Ultra-thin-layer agarose gel electrophoresis. I. Effect of the gel concentration and temperature on the separation of DNA fragments

A novel, rapid and efficient separation method is described for the analysis of double stranded (ds) DNA fragments in the form of horizontal ultra-thin-layer agarose gel electrophoresis. This separation technique combines the multilane, high-throughput separation format of agarose slab gel electrophoresis with the excellent performance of capillary electrophoresis. The electrophoretic separation of the fluorophore (Cy5)-labeled dsDNA molecules were imaged in real time by a scanning laser-induced fluorescence/avalanche photodiode detection system. Effects of the gel concentration (Ferguson plot) and separation temperature (Arrhenius plot) on the migration characteristics of the DNA fragments are discussed. An important genotyping application is also shown by characterizing the polymorphic region (2× or 4×48 base pair repeats) of the dopamine D₄ receptor gene (D₄DR, exon III region) for ten individuals, using PCR technology with Cy5-labeled primers and ultra-thin-layer agarose gel electrophoresis.     

Stabilizing labile DNA–protein complexes in polyacrylamide gels

The electrophoretic mobility‐shift assay (EMSA) is one of the most popular tools in molecular biology for measuring DNA–protein interactions. EMSA, as standardly practiced today, works well for complexes with association binding constants K_a>10⁹ M^?1 under normal conditions of salt and pH. Many DNA–protein complexes are not stable enough so that they dissociate while moving through the gel matrix giving smeared bands that are difficult to quantitate reliably. In this work we demonstrate that the addition of the osmolyte triethylene glycol to polyacrylamide gels dramatically stabilizes labile restriction endonuclease EcoRI complexes with nonspecific DNA sequences enabling quantitation of binding using EMSA. The significant improvement of the technique resulting from the addition of osmolytes to the gel matrix greatly extends the range of binding constants of protein–DNA complexes that can be investigated using this widely used assay. Extension of this approach to other techniques used for separating bound and free components such as gel chromatography and CE is straightforward.     

Effect of topological asymmetry on the electrophoretic mobility of branched DNA structures with and without single-base mismatches

The electrophoretic mobility of three-arm star DNA structures with varying degrees of branch length asymmetry has been investigated in polyacrylamide (PAA) hydrogels. We report the effect of single-base mismatches, adjacent to the branch point, on the mobility of branched DNA with three different arm lengths. Branched DNA structures were formed using wild-type and mutated fragments of the p53 tumor suppressor gene, which is believed to play an important role in cancer development. Branching was directed at the site of several previously characterized mutations in exon 7 of p53. At a given gel concentration, the mobility of branched DNA with fully complementary base pairing is found to increase as the degree of branch length asymmetry is increased. Ferguson analysis of the gel electrophoresis data leads to a retardation coefficient that is strongly dependent on topology. This finding can be explained in terms of a minimum molecular cross-section for each molecule. Specifically, we show that structures with the smallest molecular cross-section can access more pores in the gel, which leads to higher mobility. Our results can also be understood by considering the rotational diffusivity of branched DNA. Asymmetric DNA stars with higher calculated rotational diffusivities also have higher mobilities. When a mutated base is present in junctions with low degrees of branch length asymmetry, adjacent to the branch point, the mobility increases in comparison to the fully complementary molecules. The reason for this increased mobility is unclear, here, we propose that the mismatched base introduces additional flexibility to the arm containing the mutation leading to higher conformational freedom and enhanced mobility in gels. When a mismatched base is present in junctions with high degrees of branch length asymmetry, the opposite result is obtained. Here, the mutated species has a lower mobility. This result is argued to arise from incomplete hybridization and/or frayed ends. Finally, we have shown that by using two of the branch point oligonucleotides as probe molecules, mutations known to occur at specific sites can be detected through the mobility shift. If the sequences of the probe chains are changed in a controlled manner, the location and base of the mutant can also be determined.     

