Pulsed field sequencing gel electrophoresis.

The effect of pulsed fields on sequencing gel electrophoresis is investigated, using DNA fragment markers ranging in size from 20 to 6557 bases. For high continuous electric fields (5000 V/55 cm) band inversion is observed in which fragments larger than 4000 bases migrate faster than those of 800-1000 bases. The use of one-dimensional pulsed field gel electrophoresis (ODPFGE) eliminates band inversion and extends the monotonic size-mobility relationship of the DNA markers up to about 4000 bases. The relevance of these results, obtained using a manual sequencing process with autoradiographic detection, to automated sequences is discussed.     

Recognition of DNA three-way junctions by metallosupramolecular cylinders: gel electrophoresis studies

The interaction of metallosupramolecular cylinders with DNA three-way junctions has been studied by gel electrophoresis. A recent X-ray crystal structure of a palindromic oligonucleotide forming part of a complex with such a cylinder revealed binding at the heart of a three-way junction structure. The studies reported herein confirm that this is not solely an artefact of crystallisation and reveal that this is a potentially very powerful new mode of DNA recognition with wide scope. The cylinders are much more effective at stabilizing three-way junctions than simple magnesium di-cations or organic or metallo-organic tetra-cations, with the M cylinder enantiomer being more effective than P. The recognition is not restricted to three-way junctions formed from palindromic DNA with a central AT step at the junction; non-palindromic three-way junctions and those with GC steps are also stabilised. The cylinder is also revealed to stabilise other Y-shaped junctions, such as that formed at a fraying point in duplex DNA (for example, a replication fork), and other DNA three-way junction structures, such as those containing unpaired nucleotides, perhaps by opening up this structure to access the central cavity.     

Electric field gradients and band sharpening in DNA gel electrophoresis

A mathematical study of the effect of non-uniform electric fields on the width of DNA electrophoretic bands is presented. Using a simple model, we show that field gradients sharpen these bands during an experiment if the corresponding gradient of electrophoretic velocity is large enough. This is in agreement with experimental results indicating that narrower bands form when pulsed field electrophoresis is carried out in the presence of field gradients. Moreover, it is shown that there is in fact an optimal experimental duration that maximizes separation. Finally, gradients are also predicted to reduce the relative mobilities of the DNA fragments, which is a serious drawback of this technique.     

An inexpensive microslab gel DNA electrophoresis system with real-time fluorescence detection

In this paper, we describe the construction of a simple yet powerful gel electrophoresis apparatus that can be used to perform size-selective separations of DNA fragments in virtually any laboratory. This system employs a microslab gel format with a novel gel casting technique that eliminates the need for delicate combs to define sample loading wells. The compact size of the microslab gel format allows rapid separations to be performed at low voltages using submicroliter sample volumes. Real time fluorescence detection of the migrating DNA fragments is accomplished using an inexpensive digital microscope that directly connects to any PC with a USB interface. The microscope is readily adaptable for this application by replacing its white light source with a blue light-emitting diode (LED) and adding an appropriate emission filter. Both polyacrylamide and agarose gels can be used as separation matrices. Separation performance was characterized using standard dsDNA ladders, and correct sizing of a 191 bp PCR product was achieved in 15 min. The low cost and simplicity of this system makes it ideally suited for use in a variety of laboratory and educational settings.     

Preparation of plant DNA for separation by pulsed field gel electrophoresis

A method was developed for the preparation of completely intact plant DNA embedded in agarose, and suitable for restriction enzyme digestion. Digestion with restriction enzyme was carried out according to modified protocols of Anand and Kenwrick et al. The new method of DNA isolation allows the separation of high molecular weight plant DNA by pulsed field gel electrophoresis.     

