Ethidium DNA agarose gel electrophoresis: how it started

We started ethidium DNA agarose gel electrophoresis when our ultracentrifuge broke down and we needed an alternative method to check the quality of our mitochondrial DNA preparations. Agarose proved convenient for sizing DNA; ethidium in gel and buffer allowed visualization of DNA bands immediately after the run and improved the separation of the closed and open duplex forms of mitochondrial DNA circles. At smaller gel pore size mitochondrial DNA circles were excluded from the gel, whereas long linear DNAs were not. We concluded that the linear DNAs 'crawl like snakes head on through the gel'. This paper reviews some of the early experiments preceding the introduction of ethidium agarose gel electrophoresis.     

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.     

A new continuous, monodimensional electrophoretic system for the separation and quantitation of individual glycosaminoglycans

A rapid and sensitive method for agarose gel electrophoresis is described. By simply miniaturizing a conventional gel electrophoresis apparatus, we have decreased the time necessary for the separation of nucleic acid molecules by a factor of 10. The ability to detect DNA molecules by ethidium bromide fluorescence has simultaneously been increased fivefold. Transfer of DNA from these “minigels” onto nitrocellulose filters followed by hybridization using the procedure of C. M. Southern (1975, J. Mol. Biol.98, 503–517) was found to be efficient and rapid. This technique is sufficiently sensitive to detect radioactive quantities of [³²P]phosphate-labeled DNA or RNA microinjected into 500 chick embryo fibroblasts.     

Characterization of the DNA and structural proteins of entomopoxviruses from Melanoplus sanguinipes,Arphia conspirsa, and Phoetaliotes nebrascensis (Orthoptera)

Entomopoxvirus (EPV) occlusion bodies were isolated from virus infected nymphs of the grasshoppers Melanoplus sanguinipes, Arphia conspirsa, and Phoetaliotes nebrascensis. Separation of the viral structural proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave unique protein patterns for each of the three viruses. An occlusion body protein of approximately 100,000 MW was isolated from each virus. Cleavage of viral DNA with HinddIII and BamHI restriction endonucleases and separation of the fragments by agarose gel electrophoresis gave different DNA fragment patterns for each of the three entomopoxviruses. Molecular weight estimates of 120 × 10⁶ for M. sanguinipes EPV DNA, 129 × 10⁶ for A. conspirsa EPV DNA, and 125 × 10⁶ for P. nebrascensis EPV DNA were calculated from the sizes of the viral DNA fragments. Approximately 55% base sequence homology was detected by Southern hybridization of α-³²P-labeledM. sanguinipes EPV DNA with P. nebrascensis DNA. No base sequence homology was detected by Southern hybridization of labeled M. sanguinipes EPV DNA to Othnonius batesi EPV DNA (Coleoptera), Amsacta moorei EPV DNA (Lepidoptera), Euxoa auxiliaris EPV DNA (Lepidoptera), and vaccinia virus DNA fragments.     

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.     

一种有效回收小分子DNA片段的方法

介绍了一种有效回收小于200bp DNA片段的方法。用改进的冻融-离心回收法对155bp的DNA片段进行回收,并且与常规冻融-离心回收、TaKaRa回收试剂盒的结果做对比,用琼脂糖凝胶电泳检测回收结果,紫外吸收法定量分析。结果证明:改进的方法是一种经济、方便、可靠的回收小分子DNA片段的方法。     

Determination of microbial genome sizes by two-dimensional denaturing gradient gel electrophoresis.

In two-dimensional denaturing gradient gel electrophoresis, DNA is digested with a restriction endonuclease and the resulting DNA fragments are separated as a function of size by conventional agarose gel electrophoresis. Following this first dimension electrophoresis, the fragment distribution is placed at the top of a denaturing gradient slab gel and electrophoresis is carried out parallel to the gradient direction. This second dimension separation is a complex function of the base sequence of each fragment. Analysis of the DNA fragment distribution as a function of fragment size allows the DNA size to be calculated. This method has been applied to calculate three microbial genome sizes: Mycoplasma capricolum, 724 kb; Acholeplasma laidlawii, 1646 kb; and Hemophilus influenzae, 1833 kb.     