Universal interpolating function for the dispersion coefficient of DNA fragments in sieving matrices

The separation of DNA fragments by (slab or capillary) gel electrophoresis has been studied extensively. To characterize the separation achieved by such systems, one needs to understand the impact (and their dependency upon the experimental quantities) of two physical parameters: the electrophoretic mobility mu and the diffusion coefficient D. Three different regimes have been shown to exist for both mu and D: the Ogston regime, the reptation regime and the reptation with orientation regime (note that separation is only possible for the first two regimes). In the small electric field limit, both mu and D are apparently well described by theories for all three regimes. Unfortunately this results in disjointed scaling laws and no theory-based general equations can apply to all regimes. Recently, an empirical interpolating formula has been proposed that adequately fits the low electric field mobility mu of dsDNA fragments across all three regimes and is compatible with accepted theories. In this article we review and clarify the current state of knowledge regarding the size dependence of the mobility and the diffusion coefficient and propose an interpolating formula for molecular size dependence of the low field diffusion coefficient D. With formulas for both the mobility and the diffusion coefficient as a function of the experimental conditions one could, in principle, optimize any gel/polymer matrix-based electrophoresis system for a wide range of DNA molecular sizes.     

A nonradioactive electrophoretic mobility shift assay for measurement of pregnane X receptor binding activity to CYP3A4 response element

The electrophoretic mobility shift assay (EMSA) is a method for the study of specific DNA–protein interactions in vitro. The pregnane X receptor (PRX) is a key xenobiotic sensor that regulates the expression of drug‐metabolizing enzymes and many other genes. Radiolabeled ³²P‐DNA‐probes had been used in studies of PXR‐DNA interactions. There is an increasing need for nonradioactive assays, due to the health, safety and environmental issues. In the current study, we present a protocol for the nonradioactive electrophoretic mobility shift assay, allowing studying interactions between human PXR with promoter DNA sequences.     

Multiplexed DNA sizing by capillary electrophoresis using entangled polymer solutions and diode array detection

We developed a method for the analysis of multiplexed double-stranded DNA (dsDNA) samples complexed to various intercalating dyes using entangled polymer solution. A commercial single-column capillary electrophoresis (CE) instrument with diode array detection was used for multiplexed detection of DNA samples by addition of intercalating fluorescent molecules. A Phi X174HinfI and a pGEM DNA ladder (1 mg/mL) were used for the electrophoretic separation of dsDNA fragments ranging in size from 24 to 726 and 36 to 2645 bp, respectively. The results suggested that simultaneous electrophoretic separation of different DNA ladders multiplexed with different dyes could be performed in the same capillary yielding fast DNA sizing separations. CE analysis, which is often overpowered by slab gel in sample throughput, could now overcome this disadvantage by allowing multiplexed sample analysis in a fraction of the time needed for slab gel analysis. The separation efficiency of stained DNA molecules with both dyes were dramatically improved with buffers containing a large cation such as tetrapentylammonium ion (Npe(4) (+)) as the only cation in the buffer.     

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.     

The migration anomaly of DNA fragments in polyacrylamide gels allows the detection of small sequence-specific DNA structure variations

Curved DNA fragments have a reduced electrophoretic mobility in polyacrylamide gels. The retardation in gels is extremely sensitive to small structural variations which influence the DNA helix axis. This gel assay can also be used to detect very small structural variations in DNA sequences which are not curved: The noncurved sequences of interest can be combined with curved stretches in phase with the helix turn. Using such sequence constructions, even subtle influences on the DNA helix axis can be detected. Experiments of this kind allow the determination of a relative order of sequence-specific DNA twist and wedge angles.     

Activation energy of single-stranded DNA moving through cross-linked polyacrylamide gels at 300 V/cm effect of temperature on sequencing rate in high-electric-field capillary gel electrophoresis

In DNA sequencing, single-stranded DNA fragments are separated by gel electrophoresis. This separation is based on a sieving mechanism where DNA fragments are retarded as they pass through pores in the gel. In this paper, we present the mobility of DNA sequencing fragments as a function of temperature; mobility is determined in 4% T LongRanger gels at an electric field of 300 V/cm. The temperature dependence is compared with the predictions of the biased reptation model. The model predicts that the fragment length for the onset of biased reptation with stretching increases with the square of temperature; the data show that the onset of biased reptation with stretching decreases with temperature. Biased reptation fails to model accurately the temperature dependence of mobility. We analyzed the data and extracted the activation energy for passage of sequencing fragments through the gel. For fragments containing less than ca. 200 bases, the activation energy increases linearly with the number of bases at a rate of 25 J/mol per base; for longer fragments, the activation energy increases at a rate of 6.5 J/mol per base. This transition in the activation energy presumably reflects a change in conformation of the DNA fragments; small fragments exist in a random coil configuration and larger fragments migrate in an elongated configuration.     

