Electrophoresis and movements of fluorescence pattern after photobleaching of large DNA fragments in agarose gels

By combining electrophoresis with movements of fluorescence pattern after photobleaching (MOFPAP), which is abbreviated as EMOFPAP, we are able to measure electrophoretic mobilities of large DNA fragments in an agarose gel within a fairly short time scale (about 10 min or even down to 1 min). The new method represents a significant improvement in experiment time when compared with the time (typically on the order of hours) required to determine the average electrophoretic mobility of large DNA fragments in agarose gels by means of either conventional gel electrophoresis or pulsed-field gel electrophoresis. In this article, we present the EMOFPAP experimental setup and consider optical conditions, including beam profile geometry and fluorescence pattern formation. A realistic formula that can explain the parameters governing the EMOFPAP method using our present optical setup has been derived. A comparison of results between experimental and computer simulation data is made, and an optimization of the EMOFPAP method is proposed.     

Study of large DNA fragments in agarose gels by transient electric birefringence

The pulsed-field gel electrophoresis (PFG) is a newly developing technique used in the fractionation of large DNA fragments. Advances in PFG demand a better understanding in the corresponding mechanisms of DNA dynamics in the gel network. Detailed experiments are needed to verify and to extend existing theoretical predictions as well as to find optimum conditions for efficient separation of large DNA fragments. In the present study, deformation of large DNA fragments (40-70 kilobase pairs) imbedded in agarose gels were investigated by using the transient electric birefringence (TEB) technique under both singular polarity and bipolarity electric pulses at low applied electric field strengths (E less than or equal to 5 V/cm). The steady-state optical retardation (delta s) of DNA molecules is linearly proportional to E2. At a given E, the amplitude of optical retardation [delta(t)] increases monotonically with the pulse width (PW) and then reaches a plateau value [delta(t = 0) = delta s] where t = 0 denotes the time when the applied field is turned off or reversed. The field-free decay time (tau-a few minutes) is several orders of magnitudes slower than that from previous TEB observations using high electric field strengths (E-kV/cm) and short pulse widths (PW-ms). The degree of deformation (stretching and orientation) and the time of restoration to the equilibrium conformation of overall DNA chains have been related to delta and tau. In field inversion measurements, exponentially rising and linearly falling of birefringence signals in the presence of forward/inverse applied fields were observed. The rising and falling of birefringence signals were reproducible under a sequence of alternating pulses. Comparison of our results with literature findings and discussions with theories are presented.     

A study of electrophoretic mobility of DNA in agarose and polyacrylamide gels

The aim of this paper is to clarify the mechanism of gel electrophoresis of DNA under constant-field conditions. We have conducted a large number of experiments on double-stranded DNA varying in length between approximately 10 and approximately 50,000 base-pairs, in both agarose and polyacrylamide gels ranging from 0.5% to 12% concentration, and with electric field strengths ranging from 0.5 to 8 V/cm. We have made (logarithmic) plots of velocity against length of DNA for all of the various test conditions. At the left-hand side of these plots, all of the empirical curves have a unique, standard shape. When the curves are normalized so that their left-hand parts coincide, a second feature emerges in that, while for any given test the curve follows the "master curve" up to a certain point, it then "breaks away" and becomes horizontal. We describe these two patterns of behaviour as "regions 1 and 2", respectively. We find simple yet comprehensive empirical formulae that fit the observations in the two regions of behaviour: these express the velocity in terms of length of DNA, electric field strength and gel concentration. We then construct two separate theories for the two regions of behaviour. The first theory involves the statistics of motion of an object through a random array of gel obstacles, with the instantaneous speed depending on the number of obstacles with which the object is currently in contact. The second theory is based on the mechanical hypothesis (for which there is other, independent support) that the DNA moves through the gel by piling up against a barrier, which eventually breaks or deforms under the resulting force, thereby allowing the DNA to move on to the next barrier. The statistical theory is an adaptation of existing work, while the mechanical one is new. We also describe experiments on the migration of repeated-sequence, curved DNA with length up to 1500 base-pairs, and we discuss its behaviour in terms of our two theories. Our studies by electron microscopy are consistent with the view that this repeated-sequence DNA adopts a superhelical configuration. Finally, we show that a very wide range of observations may be understood clearly by means of our two theoretical schemes.     

Effect of the electric field on the apparent mobility of large DNA fragments in agarose gels

The electrophoresis of a series of DNA fragments ranging in size from 0.5 to 12 kilobase pairs, has been studied as a function of agarose gel concentration and electric field strength. The apparent mobility of all fragments decreased with decreasing electric field strength and with increasing gel concentration. When extrapolated to zero electric field strength and zero agarose concentration, the apparent mobility of all DNA fragments extrapolated to a common value (2.0 ± 0.1) × 10^?4 cm²/V s. The square roots of the retardation coefficients of the various fragments were found to be linearly related to the root-mean-square radii of gyration of the fragments, as predicted by pore-size distribution theory. As predicted by reptation theory, the molecular weights of the various fragments were found to be linearly related to the reciprocal of the apparent mobilities. An equation is given for estimating the apparent pore size of agarose gels between 0.25 and 1.5% in concentration.     