Electrophoretic analysis of covalently closed SV40 DNA: Boltzmann distributions of DNA species.

Covalently closed relaxed SV40 DNA [SV40(I')] generated by polynucleotide ligase closure of nicked circular SV40 DNA was analyzed by agarose gel electrophoresis. The DNA can be resolved into a series of bands differing in superhelical density whose intensities are approximately symmetrical about a central most intense band. Densitometric analysis of the gel pattern has revealed that the distribution of DNA species conforms to a Boltzmann distribution and has enabled us to derive an equation for the free energy of superhelix formation for SV40 DNA. We believe the observed bands reflect the time-averaged distribution of thermally induced fluctuations in DNA chain conformation in solution at the time of ligase catalyzed phosphodiester bond formation. Densitometric analysis of native supercoiled SV40 DNA, partially unwound in the presence of ethidium bromide, demonstrates that the separation between adjacent bands is approximately half that seen with SV40(I'). Agarose gel electrophoresis was also used to measure the change in average base rotation angle as a function of temperature by a procedure independent of ethidium dye binding.     

Preparation of large quantities of separated strands from simian virus 40 DNA restriction fragments by low-temperature low-salt agarose gel electrophoresis.

We have used agarose gel electrophoresis to separate complementary DNA strands obtained from simian virus 40 DNA restriction fragments produced by HindII and III or by EcoRI and HpaII digestion. By modifying existing methods we have virtually eliminated the problematic renaturation of DNA during electrophoresis. This has allowed us to recover large quantities of separated DNA strands (approximately 20 μg of DNA per 12-mm-diameter preparative tube gel). By using a combination of low temperature and low buffer concentration during electrophoresis, we have also significantly improved the resolution of DNA strands.     

Two methods that facilitate autoradiography of small 32P-labeled DNA fragments following electrophoresis in agarose gels

Two methods which permit detection by autoradiography of small 32P-labeled DNA fragments resolved by agarose gel electrophoresis are described. Agarose gel electrophoresis poses problems for autoradiography as (i) the gels are normally too thick to allow autoradiography without being dried first, and (ii) fragments of DNA of 1000 bp or less in length are readily lost during drying. In this study DNA fragments as small as 121 bp have been retained in agarose gels upon drying. This has been achieved by either (i) first fixing the DNA with the cationic detergent cetyltrimethylammonium bromide, or (ii) drying the agarose gels onto Zeta-Probe charge-modified membranes.     

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.     

Rapid separation of DNA molecules by agarose gel electrophoresis: use of a new agarose matrix and a survey of running buffer effects.

This report describes the use of a new type of agarose (FastLane agarose) for faster separation of DNA by agarose gel electrophoresis. DNA molecules separated in this agarose exhibited electrophoretic mobilities up to 30% higher than similar separations in standard analytical grade agarose. DNA molecules of all sizes examined showed higher mobilities in FastLane agarose. The mobility increase was predominantly due to the low electroendosmosis of FastLane agarose and was most pronounced in pulsed field gel electrophoresis separations. The magnitude of mobility increase varied depending on the conditions used for electrophoresis.     

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.     

Localization of Single-Chain Interruptions in Bacteriophage T5 DNA. II. Electrophoretic Studies

Upon denaturation, T5 DNA yields a large number of discrete, single-chain fragments that can be resolved by agarose gel electrophoresis. The positions of the more prominent of these fragments in the T5 duplex were determined by analyzing their sensitivity to digestion with λ exonuclease and their distribution among EcoRI fragments of T5 DNA. These experiments also provide firm evidence concerning the polarity of the strands in T5 DNA. An analogous study was carried out on the fragments produced by treating exonuclease III-degraded T5 DNA with the single-strand-specific SI endonuclease. This procedure yielded over 40 discrete duplex fragments that could be resolved with considerable precision by agarose gel electrophoresis. The positions of most of these fragments were determined by analyzing EcoRI fragments of T5st(+) and T5st(0) DNA. Over 20 sites where single-chain interruptions can occur in T5 DNA were identified, and the distribution of interruptions within the terminal repetition was shown to be identical at both ends of the molecule. A precise value for the size of the terminal repetition in T5 DNA was obtained by analyzing SI endonuclease digests of ligase-repaired, circular T5 DNA in agarose gels. The repeated segment represented 8.3% of the T5st(+) DNA. The results of this study also provide information concerning the properties of λ exonuclease. Hydrolysis by this enzyme was not terminated when single-chain interruptions were encountered either in the strand being degraded or in the complementary strand.     

