Estimation of binding constants of peptide nucleic acid and secondary-structured DNA by affinity capillary electrophoresis

An affinity capillary electrophoresis method was developed to determine a binding constant between a peptide nucleic acid (PNA) and a hairpin-structured DNA. A diblock copolymer composed of PNA and polyethylene glycol (PEG) was synthesized as a novel affinity probe. The base sequence of the probe's PNA segment was complementary to a hairpin-structured region of a 60-base single-stranded DNA (ssDNA). Upon applying a voltage, the DNA hairpin migrated slowly compared to a random sequence ssDNA in the presence of the PNA probe. This retardation was induced by strand invasion of the PNA into the DNA hairpin to form a hybridized complex, where the PEG segment received a large amount of hydrodynamic friction during electrophoresis. The binding constant between the PNA probe and the DNA hairpin was easily determined by mobility analysis. This simple method would be potentially beneficial in studying binding behaviors of various artificial nucleotides to natural DNA or RNA.     

Anisotropic elasticity of magnetically ordered agarose gel

Abstract

The physical properties of agarose gel prepared under strong magnetic fields were investigated. The storage modulus was measured by the reflection method with an ultrasonic pulse. The measurement results of the gel's elasticity indicate that agarose gel has anisotropic properties. The elasticity and its anisotropy depend on the concentration of the gel and the magnetic field to which it is exposed. The experimental results indicate that the anisotropic network structure of the gel is induced by the exposure to the magnetic field during gelation. The gelation mechanism under a magnetic field is discussed.     

Anisotropic elasticity of magnetically ordered agarose gel

The physical properties of agarose gel prepared under strong magnetic fields were investigated. The storage modulus was measured by the reflection method with an ultrasonic pulse. The measurement results of the gel''s elasticity indicate that agarose gel has anisotropic properties. The elasticity and its anisotropy depend on the concentration of the gel and the magnetic field to which it is exposed. The experimental results indicate that the anisotropic network structure of the gel is induced by the exposure to the magnetic field during gelation. The gelation mechanism under a magnetic field is discussed.     

Radial capillary array electrophoresis microplate and scanner for high-performance nucleic acid analysis

The design, fabrication, and operation of a radial capillary array electrophoresis microplate and scanner for high-throughput DNA analysis is presented. The microplate consists of a central common anode reservoir coupled to 96 separate microfabricated separation channels connected to sample injectors on the perimeter of the 10-cm-diameter wafer. Detection is accomplished by a laser-excited rotary confocal scanner with four color detection channels. Loading of 96 samples in parallel is achieved using a pressurized capillary array system. High-quality separations of 96 pBR322 restriction digest samples are achieved in < 120 s with the microplate system. The practical utility and multicolor detection capability is demonstrated by analyzing 96 methylenetetrahydrofolate reductase (MTHFR) alleles in parallel using a noncovalent 2-color staining method. This work establishes the feasibility of performing high-throughput genotyping separations with capillary array electrophoresis microplates.     

Liquid chromatographic determination of 2-thioxothiazolidine-4-carboxylic acid isolated from urine by affinity chromatography on organomercurial agarose gel

Urinary 2-thioxothiazolidine-4-carboxylic acid is a useful indicator to assess the degree of occupational exposure to carbon disulfide. A new procedure is described for the isolation of this compound from urine prior to reversed-phase high-performance liquid chromatography. It is based on liquid-liquid extraction with methyl tert-butyl ether, followed by affinity chromatography on organomercurial agarose gel. 5-Carboxythiouracil is used as internal standard. The superior selectivity of affinity chromatography for the isolation of 2-thioxothiazolidine-4-carboxylic acid from urine permits an isocratic high-performance liquid chromatographic analysis. The total recovery of 2.5 mg of 2-thioxothiazolidine-4-carboxylic acid/g of creatinine in spiked urine by liquid-liquid extraction combined with affinity chromatography was 48.0% (SD 2.0%, n = 8). Within-run and within-day relative standard deviations averaged 4.0% (means = 2.48 mg/g of urinary creatinine, n = 9) and 6.5% (means = 1.19 mg/g of urinary creatinine, n = 15), respectively. The detection limit of the method was estimated at 0.05 mg of 2-thioxothiazolidine-4-carboxylic acid/g of urinary creatinine. The identity and spectral purity of 2-thioxothiazolidine-4-carboxylic acid detected in the urine extracts were confirmed by diode-array UV-vis detection. Typical 2-thioxothiazolidine-4-carboxylic acid levels in individual workers exposed to carbon disulfide ranged from nondetectable to 11 mg/g of urinary creatinine, several of which exceeded the generally accepted biological exposure index of 5 mg/g of urinary creatinine.     

