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.     

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.     

Purification of DNA from agarose gels

Summary We describe a rapid and easily reproducible modification of the freeze-squeeze method of separating DNA from agarose gels. Our method involves slicing out the agarose gel portion which contains the DNA of interest, freezing this gel slice at –20°C, then centrifuging the frozen slice in a filtration unit which contains a cellulose acetate filter. The agarose is retained on the filter and the filtrate contains the DNA. DNA purified in this manner could be completely digested with restriction endonucleases and completely ligated with DNA ligase, without further purification. The percentages of recovery for various sizes of linear and plasmid double-stranded DNA ranged from 57 to 69%. The procedure takes less than 30 minutes to perform.     

Orientation of DNA in agarose gels.

An orientation of the lambda DNA during the electrophoresis in agarose gels was measured by a microscopic linear dichroism technique. The method involved staining the DNA with the dye ethidium bromide and measuring under the microscope the polarization properties of the fluorescence field around the electrophoretic band containing the nucleic acid. It was first established that the fluorescence properties of the ethidium bromide-DNA complex were the same in agarose gel and in a solution. Then the linear dichroism method was used to measure the dichroism of the absorption dipole of EB dye bound to lambda DNA. In a typical experiment the orientation of two-tenth of a picogram (2 x 10(-13)g) of DNA was measured. When the electric field was turned on, the dichroism developed rapidly and assumed a steady state value which increased with the strength of the field and with the size of DNA. A linear dichroism equation related the measured dichroism of fluorescence to the mean orientation of the absorption dipole of ethidium bromide and to an extent to which the orientation of this dipole deviated from the mean. The observed development of dichroism in the presence of an electric field was interpreted as an alignment of DNA along the direction of the field. The increase in the steady state value of dichroism with the rise in the strength of the field and with the increase of the size of DNA was interpreted as a better alignment of DNA along the direction of the field and as a smaller deviation from its mean orientation.     

Electrophoresis of DNA in oriented agarose gels

Oriented agarose gels were prepared by applying an electric field to molten agarose while it was solidifying. Immediately afterwards, DNA samples were applied to the gel and electrophoresed in a constant unidirectional electric field. Regardless of whether the orienting field was applied parallel or perpendicular to the eventual direction of electrophoresis, the mobilities of linear and supercoiled DNA molecules were either faster (80% of the time) or slower (20% of the time) than observed in control, unoriented gels run simultaneously. The difference in mobility in the oriented gel (whether faster or slower) usually increased with increasing DNA molecular weight and increasing voltage applied to orient the agarose matrix. In perpendicularly oriented gels linear DNA fragments traveled in lanes skewed toward the side of the gel; supercoiled DNA molecules traveled in straight lanes. If the orienting voltage was applied parallel to the direction of electrophoresis, both linear and supercoiled DNA molecules migrated in straight lanes. These effects were observed in gels cast from different types of agarose, using various agarose concentrations and two different running buffers, and were observed both with and without ethidium bromide incorporated in the gel. Similar results were observed if the agarose was allowed to solidify first, and the orienting electric field was then applied to the gel for several hours before the DNA samples were added and electrophoresed. The results suggest that the agarose matrix can be oriented by electric fields applied to the gel before and probably during electrophoresis, and that orientation of the matrix affects the mobility and direction of migration of DNA molecules. The skewed lanes observed in the perpendicularly oriented gels suggest that pores or channels can be created in the matrix by application of an electric field. The oriented matrix becomes randomized with time, because DNA fragments in oriented and unoriented gels migrated in straight lanes with identical velocities 24 hours later.     

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.     

Simple DNA elution from agarose gels

We developed a simple DNA elution method from agarose gels. After electrophoresis of DNA in an agarose gel, the DNA fragment to be recorved was excised out of gel with a scalpel. The excised gel was placed in the middle of small Parafilm piece, and the Parafilm was folded over the gel piece. Using the petriplate, or thumb, the gel piece was pressed between the Parafilm. Upon squeezing, the DNA inside of the gel gets extruded along with the buffer. The droplets were collected with a pipet. The DNA was then purified by conventional phenol: chloroform extraction method. Typical yields are greater than 50% as determined by UV absorbance.     