从琼脂糖凝胶中高效回收DNA技术的探讨

用两只离心管制成的凝胶过滤装置,从电泳后的琼脂糖凝胶中回收DNA片段的简易方法。它依次包括以下步骤：凝胶过滤装置的制作、凝胶切割、凝胶低温冷冻、低温高速离心、ddH20洗胶、DNA纯化和回收效果检测等。用此方法回收的DNA片段产率高、质量纯,可直接用于分子生物学实验的后续操作,如载体连接、PCR模板获得、DNA探针制备、基因测序等。其优点是：DNA片段的回收率高（90％以上）,质量好;操作简便,耗时短;回收装置简单,成本低廉,可进行商品化开发。     

A polarized photobleaching study of DNA reorientation in agarose gels

Polarized fluorescence recovery after photobleaching (pFRAP) has been used to study the internal dynamics of relatively long DNA molecules embedded in gels that range in concentration from 1% to 5% agarose. The data indicate that, even in very congested gels, rapid internal relaxation of DNA is largely unhindered; however, interactions with gel matrices apparently do perturb the larger amplitude, more slowly (microseconds to milliseconds) relaxing internal motions of large DNAs. The relationship between this work and recent studies which indicate that internal motions of DNA play an important role in the separation achieved with pulsed-field gel electrophoresis techniques is discussed. The polarized photobleaching technique is also analyzed in some detail. In particular, it is shown that "reversible" photobleaching phenomena are probably related to depletion of the ground state by intersystem crossing to the triplet state.     

The influence of agarose--DNA affinity on the electrophoretic separation of DNA fragments in agarose gels

The effects of DNA concentration, buffer composition, added "carrier" DNA, and chemical modification of agarose on the electrophoretic separation of DNA restriction fragments in agarose gels were tested. Electrophoretic zones of migrating DNA were found to broaden by trailing as sample load was decreased, and this effect was found to be more pronounced for species of higher molecular weight. As DNA sample load was increased, DNA fragments were found to move faster in the direction of electrophoresis (front forward). Sharp, well-resolved electrophoretic zones were obtained at very low DNA loads only when a high-salt, high-pH, high-EDTA buffer was employed or when "carrier DNA" having a broad and uniform molecular weight distribution was included in the sample. Moreover, DNA in high concentration was found to displace DNA in low concentration from a given gel region. Unmodified agaroses were found to differ only slightly in their effectiveness in retarding DNA fragments at a given agarose concentration. However, hydroxyethylated agarose was much more effective in retarding DNA, at a given gel concentration, than the unmodified agaroses tested. These results show that it is useful to consider the agarose gel matrix as possessing the properties of both a molecular sieve and a chromatographic adsorbent when designing electrophoretic separation techniques for DNA. A model for these separations which includes the effects of DNA-agarose interaction and molecular sieving is discussed.     

Improved resolution of DNA fragments in polysaccharide-supplemented agarose gels

The electrophoretic separation of nucleic acids, including small DNA fragments in the range 50-1000 bp, is presently carried out in polyacrylamide gels or in gels containing high concentrations of agarose. We have developed an alternative gel matrix composition which is inexpensive, nontoxic, easy to prepare, and highly transparent to visible and uv light. The composition combines a soluble nonionic polysaccharide such as hydroxyethylcellulose, methylcellulose, or galactomannan with a minimum but sufficient concentration of agarose to form a gel which immobilizes the "liquid phase sieve." These mixtures do not replace polyacrylamide for resolving fragments smaller than approximately 75 nucleotides. However, the new gels show DNA fragment resolution (band separation versus distance traveled) and optical clarity superior to those of conventional agarose.     

The orientation of DNA fragments in the agarose gels

A microscopic method of measuring the orientation of nucleic acids in the agarose gels is described. A nucleic acid undergoing electrophoresis is stained with the dye ethidium bromide and is viewed under high magnification with a polarization microscope. A high-numerical-aperture microscope objective is used to illuminate and to collect the fluorescence signal, and therefore the orientation of the minute quantities of nucleic-acid can be measured: in a typical experiment we can detect the orientation of one-tenth of a picogram (10(13)g) of DNA. Polarization properties of the fluorescent light emitted by the separate bands corresponding to different molecular weights of the DNA are examined. A linear dichroism equation relates the measured fluorescence to the mean orientation of the absorption dipole of the ethidium bromide (and therefore DNA) and to the extent to which it is disorganized. As an example, we measured the orientation of phi X174 DNA RF/HaeIII fragments undergoing electrophoresis in a field of 10 V/cm. Ethidium bromide bound to the fragments with an angle of the absorption dipole largely perpendicular to the direction of the electrophoretic current. The dichroism declined as the molecular weight of the fragments decreased which is interpreted as an increase in the degree of disorder for shorter DNA.     