Label-free fluorescent assay for real-time monitoring site-specific DNA cleavage by EcoRI endonuclease

DNA cleavage reaction catalyzed by nucleases is essential in many important biological processes and medicinal chemistry. Therefore, it is important to develop reliable and facile methods to assay nuclease activity. With this goal in mind, we report a fluorescent assay for label-free, facile, and real-time monitoring of DNA cleavage by EcoRI endonuclease using SYBR Green I (SGI) as a signal probe. The fluorescence of SGI dramatically increased when the free SGI was mixed with double-stranded DNA (dsDNA) substrate. Upon interacting with EcoRI, which cleaves the dsDNA into small fragments, the weakened interaction between SGI and the shortened DNA fragments caused a decrease in fluorescence of SGI. EcoRI-DNA interaction was real-time studied by monitoring fluorescence change with the prolonging of interaction time. The important kinetic parameters, including Michaelis-Menten constant (K(M)) and maximum initial velocity (V(max)), were accurately calculated, which is consistent with previously reported studies. Site-specific DNA cleavage by EcoRI endonuclease has also been verified by gel electrophoresis analysis, which indicated that this method is a simple and effective approach to assay DNA cleavage reaction. Specificity investigation demonstrated that EcoRI-DNA interactions can be studied with high selectivity. Compared with previously reported methods, this approach is selective, simple, convenient and cost-efficient without any labeling of the probe or of the target.     

Simultaneous immunoblotting analysis with activity gel electrophoresis in a single polyacrylamide gel

We describe here that a simple diffusion blotting method can couple immunoblotting analysis with another biochemical technique in a single polyacrylamide gel. The efficiency of protein transfer was evaluated by serial dilutions of nephrosin, a metalloproteinase of the astacin family, and by immunodetection. It is estimated that diffusion blotting produces 25-50% of the signal intensity compared to the classical electrophoretic transfer method. However, with diffusion blotting it is possible to generate several replicas from a single gel. In addition, a protein blot can be obtained from a sodium dodecyl sulfate (SDS)-polyacrylamide gel for zymography assay or from a native polyacrylamide gel for electrophoretic mobility shift assay (EMSA). In this regard, a particular signal in zymography or EMSA can be confirmed by simultaneous immunoblotting analysis with a corresponding antiserum. Therefore, diffusion blotting allows a direct comparison of signals between gels and replicas in zymography assay and EMSA. These advantages make diffusion blotting desirable when partial loss of transfer efficiency can be tolerated or be compensated by a more sensitive immunodetection reaction using enhanced chemiluminescence substrates.     

Influence of branch length asymmetry on the electrophoretic mobility of rigid rod-like DNA

The electrophoretic mobility of three-arm asymmetric star DNA molecules, produced by incorporating a short DNA branch at the midpoint of rigid-rod linear DNA fragments, is investigated in polyacrylamide gels. We determine how long the added branch must be to separate asymmetric star DNA from linear DNA with the same total molecular weight. This work focuses on two different geometric progressions of small DNA molecules. First, branches of increasing length were introduced at the center of a linear DNA fragment of constant length. At a given gel concentration, we find that relatively small branch lengths are enough to cause a detectable reduction in electrophoretic mobility. The second geometric progression starts with a small branch on a linear DNA fragment. As the length of this branch is increased, the DNA backbone length is decreased such that the total molar mass of the molecule remains constant. The branch length was then increased until the asymmetric branched molecule becomes a symmetric three-arm star polymer, allowing the effect of molecular topology on mobility to be studied independent of size effects. DNA molecules with very short branches have a mobility smaller than linear DNA of identical molar mass. The reason for this change in mobility when branching is introduced is not known, however, we explore two possible explanations in this article. (i) The branched DNA could have a greater interaction with the gel than linear DNA, causing it to move slower; (ii) the linear DNA could have modes of motion or access to pores that are unavailable to the branched DNA.     