High-throughput separation of DNA and proteins by three-dimensional geometry gel electrophoresis: feasibility studies

We report a novel separation method that is applicable to both DNA and protein samples, based on electrophoresis in a three-dimensional (3-D) geometry. In contrast to conventional electrophoresis, samples are applied in a two-dimensional, planar array to one of the surfaces of a 3-D geometry separation medium. Loading onto a plane results in a very high sample capacity. Sample migration and separation occur along the third spatial dimension, which is perpendicular to the loading plane. The key problem of electrophoresis in a 3-D geometry separation setup is that temperature gradients are caused by Joule's heat, affecting the electrical conductivity and viscosity of the separation medium. A means of achieving straight sample migration under these circumstances is to force heat flow through the separation medium parallel to the axis of sample migration. This can be done by dissipating the heat via the electrode sides of the gel and blocking any other heat transfer. The separation of DNA and proteins by this method has been tested using agarose gel electrophoresis, polyacrylamide gel electrophoresis, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Data were acquired off-line by conventional staining methods as well as on-line by detection of laser-induced fluorescence. We describe how to excise samples from the separation medium for preparative purposes. Possible unique applications of this 3-D geometry electrophoresis separation method are also discussed.     

Discontinuous buffer system for polyacrylamide and agarose gel electrophoresis of DNA fragments

DNA fragments up to 9 kb in size were stacked and separated by polyacrylamide gel electrophoresis, and those up to 50 kb in size by agarose gel electrophoresis, using a discontinuous buffer system. Polyacrylamide gels at pH 8.9, 2 degrees C, 0.01 M ionic strength, yielded sharp bands with DNA loads of 8 micrograms/cm2 of gel of a mixture of 19 DNA fragments in the size range of 72-23130 bp, while agarose gels at pH 8.5, 25 degrees C, provided well-resolved, unperturbed bands at 0.04 M ionic strength with DNA loads of 1 microgram/cm2 of the same mixture. Note that the ionic strength of the agarose gels is comparable to the conventionally used 0.5 x TBE (Tris-borate-EDTA) buffer, while that successfully applied to polyacrylamide is seven-fold less than the ionic strength of conventionally used 1 x TBE buffer, with a substantially shorter duration of electrophoresis as a result. The application of a discontinuous buffer system to the gel electrophoresis of DNA results in (i) Band identification by Rf, the migration distance relative to a sharply defined "buffer front" (moving boundary). This is sufficiently labor saving, compared to determining absolute mobilities, so as to render practical the expression of bands as numbers, with benefits for data storage, statistical manipulations and physico-chemical exploitation of mobility data. The use of Rf's also circumvents loss of precision in mobility measurement resulting from progressive band spreading of dye bands used as a front. (ii) A uniformly and highly concentrated starting zone, beneficial to resolution, is obtained, without the losses by which separate concentration steps are usually burdened.(ABSTRACT TRUNCATED AT 250 WORDS)     

Capillary gel electrophoresis of oligonucleotides: prediction of migration times using base- specific migration coefficients

Chemically synthesized oligodeoxyribonucleotides were subjected to capillary gel electrophoresis on three different polyacrylamide-based matrices. Analysis of about 1000 samples over a 1-year period showed that the gel matrix evolved with time resulting in shifting migration times, making it essential to use an internal standard. Cross-linked polyacrylamide matrices had the highest stability, allowing an average of 100 injections on the same capillary. Computer- aided prediction of migration times was subsequently evaluated to confirm the size and base composition of oligonucleotides more accurately. A number of problems were noted when using this approach on a routine basis, such as insufficient stability of the gel matrices, effects of secondary structure on migration and insufficient differences in migration times for oligonucleotides containing>50 bases. Capillary gel electrophoresis at pH 3.5 in replaceable gels showed that migration was mainly dependent on the charge per base ratio resulting in separations of significantly altered selectivity which complemented analyses under the commonly used basic pH conditions.     