A rapid method for extracting DNA from agarose gels

A method for obtaining high recovery of deoxyribonucleic acid (DNA) from agarose gels using an agarase extraction procedure is presented. This DNA is physically intact and biologically active. The DNA obtained with this procedure should be useful for a wide range of applications.     

Recovery of DNA segments from agarose gels

After electrophoresis, DNA can be efficiently recovered by solubilization of agarose gels with NaClO₄, followed by retention of DNA on glass fiber filters. After removal of the NaClO₄ by ethanol, the DNA can be extracted with a low salt buffer.     

Direct hybridization of labeled DNA to DNA in agarose gels

A naringinase assay capable of distinguishing between the content of naringin, prunin, and naringenin present in the incubation mixture, is described. The amount of these compounds can be estimated by combining two spectrophotometric procedures. (a) Treatment with strong alkali to determine the amount of nargingenin as well as the sum of naringin and prunin. (b) Assay of the liberated aldohexoses with o-aminodiphenyl. From the data thus obtained, the amount of the remaining substrate, the amount of the intermediate as well as the product at any given time can be calculated.     

A freeze-squeeze method for recovering long DNA from agarose gels

Long DNA can be recovered from agarose gels after electrophoresis by freezing the gel slices and manually squeezing out liquid containing the DNA. With this method the recoveries of phage T₇ DNA (molecular weight 25 × 10⁶) and the open and closed forms of circular phage PM2 DNA (molecular weight 6 × 10⁶) were about 70%. Sedimentation analysis shows that the extruded DNA has not sustained double- or single-stranded breaks. The extruded DNA can be used without further purification as substrate for the restriction endonuclease Hind_II,III, from Hemophilus influenzae, for DNA·DNA hybridization and for electron microscopy.     

Quantitation of protein and DNA in silver-stained agarose gels

A silver stain for both proteins and DNA in agarose gels is described. Quantitation of proteins with this stain is possible, with individual proteins exhibiting characteristic responses, as observed with other stains. The advantage of the silver stain over Coomassie blue is its increased (50- to 100-fold) sensitivity, which allows samples containing very low protein concentrations to be analyzed without prior concentration. This silver stain, when applied to DNA, is at least as sensitive as ethidium bromide, and gives a linear response for the type of DNA and fragment sizes studied.     

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.     

Rapid isolation of high-molecular-weight DNA from agarose gels

We have developed a simple, reliable, and rapid method for recovering DNA from agarose gels. While many methods for DNA extraction have already been described, few provide quantitative recovery of large DNA molecules. These procedures generally require costly apparatus, extended elution times, or considerable handling of the sample after elution. Our method employs a novel electroelution chamber constructed from acrylic plastic. Gel slices containing DNA are placed in the chamber between platinum electrodes. Voltage is applied and a continuous flow of buffer sweeps the eluted DNA from the chamber into an external receptacle. Elution is complete in 7 min. Concentrated DNA is obtained by butanol extraction and alcohol precipitation in 1 h. Recoveries, quantitated by counting radiolabeled DNA or by densitometry of analytical gels, were 94 to 100% for fragments of 4 to 50 kb. The eluted DNA was undegraded and could be digested with restriction enzymes, ligated, end-labeled, or used to transform cells as efficiently as noneluted DNA. Complete elution of a 100-kb plasmid, a 194-kb concatemer of bacteriophage lambda, and of 440- and 550- chromosomes of Saccharomyces cerevisiae was also achieved using the same process. This method is suitable for routine use in a wide range of cloning applications, including the electrophoretic isolation of large DNA molecules.     

A method for the recovery of DNA from agarose gels.

We describe a quick and versatile method for the isolation of DNA from agarose gels. The DNA is electrophoresed into a trough containing hydroxyapatite, where it is bound. The hydroxyapatite is taken out and the DNA eluted with phosphate buffer. By putting the hydroxyapatite on a small column of Sephadex G50, elution and subsequent removal of phosphate can be performed in one step. The DNA recovered can be used equally well in enzymatic incubations as DNA not purified through agarose gel electrophoresis. Several applications of this technique are described.     

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.