Synthesis and properties of nicked dumbbell and dumbbell DNA conjugates having stilbenedicarboxamide linkers

The synthesis and properties of nicked dumbbell and dumbbell DNA conjugates having A-tract base pair domains connected by rod-like stilbenedicarboxamide linkers are reported. The nicked dumbbells have one to eight dA-dT base pairs and are missing a sugar-phosphate bond either between the linker and a thymine nucleoside residue or between two thymine residues. Chemical ligation of all of the nicked dumbbells with cyanogen bromide affords the dumbbell conjugates in good yield, providing the smallest mini-dumbbells prepared to date. The dumbbells have exceptionally high thermal stability, whereas the nicked dumbbells are only marginally more stable than the hairpin structures on either side of the nick. The structures of the nicked dumbbells and dumbbells have been investigated using a combination of circular dichroism spectroscopy and molecular modeling. The base pair domains are found to adopt normal B'-DNA geometry and thus provide a helical ruler for studies of the distance and angular dependence of electronic interactions between the chromophore linkers.     

The electric field dependence of DNA mobilities in agarose gels: a reinvestigation

The electric field dependence of the electrophoretic mobility of linear DNA fragments in agarose gels was reinvestigated in order to correct the observed mobilities for the different temperatures actually present in the gel during electrophoresis in different electric field gradients. When corrected to a common temperature, the electrophoretic mobilities of DNA fragments less than or equal to 1 kilobase pairs (kbp) in size were independent of electric field strength at all field strengths from 0.6 to 4.6 V/cm if the gels contained less than or equal to 1.4% agarose. The mobilities of larger DNA fragments increased approximately linearly with electric field strength. If the agarose concentration was higher than 2%, the mobilities of all DNA fragments increased with increasing electric field strength. The electric field dependence of the mobility was larger in gels cast and run in Tris-borate buffer (TBE) than in gels cast and run in Tris-acetate buffer (TAE), and was more pronounced in gels without ethidium bromide incorporated in the matrix. Ferguson plots were constructed for the various DNA fragments, both with and without extrapolating the temperature-corrected mobilities to zero electric field strength. Linear Ferguson plots were obtained for all fragments less than or equal to 12 kbp in size in agarose gels less than or equal to 1.4% in concentration if the mobilities were first extrapolated to zero electric field strength. Concave upward curvature of the Ferguson plots was observed for DNA fragments greater than or equal to 2 kbp in size at finite electric field strengths. Convex downward curvature of the Ferguson plots was observed for DNA fragments greater than or equal to 1 kbp in size in agarose gels greater than or equal to 2% in concentration. The mobilities of the various DNA fragments, extrapolated to zero agarose concentration and zero electric field strength, decreased with increasing DNA molecular weight; extrapolating to zero molecular weight gave an "intrinsic" DNA mobility of 2.7 x 10(-4) cm2/Vs at 20 degrees C. The pore sizes of LE agarose gels cast and run in TAE and TBE buffers were estimated from the mobility of the DNA fragments.(ABSTRACT TRUNCATED AT 400 WORDS)     

The electrophoretic mobility of DNA fragments differing by a single 3′‐terminal nucleotide in an automated capillary DNA sequencer

The electrophoretic mobility of DNA fragments that differ by a single 3′‐terminal nucleotide was assessed by capillary electrophoresis. This was accomplished using dideoxy sequencing with a 5′‐fluorescently labelled primer to generate DNA fragments with 3′‐hydrogen ends. The resulting DNA fragments were electrophoresed on the ABI 3730 automated capillary sequencer, and the data were analysed with the GeneMapper software to determine the electrophoretic mobility differences on addition of a 3′‐terminal nucleotide. It was found that the 3′‐terminal nucleotide gave rise to different electrophoretic mobility profiles depending on the identity of the terminal nucleotide. The apparent electrophoretic mobility was (faster) –C > ?A > ?T > ?G (slower). The C‐terminated fragments were the fastest and the G‐terminated fragments the slowest, relative to other nucleotides. It was proposed that the terminal nucleotide effect was due to changes in partial net charges on the nucleotides that resulted in alterations in the electrophoretic mobility of the DNA fragments in the automated capillary DNA sequencer. Other alternative explanations are also discussed. Copyright © 2012 John Wiley & Sons, Ltd.     

The electrophoretic mobility of DNA three-way junctions is affected by the sequence of overhanging single-stranded ends

The folding of three- and four-way DNA junctions is often assessed by comparing the electrophoretic mobility of restriction enzyme fragments, using the long-short arm assay. We have compared the mobility of synthetic three-way junctions that contain identical branch point sequences, but different restriction sites in the arms. We show that the mobility of fragments is affected by the sequence of the overhanging ends. In general, GC-rich overhangs produce fragments with anomalous mobilities. These anomalies can be prevented by treating the cleaved junctions with mung bean endonuclease, elevating the electrophoresis temperature or using blunt cleaving restriction endonucleases.     