Pulsed amperometric detection of DNA with an ssDNA/polypyrrole-modified electrode

Pulsed amperometric detection (PAD) of target DNA with platinum electrodes modified by single-stranded DNA (ssDNA) entrapped within polypyrrole (ssDNA/Ppy) is reported for the first time. Single-stranded DNA 20-mers complementary to the target DNA were used to construct the DNA biosensors. Polymerase chain reaction (PCR) amplified bovine leukaemia virus (BLV) provirus DNA was used as target DNA. Electrochemical impedance spectroscopic (EIS) investigation of ssDNA/Ppy before and after incubation in target DNA-containing sample revealed significant changes in terms of an imaginary (Z) vs. a real (Z) component. The PAD results were in good agreement with EIS investigations. The PAD method was selected, because it does not require such sophisticated equipment as it is used to perform EIS and the results obtained can be more easily estimated. Optimum conditions for performing PAD and evaluating an analytical signal were elaborated. No label-binding step was necessary for detection of target DNA in PCR-amplified amplicons and detection time was reduced by as much as 30–35 min. The changes of PAD signals were at least 6–7 times higher if ssDNA/Ppy-modified electrodes instead of blank Ppy-modified electrodes were incubated in the target DNA solutions. If ssDNA/Ppy modified electrodes were incubated in non-complementary (control) DNA solution changes in PAD signals were smaller than those detected after incubation in complementary (target) DNA-containing solution by a factor of at least 6–8.     

Pulsed field electrophoresis in contour-clamped homogeneous electric fields for the resolution of DNA by size or topology

The electrophoretic separation of DNA molecules can be controlled by the use of contour-clamped homogeneous electric fields (CHEF). This paper describes an improved CHEF apparatus with negligible distortion in the electric fields. When the electric field was periodically reoriented, DNA molecules up to 2 megabases were resolved in a highly uniform manner. Furthermore, when the field strength was changed with orientation, topological variations of conventional-sized DNA molecules were resolved.     

Modeling the gel electrophoresis of short duplex DNA by Brownian dynamics: cubic gel lattice with direct interaction

The technique of Brownian dynamics is used to model the electrophoretic mobility of spherical and rod-like particles in a three-dimensional cubic gel lattice. In addition to excluded volume interactions between the migrating particle and the gel, direct interactions are also included. The methodology is first applied to spherical particles in the absence of direct interactions and the resulting mobilities are shown to agree with independent studies. The methodology is then applied to rod-like models of short duplex DNA fragments 10-50 base pairs in length. In the absence of direct interactions between gel and DNA, calculated mobilities show a much weaker dependence on gel concentration than observed in experiments of DNA in Tris-acetate buffer and polyacrylamide gels. When an attractive interaction between gel and DNA of approximately -0.3 k(B)T per base pair at contact is included, good agreement between calculated and experimental mobilities is achieved.     

Capillary gel electrophoresis of single-stranded DNA fragments with UV detection

Capillary gel electrophoresis has proven to be a powerful tool in biomedical research. We report our investigation of some of the critical parameters affecting separations of single-stranded DNA fragments as monitored by ultraviolet (UV) absorbance detection. Although not as sensitive as laser-induced fluorescence (LIF), UV absorbance detection allows one to calculate quite accurately, and inexpensively, the molarity of each separated DNA fragment and, moreover, the signal “fading” effect normally observed with LIF detection can be, in many cases, substituted for fluorescence to detect the many different single-stranded DNAs, as well as for detection of sequencing reactions.     

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.     

Influence of electric field intensity, ionic strength, and migration distance on the mobility and diffusion in DNA surface electrophoresis

In order to increase the separation rate of surface electrophoresis while preserving the resolution for large DNA chains, e.g., genomic DNA, the mobility and diffusion of Lambda DNA chains adsorbed on flat silicon substrate under an applied electric field, as a function of migration distance, ionic strength, and field intensity, were studied using laser fluorescence microscope. The mobility was found to follow a power law with the field intensity beyond a certain threshold. The detected DNA peak width was shown to be constant with migration distance, slightly smaller with stronger field intensity, but significantly decreased with higher ionic strength. The molecular dynamics simulation demonstrated that the peak width was strongly related with the conformation of DNA chains adsorbed onto surface. The results also implied that there was no diffusion of DNA during migration on surface. Therefore, the Nernst-Einstein relation is not valid in the surface electrophoresis and the separation rate could be improved without losing resolution by decreasing separation distance, increasing buffer concentration, and field intensity. The results indicate the fast separation of genomic DNA chains by surface electrophoresis is possible.     

A modified method of genomic DNA preparation in agarose inserts for pulse field gel electrophoresis.