Staining method for protein analysis by capillary gel electrophoresis

A novel staining method and the associated fluorescent dye were developed for protein analysis by capillary SDS-PAGE. The method strategy is to synthesize a pseudo-SDS dye and use it to replace some of the SDS in SDS-protein complexes so that the protein can be fluorescently detected. The pseudo-SDS dye consists of a long, straight alkyl chain connected to a negative charged fluorescent head and binds to proteins just as SDS. The number of dye molecules incorporated with a protein depends on the dye concentration relative to SDS in the sample solution, since SDS and dye bind to proteins competitively. In this work, we synthesized a series of pseudo-SDS dyes, and tested their performances for capillary SDS-PAGE. FT-16 (a fluorescein molecule linked with a hexadodecyl group) seemed to be the best among all the dyes tested. Although the numbers of dye molecules bound to proteins (and the fluorescence signals from these protein complexes) were maximized in the absence of SDS, high-quality separations were obtained when co-complexes of SDS-protein-dye were formed. The migration time correlates well with protein size even after some of the SDS in the SDS-protein complexes was replaced by the pseudo-SDS dye. Under optimized experimental conditions and using a laser-induced fluorescence detector, limits of detection of as low as 0.13 ng/mL (bovine serum albumin) and dynamic ranges over 5 orders of magnitude in which fluorescence response is proportional to the square root of analyte concentration were obtained. The method and dye were also tested for separations of real-world samples from E. coli.     

Comprehensive interpretation of gel electrophoresis data

From temperature analysis of polyacrylamide gel electrophoresis data for rigid-rod DNA analytes, it is proposed that an entropic force term is responsible for the discrepancy between Ogston-Morris-Rodbard-Chrambach model predictions and experimental results. This entropic force originates from reduction of the orientational freedom of anisotropic analytes in small pores of polyacrylamide gels. Time-dependent fluorescence anisotropy decay measurements confirm that, even in the absence of an external field, orientation of anisotropic analytes is restricted in polyacrylamide gels. A new comprehensive model is proposed that takes this effect into consideration. Predictions based on this model are found to compare favorably with experimental data for linear and three-arm asymmetrically branched rigid-rod DNA analytes covering a broad range of molecular aspect ratios and sizes. A new length scale is also proposed for describing the effect of analyte topology on electrophoretic mobility. This length scale reduces to the analyte radius of gyration in the limiting cases of spherically symmetric and linear rigid-rod species. Based on these results, a general approach is proposed for interpreting gel electrophoresis data of charged analytes possessing simple and complex topologies.     

Separation of nanoparticles by gel electrophoresis according to size and shape

We demonstrate the separation of gold and silver nanoparticles according to their size and shape by agarose gel electrophoresis after coating them with a charged polymer layer. The separation is monitored optically using the size- and shape-dependent plasmon resonance of noble metal particles and confirmed by transmission electron microscopy (TEM). Electrophoretic mobilities are quantitatively explained by a model based on the Henry formula, providing a theoretical framework for predicting gel mobilities of polymer coated nanoparticles.     

Identification of proteins in complexes by solid-phase microextraction/multistep elution/capillary electrophoresis/tandem mass spectrometry.