Separation of large circular DNA by electrophoresis in agarose gels

The electrophoresis of circular DNA, ranging in size from 4.4 kilobase pairs (kbp) to 220 kbp, was studied in agarose gels. Bacterial artificial chromosome (BAC) DNA was used as a source of large supercoiled and open circular (relaxed) forms. The open circles above approximately 50 kbp were trapped at the sample wells of 1% agarose gels during electrophoresis at 3 V/cm. Field inversion gel electrophoresis (FIGE) was used to relieve the trapping of the open circles in the gels. Using FIGE (30 s forward pulse time), open circles with sizes of 115 and 220 kbp required reverse pulse times of 3 and 6 s, respectively, to free the circles from open-ended gel fibers. A minimum in the gel velocity of the open circles was measured at approximately 20 kbp. Open circles below approximately 20 kbp migrated slower than the supercoiled forms, and above 20 kbp the order was reversed. These results indicate that when the size of the open circles exceeded the average pore size of a gel and it was forced to span multiple pores, the open circles gained a mobility advantage. Decreasing the ionic strength of the electrophoresis buffer significantly decreased the mobility of the smaller circles and slightly increased the mobility of the larger circles.     

Purification and cloning of DNA fragments fractionated on agarose gels

Purification of DNA fragments from acrylamide or agarose gels is a commonly used technique in the molecular biology laboratory. This article describes a rapid, efficient, and inexpensive method of purifying DNA fractions from an agarose gel. The purified DNA is suitable for use in a wide range of applications including ligation using DNA ligase. The procedure uses standard high-melting-temperature agarose and normal TBE electrophoresis buffer. In addition, the protocol does not involve the use of highly toxic organic solvents such as phenol.     

Simultaneous electroelution and concentration of DNA fragments from agarose gels

A rapid and inexpensive method for the electroelution of DNA fragments from agarose gels is described. DNA fragments were separated by agarose gel electrophoresis and visualized by staining with ethidium bromide. Selected DNA fragments were placed into electroeluter tubes capped with dialysis membrane and electroeluted into a small volume of buffer using a conventional horizontal gel electrophoresis apparatus. The method successfully eluted and concentrated DNA fragments with molecular weights ranging from 2.7 to 13.9 MDa in 3 h.     

Lateral mobility in membranes as detected by fluorescence recovery after photobleaching.

The evaluation of lateral diffusion coefficients of membrane components by the technique of fluorescence recovery after photobleaching (FRAP) is often complicated by uncertainties in the values of the intensities F(O), immediately after bleaching, and F(infinity), after full recovery. These uncertainties arise from instrumental settling time immediately after bleaching and from cell, tissue, microscope, or laser beam movements at the long times required to measure F(infinity). We have developed a method for precise analysis of FRAP data that minimizes these problems. The method is based on the observation that a plot of the reciprocal function R(tau) = F(infinity)/[F(infinity)-F(tau)] is linear over a large time range when (a) the laser beam has a Gaussian profile, (b) recovery involves a single diffusion coefficient, and (c) there is no membrane flow. Moreover, the ratio of intercept to slope of the linear plot is equal to tau 1/2, the time required for the bleached fluorescence to rise to 50% of the full recovery value, F(infinity). The lateral diffusion coefficient D is related to tau 1/2 by tau 1/2 = beta w2/4D where beta is a defined parameter and w is the effective radius of the focused laser beam. These results are shown to indicate that the recovery of fluorescence F(tau) can be represented over a large range of percent bleach, and recovery time tau by the relatively simple expression F(tau) = [ F(o) + F(infinity) (tau/tau 1/2)]/[1 + tau/tau 1/2)]. FRAP data can therefore be easily evaluated by a nonlinear regression analysis with this equation or by a linear fit to the reciprocal function R(tau). It is shown that any error in F(infinity) can be easily detected in a plot of R(tau) vs. tau which deviates significantly from a straight line when F(infinity) is in error by as little as 5%. A scheme for evaluating D by linear analysis is presented. It is also shown that the linear reciprocal plot provides a simple method for detecting flow or multiple diffusion coefficients and for establishing conditions (data precision, differences in multiple diffusion coefficients, magnitude of flow rate compared to lateral diffusion) under which flow or multiple diffusion coefficients can be detected. These aspects are discussed in some detail.     