Effect of UV irradiation at defined wavelengths on the tertiary structure of double-stranded covalently closed circular DNA

Double-stranded, covalently closed, supercoiled circular DNA from phage fd (replicative form) was irradiated with increasing doses of UV light at 254 nm, 290 nm, 313 nm and 365 nm, and subjected to electrophoresis on agarose slab gels. Increasing the doses of UV light at 254 and 290 nm promotes a smooth reduction in the electrophoretic mobility of the sample, as would be expected if the major effect of light at these two wavelengths were to induce the formation of photoproducts leading to the unwinding of the double strand. At high doses, UV light at 290 nm introduces single-strand breaks (1.2 kJ m-2 per nick per million phosphodiester bonds). UV light at 313 nm promotes an abrupt change in the electrophoretic mobility, as would be expected if the effect of this wavelength were to induce single-strand breaks, leading to the transformation of the supercoiled molecules in their relaxed form (23 kJ m-2 in order to introduce one nick per million phosphodiester bonds). UV light at 365 nm also promotes single-strand breaks in DNA (140 kJ m-2 per nick per million phosphodiester bonds).     

Quantitative experimental determination of primer-dimer formation risk by free-solution conjugate electrophoresis

DNA barcodes are short, unique ssDNA primers that "mark" individual biomolecules. To gain better understanding of biophysical parameters constraining primer-dimer formation between primers that incorporate barcode sequences, we have developed a capillary electrophoresis method that utilizes drag-tag-DNA conjugates to quantify dimerization risk between primer-barcode pairs. Results obtained with this unique free-solution conjugate electrophoresis approach are useful as quantitatively precise input data to parameterize computation models of dimerization risk. A set of fluorescently labeled, model primer-barcode conjugates were designed with complementary regions of differing lengths to quantify heterodimerization as a function of temperature. Primer-dimer cases comprised two 30-mer primers, one of which was covalently conjugated to a lab-made, chemically synthesized poly-N-methoxyethylglycine drag-tag, which reduced electrophoretic mobility of ssDNA to distinguish it from ds primer-dimers. The drag-tags also provided a shift in mobility for the dsDNA species, which allowed us to quantitate primer-dimer formation. In the experimental studies, pairs of oligonucleotide primer barcodes with fully or partially complementary sequences were annealed, and then separated by free-solution conjugate CE at different temperatures, to assess effects on primer-dimer formation. When less than 30 out of 30 base-pairs were bonded, dimerization was inversely correlated to temperature. Dimerization occurred when more than 15 consecutive base-pairs formed, yet non-consecutive base-pairs did not create stable dimers even when 20 out of 30 possible base-pairs bonded. The use of free-solution electrophoresis in combination with a peptoid drag-tag and different fluorophores enabled precise separation of short DNA fragments to establish a new mobility shift assay for detection of primer-dimer formation.     

Spatial open-network formed by mixed triblock copolymers as a new medium for double-stranded DNA separation by capillary electrophoresis

A mixture of two polyoxybutylene-polyoxyethylene-polyoxybutylene (BEB) triblock copolymers (B6E46B6 and B10E271B10, respectively) was used as a new separation medium for separating double-stranded DNA (dsDNA) fragments by capillary electrophoresis (CE). The two block copolymer mixtures were designed to form mixed flower-like micelles in dilute solution and a homogeneous gel-like open-network with hydrophobic clusters as cross-linking points at higher polymer concentrations. Being a polyoxyalkylene block copolymer gel, the separation medium has some special advantages, including the temperature-dependent sol-gel transition that makes sample injection easy, and the self-coating of the inner capillary wall that makes experimental procedures simple and reproducible. Furthermore, it can shorten the elution time and further improve the separation resolution, especially for small dsDNA fragments, when compared with EPE-type separation media, e.g., F127 (E99P69E99, with P being polyoxypropylene) block copolymer gels formed by the closed packing of spherical micelles. Single base pair resolution can be achieved by using the new separation medium for dsDNA fragments up to over 100 base pairs.