A method to directly identify proteins in complex mixtures by solid-phase microextraction (micro-SPE)/multistep elution/capillary electrophoresis (CE)/tandem mass spectrometry (MS/MS) is described. A sheathless liquid-metal junction interface is used to interface CE and electrospray ionization MS/MS. A subfemtomole detection limit is achieved for protein identification through database searching using MS/MS data. The SPE serves as a semiseparation dimension using an organic-phase step-elution gradient in combination with the second separation dimension for increased resolving power of complex peptide mixtures. This approach improves the concentration detection limit for CE and allows more proteins in complex mixtures to be identified. A 75-protein complex from yeast ribosome is analyzed using this method and 80-90% of the proteins in the complex can be identified by searching the database using the MS/MS data from a complete analysis. This multidimensional CE/MS/MS methodology provides an alternative to multidimensional liquid chromatography/MS/MS for direct identification of small amounts of protein in mixtures.     

Amperometric detection of nucleic acid at femtomolar levels with a nucleic acid/electrochemical activator bilayer on gold electrode

Cationic redox polymers containing osmium-bipyridine complexes strongly interact with anionic enzymes, such as glucose oxidase and peroxidases, and electrochemically "activate" the enzymes. On the basis of these observations, attempts were made to develop an ultrasensitive nucleic acid biosensor. A mixed monolayer of single-stranded oligonucleotide capture probe and 16-mercaptohexadecanoic acid was formed on a gold electrode through self-assembly. Following hybridization with a complementary nucleic acid and glucose oxidase labeled oligonucleotide detection probe, a cationic redox polymer (electrochemical activator) overcoating was applied to the electrode through layer-by-layer electrostatic self-assembly. The formation of an anionic-cationic bilayer brought the glucose oxidase in electrical contact with the redox polymer, making the bilayer an electrocatalyst for the oxidation of glucose. Thus, nucleic acid molecules were quantified amperometrically at femtomolar levels. The effect of experimental variables on the amperometric response was investigated and optimized to maximize the sensitivity and speed up the assay time. A detection limit of 1.0 fmol/L in 1.0-microL droplets and a linear current-concentration relationship up to 800 fmol/L were attained following a 30-min hybridization. The biosensor was applied to the detection of the 16S gene in a mixture of Escherichia coli 16S + 32S rRNA and a full-length rat housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), of a RT-PCR product.     

Study of a fiber optic humidity sensor based on agarose gel

An optical fiber humidity sensor was fabricated using a hydrophilic gel (agarose) deposited on the tapered plastic optical fiber (POF). The sensing element, agarose, can absorb and exude moisture from/to the ambience, thereby altering its refractive index and changing its ability to modulate the intensity of light that propagates through the fiber. Thus, the operating principle of the sensor is based on the intensity modulation technique, which utilizes a tapered POF probe coated with agarose that is sensitive to humidity. The POF, which was fabricated using an etching method, has a waist diameter of 0.45 mm and tapering length of 10 mm. As the relative humidity varies from 50% to 80%, the output voltage of the sensor with agarose gel of 0.5% weight content decreases linearly from 2.24 mV to 1.55 mV. The agarose-based sensor produces a sensitivity of 0.0228 mV/%, with a slope linearity of more than 98.36%. The tapered fiber with agarose gel of 1% weight content produces a sensitivity of 0.0103 mV/% with a slope linearity of more than 94.95% and a limit of detection of 2.635%, while the tapered fiber with agarose gel of 1.5% weight content produces a sensitivity of 0.0079 mV/% with a slope linearity of more than 98.53% and a limit of detection of 6.853%. The fiber with agarose gel of 0.5% weight content shows higher sensitivity compared to that of 1% and 1.5% due to the effect of pore size, which changes with concentration. The results demonstrate that agarose-based optical fiber sensors are both sensitive and efficient for economical and flexible measurements of humidity.     

Noninvasive imaging of nanogram quantities of DNA in agarose electrophoresis gels.

Image processing techniques which separate the DNA Kerr effect and induced electrokinetic distortion contributions to electric birefringence images of agarose nucleic acid electrophoresis gels are described. Under standard electrophoresis conditions, detection limits of 10 ng of DNA per well are obtained in hydroxyethylated agarose without signal averaging or 7.5 ng with averaging of four measurements. Maintaining constant gel temperature is shown to improve the quality of the images. Monochromatic light (589 nm) is shown to give a small increase in sensitivity compared to polychromatic (650 +/- 20 nm) light.     