According to standard protocol, DNA in agarose inserts is prepared by first embedding the cell in agarose. This is then incubated in the required enzyme (lysozyme, lysostaphin, or zymolase) depending on the cell type (bacterial or plants), for spheroplast formation. Subsequent treatment of the spheroplast with proteinase K allows the isolation of large genomic DNA in agarose suitable for pulse field gel electrophoresis. An efficient and rapid method of preparation of spheroplast is described. In this method a low concentration of enzyme required for spheroplast formation was added before embedding the cell in agarose, which facilitated the digestion of cell wall by the enzyme and allowed use of a low amount of enzyme. Digestion of DNA in agarose inserts prepared by this method, with rare cutting restriction enzyme and pulse field gel electrophoresis, showed that the quality of DNA was as good as obtained by the standard method.     

Prediction of migration behavior of oligonucleotides in capillary gel electrophoresis.

The influence of the primary structure (base composition) on the electrophoretic migration properties of single-stranded oligodeoxyribonucleotides in capillary polyacrylamide gel electrophoresis was investigated using homo- and heterooligomers under denaturing and non-denaturing conditions. Homooligodeoxyribonucleotides of equal chain lengths but of different base composition showed significant differences in mobility. In addition, the migration properties of heterooligomers were found to be highly dependent on their base composition. A simple equation is presented for predicting relative migration times using denaturing and non-denaturing polyacrylamide capillary gel electrophoresis. Orange-G was used as an internal standard and as the basis of the relative migration time calculations. Examples are presented using homo- and heterooligomers in the 10-20-mer range to show the correlation of the primary structure and their predicted and observed migration rates.     

Rapid microwell polymerase chain reaction with subsequent ultrathin-layer gel electrophoresis of DNA

Large-scale genotyping, mapping and expression profiling require affordable, fully automated high-throughput devices enabling rapid, high-performance analysis using minute quantities of reagents. In this paper, we describe a new combination of microwell polymerase chain reaction (PCR) based DNA amplification technique with automated ultrathin-layer gel electrophoresis analysis of the resulting products. This technique decreases the reagent consumption (total reaction volume 0.75-1 microL), the time requirement of the PCR (15-20 min) and subsequent ultrathin-layer gel electrophoresis based fragment analysis (5 min) by automating the current manual procedure and reducing the human intervention using sample loading robots and computerized real time data analysis. Small aliquots (0.2 microL) of the submicroliter size PCR reaction were transferred onto loading membranes and analyzed by ultrathin-layer gel electrophoresis which is a novel, high-performance and automated microseparation technique. This system employs integrated scanning laser-induced fluorescence-avalanche photodiode detection and combines the advantages of conventional slab and capillary gel electrophoresis. Visualization of the DNA fragments was accomplished by "in migratio" complexation with ethidium bromide during the electrophoresis process also enabling real time imaging and data analysis.     

Characterization of single-stranded DNA separation by capillary gel electrophoresis

We have investigated the effect of polymer gel reconditioning, the shape of the capillary, the applied electric field, and the capillary length for single-stranded DNA. The polyethylene oxide gel had deformed under the high electric field causing the degradation of the separation power. By the reintroduction of the fresh polyethylene oxide gel for the next run, one-base resolution was recovered. It turned out that the tip of the capillary at the injection side needed to be clean and symmetric for much improved resolution. Changing DNA motion by the pulsed electric field resulted in the separation of DNA far more than 500 bases.     

Irreversible trapping of DNA during crossed-field gel electrophoresis.

Using an original protocol with a rotating gel electrophoresis apparatus, it is shown that duplex DNA undergoing crossed-field electrophoresis in agarose gets trapped in the gel when the field is increased above a threshold value which decreases with the chain length and depends on the angle between the fields in a non-monotonous manner. This trapping is irreversible, i.e. once trapped at a high field strength, chains are unable to resume their motion when the field is returned to a lower value at which they moved prior to trapping. A model of trapping by "tight knots" is proposed. It predicts a trapping threshold proportional to the inverse square of the electric field, in qualitative agreement with the data. The implications of our results for the separation of large DNA molecules are discussed.