Characterization of a cytoplasmic polyhedrosis virus from Estigmene acrea (Lepidoptera)

A cytoplasmic polyhedrosis virus (CPV) containing a segmented double-stranded RNA genome was isolated from Estigmene acrea larvae by isopycnic centrifugation in a sucrose density gradient. Ten double-stranded RNA segments with molecular weights (MW) from 2.8 to 0.67 × 10⁶ were separated by agarose gel electrophoresis. A total of ten virus proteins ranging from 14,000 to 128,000 MW were detected after separation by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. A MW of 28,500 was determined for E. acrea CPV occlusion body protein.     

Amplified fragment length polymorphism analysis of Listeria monocytogenes by Experion™ automated microfluidic electrophoresis

Fifty Listeria monocytogenes strains were genotyped by sAFLP and PCR products were separated by agarose gel and automated chip-based microfluidic electrophoresis. A high congruency of results was observed comparing the two techniques, although for some cultures a better separation of sAFLP fragments was achieved with microfluidic system, which proved to be a highly reliable and reproducible tool to improve the molecular typing of L. monocytogenes, requiring lower volumes of samples and reducing significantly analysis time.     

Mapping genomic organization by field inversion and two-dimensional gel electrophoresis: application to the murine T-cell receptor gamma gene family.

A new two-dimensional gel electrophoresis technique has been developed for the mapping of multigene families. Resolution in the first dimension is based on the generation of large size DNA fragments by infrequently-cutting restriction enzymes, and separation of these fragments by field inversion gel (FIG) electrophoresis. A second restriction enzyme digestion is then carried out with the separated DNA fragments in the agarose gel. Standard gel electrophoresis in the second dimension allows one to estimate the number of hybridizing genes contained in each large DNA fragment. We have also developed a novel method to increase the separation, resolution and hybridization signal in the second dimension by condensing the bands from the first dimension into spots. As an example, we have applied these techniques to determine the organization of the murine T-cell receptor gamma locus. The murine gamma gene family was found to be contained on two DNA fragments encompassing 195 kilobases of DNA. The two-dimensional gel electrophoresis method is particularly useful in the analysis of the organization of multigenic families where single copy probes are not readily available, and should extend the potential usefulness of field inversion gel electrophoresis in gene mapping.     

Restriction endonuclease map of the chloroplast DNA of Chlamydomonas reinhardii

The chloroplast DNA of Chlamydomonas reinhardii has been examined by restriction endonuclease analysis. EcoRI, BamHI and BglII produce 30, 17 and 12 fragments, respectively, whose sites have been determined by electron microscopy and by comparative gel electrophoresis. These fragments have been ordered into a circular map which corresponds to a genome size of M_r = 126 × 10⁶. The map was established by comparing the double digests of individual restriction fragments and by hybridizing purified labelled fragments to restriction enzyme digests of chloroplast DNA. The restriction fragments were isolated by molecular cloning or by preparative agarose gel electrophoresis.The two sets of chloroplast ribosomal RNA genes are contained within two inverted repeats of 13 × 10⁶ molecular weight, which are located nearly at opposite sides of the map. In addition, the mapping studies have revealed the presence of short repeated base sequences which are interspersed throughout the chloroplast genome.     

Construction of a SalI/PstI restriction map of spinach chloroplast DNA using low-gelling-temperature-agarose electrophoresis

The restriction endonucleases SalI and PstI cleave circular chloroplast DNA of spinach (Spinacia oleracea) into 12 and 10 fragments, respectively. The sum of the fragment sizes in each of the series is equivalent to the contour length of the molecule (about 95 Md). A physical map was constructed by sequential digestions using low-gelling-temperature agarose to avoid the necessity of extracting the fragments from the gel. The circular DNA molecule of spinach chloroplasts consists of two identical sequences (each about 15 Md) arranged as an inverted repeat separated by two single-copy regions of different sizes (about 52 and 13 Md).     

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.