Measurement of the lateral mobility of cell surface components in single living cells by fluorescence recovery after photobleaching

The use of fluorescence recovery after photobleaching (FRAP) techniques to monitor the lateral mobility of plant lectin-receptor complexes on the surface of single, living mammalian cells is described in detail. FRAP measurements indicate that over 75% of the wheat germ agglutinin receptor (WGA-receptor) complexes on the surface of human embryo fibroblasts are mobile. These WGA-receptor complexes diffuse laterally (as opposed to flow) on the cell surface with a diffusion coefficient in the range of 2 × 10^?11 to 2 × 10^?10 cm²/sec. Both the percentage of mobile WGA-receptor complexes and the mean diffusion coefficient of these complexes are higher than that obtained from earlier FRAP measurements of the mobility of concanavalin A-receptor (Con A-receptor) complexes in a variety of cell types. The possible reasons for the differing mobilities of WGA and Con A receptors are discussed.     

The isolation of DNA from agarose gels by electrophoretic elution onto malachite green-polyacrylamide columns

Electrophoretic elution of DNA coupled with direct adsorption onto malachite green-polyacrylamide columns was used to isolate double- and single-stranded DNA from agarose gels. Subsequently, DNA was eluted with a high salt buffer and filtered through Sephadex which permitted recovery of the DNA in a low salt buffer at concentrations suitable for heteroduplex analysis by electron microscopy. This method was tested by examining hetero-duplexes formed from the isolated complementary single strands of T7 wild type DNA and a T7 deletion mutant. More than 80% of the reannealed molecules were intact heteroduplexes showing the deletion loop. Irradiation of single-stranded DNA with 254 nm light resulted in distorted, convoluted heteroduplexes while 366 nm light did not show this effect.     

Electrophoretic mobility of DNA in gels. II. Systematic experimental study in agarose gels

A systematic study has been undertaken to prove or disprove the predictions of a revised reptation model, biased reptation with fluctuations (BRF). Our data, which scan about two orders of magnitude of DNA sizes and of electric fields, and a fourfold range of gel concentrations, are in qualitative and quantitative agreement with the model and support the applicability of this theory to DNA gel electrophoresis. In particular, we show that the mobility in the compression zone scales as the first power of the electric field, and that the limit of separation scales as the inverse first power of the electric field, for low enough fields. © 1994 John Wiley & Sons, Inc.     

Electrophoretic mobility of lambda phage HIND III and HAE III DNA fragments in agarose gels: a detailed study

The electrophoretic mobility (μ) of DNA fragments from λ phage and ΦX 174, split by restriction enzyme to molecular lengths from 3 × 10² to 2.36 × 10⁴ base pairs, has been investigated in 0.6–4% agarose gels at various field strengths, ionic strengths, and temperatures. As already observed, μ is seen to be very sensitive to the field, increasing with field strength. The sensitivity increases with the molecular length of the DNA and decreases at high gel concentration. Our data are in qualitative agreement with recent theoretical predictions that concern the influence of the electric field on electrophoretic mobility. Mobility data have been extrapolated to zero field. This enables a comparison of our experimental results with theoretical predictions on the dependence of μ on the molecular weight of the DNA fragments. Our data fit, quite closely, a reptation model, where the tube path is described as a semiflexible entity with a persistence length equal to the pore diameter. The influence of the agarose concentration and the ionic strength of the buffer on the two parameters of the model—intrinsic electrophoretic mobility (μ₀) and the number of base pairs per element of the tube (g)—are well described by the model. The temperature dependence of the electrophoretic mobility, together with the influence of the agarose concentration on μ₀, indicate that the hydrodynamic drag is the leading frictional force on the DNA molecules in the gel.     

Electrophoretic mobility of high-molecular-weight, double-stranded DNA on agarose gels.

A new theoretical model for the migration of high-molecular-weight, double-stranded DNA on agarose gels is presented. This leads to the prediction that under certain conditions of electrophoresis, a linear relationship will exist between the molecular weight of a DNA molecule, raised to the (-2/3) power, and its electrophoretic mobility. Agarose gel electrophoresis of the fragments of bacteriophage lambda DNA produced by several restriction endonucleases confirms this relationship, and establishes some of the limits on its linearity. For this work, a polyacrylamide slab gel apparatus was modified for use with agarose gels. This apparatus has several advantages over others commercially available for agarose gel electrophoresis, including the abilities to run a larger number of samples at one time, to use lower-concentration gels, and to maintain better temperature stability across the width of the gel. The validation of the relationship developed here between molecular weight and electrophoretic mobility should make this a useful method for determining the molecular weights of DNA fragments.     

Quantitative electrophoretic transfer of DNA from polyacrylamide or agarose gels to nitrocellulose

A method for efficient electrophoretic transfer of DNA fragments from polyacrylamide gels to nitrocellulose sheets was developed. Hybridization to these fragments can be performed by standard techniques. The method is also applicable to agarose gels, allowing this transfer method to be used for DNA ranging from 40 to at least 23,000 bp.