knots. The results also indicate that linear dichroism on field-aligned molecules can be used to measure the supercoiling angle, if relaxed DNA circles are used as controls for the global degree of orientation.     

High-sensitivity DNA detection with a laser-excited confocal fluorescence gel scanner

A high-sensitivity, laser-excited confocal fluorescence gel scanner has been developed and applied to the detection of fluorescently labeled DNA. An argon ion laser (1-10 mW at 488 nm) is focused in the gel with a high-numerical aperture microscope objective. The laser-excited fluorescence is gathered by the objective and focused on a confocal spatial filter, followed by a spectral filter and photodetector. The gel is placed on a computer-controlled scan stage, and the scanned image of the gel fluorescence is stored and analyzed in a computer. This scanner has been used to detect DNA separated on sequencing gels, agarose mapping gels and pulsed field gels. Sanger sequencing gels were run on M13mp18 DNA using a fluoresceinated primer. The 400-microns-thick gels, loaded with 30 fmol of DNA fragments in 3-mm lanes, were scanned at 78-microns resolution. The high resolution of our scanner coupled with image processing allows us to read up to approximately 300 bases in four adjacent sequencing lanes. The minimum band size that could be detected and read was approximately 200 microns. This instrument has a limiting detection sensitivity of approximately 10 amol of fluorescein-labeled DNA in a 1 x 3-mm band. In applications to agarose mapping gels, we have exploited the fact that DNA can be prestained with ethidium homodimer, followed by electrophoresis and fluorescence detection to achieve picogram sensitivity. We have also developed methods using both ethidium homodimer and thiazole orange staining which permit two-color detection of DNA in one lane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Contour-clamped homogeneous electric field electrophoresis of Staphylococcus aureus

Contour-clamped homogeneous electric field (CHEF) electrophoresis is a technique of pulsed-field gel electrophoresis that enables the resolution of large fragments of DNA that cannot be resolved by conventional gel electrophoresis. The procedure involves the application of controlled electric fields that change direction at a predetermined angle to samples of DNA that have been embedded in an agarose gel matrix and digested with a restriction endonuclease. Adjustment of the electrophoresis conditions enables the separation of DNA fragments with lengths from 10 kilobases up to 9 megabases in a size-dependent manner in agarose gels. The banding patterns can be used for epidemiological typing, the separated DNA can be immobilized onto a membrane and used for genetic mapping, or individual fragments can be extracted and used for downstream genetic manipulations. The protocol requires specialized equipment and can be completed in a maximum of 7 days.     

Orientation of DNA molecules in agarose gels by pulsed electric fields

The electric birefringence of DNA restriction fragments of three different sizes, 622, 1426, and 2936 base pairs, imbedded in agarose gels of different concentrations, was measured. The birefringence relaxation times observed in the gels are equal to the values observed in free solution, if the median pore diameter of the gel is larger than the effective hydrodynamic length of the DNA molecule in solution. However, if the median pore diameter is smaller than the apparent hydrodynamic length, the birefringence relaxation times increase markedly, becoming equal to the values expected for the birefringence relaxation of fully stretched DNA molecules. This apparent elongation indicates that end-on migration, or reptation is a likely mechanism for the electrophoresis of large DNA molecules in agarose gels. The relaxation times of the stretched DNA molecules scale with molecular weight (or contour length) as N2.8, in reasonable agreement with reptation theories.     

Electrophoretic mobility of supercoiled,catenated and knotted DNA molecules

We systematically varied conditions of two-dimensional (2D) agarose gel electrophoresis to optimize separation of DNA topoisomers that differ either by the extent of knotting, the extent of catenation or the extent of supercoiling. To this aim we compared electrophoretic behavior of three different families of DNA topoisomers: (i) supercoiled DNA molecules, where supercoiling covered the range extending from covalently closed relaxed up to naturally supercoiled DNA molecules; (ii) postreplicative catenanes with catenation number increasing from 1 to ∼15, where both catenated rings were nicked; (iii) knotted but nicked DNA molecules with a naturally arising spectrum of knots. For better comparison, we studied topoisomer families where each member had the same total molecular mass. For knotted and supercoiled molecules, we analyzed dimeric plasmids whereas catenanes were composed of monomeric forms of the same plasmid. We observed that catenated, knotted and supercoiled families of topoisomers showed different reactions to changes of agarose concentration and voltage during electrophoresis. These differences permitted us to optimize conditions for their separation and shed light on physical characteristics of these different types of DNA topoisomers during electrophoresis.     

Dependence of the electrophoretic mobility of DNA in gels on field intermittency

The electrophoretic mobility of double helical DNA in agarose and polyacrylamide gels increases as a function of time after the electric field is applied to the gel and decreases after the field is terminated. The changes are large for long (more than 10 kb) molecules. The effects of other variables are indicated.     

Electrophoresis of DNA restriction fragments in poly-N-acryloyl-tris gels

Poly-N-acryloyl-tris(hydroxymethyl)aminomethane (NAT) gels were evaluated as a matrix for DNA electrophoresis. The resolution of DNA restriction fragments in three poly(NAT)-N,N'-methylenebisacrylamide (Bis) gels (4, 5, and 6%) was compared with the resolution in polyacrylamide (AA)-Bis gels of the same percentage. Poly(NAT) gels were found to give a substantially improved separation of DNA fragments larger than 200 bp. In contrast to poly(AA) gels, DNA fragments of up to 4 kbp were well resolved in the new matrix. By pulse-field electrophoresis the useful separation range of poly(NAT) gels was expanded to at least 23 kbp. For DNA fragments below 10 kbp, the resolution was better than that in a 0.7% agarose gel. Thus poly(NAT) gels are most suitable for the electrophoretic separation of DNA molecules whose size is out of the optimal fractionation range of poly(AA) or agarose gels.     

Orientation of DNA Molecules in Agarose Gels By Pulsed Electric Fields

Abstract

The electric birefringence of DNA restriction fragments of three different sizes, 622,1426, and 2936 base pairs, imbedded in agarose gels of different concentrations, was measured. The birefringence relaxation times observed in the gels are equal to the values observed in free solution, if the median pore diameter of the gel is larger than the effective hydrodynamic length of the DNA molecule in solution. However, if the median pore diameter is smaller than the apparent hydrodynamic length, the birefringence relaxation times increase markedly, becoming equal to the values expected for the birefringence relaxation of fully stretched DNA molecules. This apparent elongation indicates that end-on migration, or reptation is a likely mechanism for the electrophoresis of large DNA molecules in agarose gels. The relaxation times of the stretched DNA molecules scale with molecular weight (or contour length) as N^2.8, in reasonable agreement with reptation theories.