Oligomeric separation of ionic and nonionic ethoxylated polymers by capillary gel electrophoresis

Capillary gel electrophoresis (CGE) has proven itself as a superior, high-resolution technique for separating proteins, peptides, oligonucleotides, and other naturally occurring molecules. In the years since its inception, few applications of CGE to nonbiological synthetic polymers have been reported. CGE has been applied to the separation of ionic and nonionic ethoxylated surfactants and poly(ethylene glycol) (PEG) oligomers. Oligomer distributions of several sulfated and phosphated alkylphenol ethoxylate surfactants have been baseline resolved with CGE on commercial cross-linked polyacrylamide gel columns. Nonionic surfactants and PEG oligomers were derivatized with phthalic anhydride in order to provide charge and detectability. PEG oligomers ranging from ethylene glycol to species containing more than 120 ethylene oxide units have been resolved. A linear relationship between migration time and molecular weight was found, which indicates that the separation mechanism is not simply based on molecular size but is also influenced by the electrophoretic mobility of the oligomers. The main drawbacks of CGE include relatively long analysis times and somewhat fragile and expensive columns.     

Surface-enhanced Raman spectroscopy of chernozem humic acid and their fractions obtained by coupled size exclusion chromatography-polyacrylamide gel electrophoresis (SEC-PAGE)

A humic acid extracted from a chernozem soil was fractionated combining size exclusion chromatography and polyacrylamide electrophoresis (SEC-PAGE). Three fractions named A, B, and C+D, with different electrophoretic mobilities and molecular sizes (MS), were obtained and subsequently characterized by thermochemolysis and surface-enhanced Raman spectroscopy (SERS). The data confirmed that fraction A, with the higher MS, was more aliphatic than fractions B and C+D and, in turn, fractions with lower MS (B and C+D) denoted an enrichment in lignin residues. These structural features explain conformational changes when varying the pH in the humic fraction A and indicated that combination of the two techniques is a good approach for characterizing humic substances.     

Capillary gel affinity electrophoresis of DNA fragments.

The incorporation of an affinity ligand within a polyacrylamide gel provides a general means of manipulating the selectivity of capillary gel electrophoresis separations. As an example of this approach, high resolution of DNA restriction fragments by capillary gel affinity electrophoresis has been achieved by adding a soluble intercalating agent, ethidium bromide, to the gel-buffer system. A migration model has been developed that can be used for selectivity optimization. Various parameters, such as ligand concentration and applied electric field, have been examined in terms of their influence on retention and selectivity of different-size DNA molecules. From this study, high-resolution separations have been developed with efficiencies as high as 10(7) theoretical plates per meter.     

Small-scale analysis of O-linked oligosaccharides from glycoproteins and mucins separated by gel electrophoresis

A technique with subpicomolar sensitivity was developed for analyzing O-linked oligosaccharides released from glycoproteins separated by gel electrophoresis. The protocol involves gel electrophoresis, electroblotting to poly-(vinylidene fluoride) membrane, reductive beta-elimination, and analysis of released oligosaccharides by liquid chromatography coupled to negative ion electrospray mass spectrometry. It was also found that N-linked oligosaccharides could be recovered under the same conditions, found both as free oligosaccharides and as distinct glycopeptides created from reductive cleavage of the protein backbone, giving some information on site-specific glycosylation. The method was used to demonstrate that the difference between human alpha-2HS-glycoprotein isoforms separated by 2D-gel electrophoresis was partially due to sialylation of both O-linked and N-linked oligosaccharides. It was also shown that both acidic and neutral oligosaccharides could be recovered and analyzed simultaneously from high molecular mass (200,000-5,000,000 Da) highly glycosylated mucin glycoproteins collected from small intestine and saliva and separated by sodium dodecyl sulfate-agarose/polyacrylamide composite gels. Mass spectrometric data not only gave information about the mass distribution of the heterogeneous mixtures of oligosaccharides from [M - xH](x-) ions but also gave information about the isomeric heterogeneity of the oligosaccharides from their resolution by porous graphitized carbon chromatography. Tandem mass spectrometry was explored as a technique for distinguishing between oligosaccharide isomers with different sequences and also between oligosaccharides with the same sequence but with different linkage configurations.