Capillary zone electrophoresis for the characterization of latex particles

Capillary zone electrophoresis (CZE) was applied to the separation of acrylic styrene copolymer emulsion particles. Fast separations could be performed on samples containing chemically identical latex particles of different size, as well as on samples with particles of the same size but differing in chemical composition. The developed method was also used for the analysis of water soluble fractions of urethane dispersions. Additionally, the physical interaction between different particles (e.g., acrylic and urethane particles) could be studied using this method. The separation mechanism is based on the zeta potential of the particles and the relaxation effect under the applied analytical conditions.     

Capillary electrophoresis investigation of a partially unfolded conformation of beta(2)-microglobulin

Dialysis-related amyloidosis is a disease in which partial unfolding of beta(2)-microglobulin plays a key pathogenetic role in the formation of the amyloid fibrils. We have recently demonstrated that a partially unfolded conformer of beta(2)-microglobulin is involved in fibrillogenesis and that this species is significantly populated under physiological conditions. In this work capillary electrophoresis has been used to measure the equilibrium between the native protein and this conformer in samples known to have a higher or lower amyloidogenic potential, namely full-length beta(2)-microglobulin, two truncated species and a mutant, created by replacing histidine in position 31 with thyrosine. In addition, for all protein species folding stability experiments have been carried out by monitoring the secondary structure by circular dichroism at increasing concentrations of guanidinium chloride. The values of free energy of unfolding in the absence of denaturant, obtained by elaboration of these experiments, were found to be inversely correlated to the area percent of the partially unfolded conformer, as measured by capillary electrophoresis. Affinity capillary electrophoresis experiments have been also carried out under nondenaturing conditions to assess the affinity of copper and suramin to either the native form or the conformational intermediate of full-length beta(2)-microglobulin.     

Capillary electrophoresis for detection of inherited disorders of purine and pyrimidine metabolism: a selective approach

We developed a capillary electrophoresis method as a diagnostic tool for purine and pyrimidine metabolic disorders. Optimal conditions allowed the separation of the major diagnostic metabolites in urine samples within an analysis time of 10 min and with a separation efficiency of about 350,000 theoretical plates/m. The diagnostically important metabolites (adenine, adenosine, 2-deoxyadenosine, 2-deoxyguanosine, 2,8-dihydroxyadenine, guanosine, hypoxanthine, orotidine, orotic acid, and creatinine) were detectable at concentrations of 1.0-5.7 micromol/L. The method gives a linear calibration curve for tested purine and pyrimidine derivatives within the range of 5-500 micromol/L (r > 0.996) The coefficients of variation for the within- and between-day imprecisions were less than 3.2 and 5.8%, respectively. Characteristic abnormalities were detected in the electropherograms of urine samples from patients with purine and pyrimidine enzyme deficiencies. We provide electrophoretic and spectral characteristics of intermediates in purine and pyrimidine metabolism and possible artifacts from medication and their UV-absorbing compounds. Our method allows the detection of the majority of inborn errors of purine and pyrimidine metabolism.     

Optimization of capillary array electrophoresis single-strand conformation polymorphism analysis for routine molecular diagnostics

Mutation screening is widely used for molecular diagnostics of inherited disorders. Furthermore, it is anticipated that the present and future identification of genetic risk factors for complex disorders will increase the need for high-throughput mutation screening technologies. Capillary array electrophoresis (CAE) SSCP analysis is a low-cost, automated method with a high throughput and high reproducibility. Thus, the method fulfills many of the demands to be met for application in routine molecular diagnostics. However, the need for performing the electrophoresis at three temperatures between 18 degrees C and 35 degrees C for achievement of high sensitivity is a disadvantage of the method. Using a panel of 185 mutant samples, we have analyzed the effect of sample purification, sample medium and separation matrix on the sensitivity of CAE-SSCP analysis to optimize the method for molecular diagnostic use. We observed different effects from sample purification and sample medium at different electrophoresis temperatures, probably reflecting the complex interplay between sequence composition, electrophoresis conditions and sensitivity in SSCP analysis. The effect on assay sensitivity from three different polymers was tested using a single electrophoresis temperature of 27 degrees C. The data suggest that a sensitivity of 98-99% can be obtained using a 10% long chain poly-N,N-dimethylacrylamide polymer.     

A new approach for separating low-molecular-weight RNA molecules by staircase electrophoresis in non-sequencing gels

Low-molecular-weight (LMW) RNA profiles, which include ribosomal and transfer RNA molecules with similar small sizes, are molecular signatures of microorganisms with a great potential in microbial identification. The greatest resolution of these profiles was achieved by staircase electrophoresis in sequencing gels. Nevertheless, this technique is difficult to use because it takes 7 h, the gels have large sizes and it is necessary to heat the system and to recycle the buffer to maintain the denaturing conditions and avoid smile effects. Most available sequencing slabs have no internal temperature control or homogenizing devices, which by contrast are present in some newly designed non-sequencing slabs. Nevertheless, these slabs present two important problems for separating LMW RNA molecules, the size of gels is only 20 cm (instead of 40 cm) and the maximum voltage that can be reached is only 840 V (instead 2400 V). Staircase electrophoresis follows a model in which the external polarization is incrementally modified with a constant time step value. In the present work, we experimentally confirmed that by reducing the time step and increasing the total number of steps a suitable resolution is achieved. Under these conditions, despite the smaller size of the gels and the lower values of the electric field, the intensity reaches higher values than in sequencing gels and the LMW RNA profiles are correctly separated in 5 h. The resolution of these profiles obtained in non-sequencing gels is similar to that obtained in sequencing ones facilitating the analysis of large populations of microorganisms in any laboratory.     

Adaption of a fragment analysis technique to an automated high-throughput multicapillary electrophoresis device for the precise qualitative and quantitative characterization of microbial communities

The analysis of microbial communities is of increasing importance in life sciences and bioengineering. Traditional techniques of investigations like culture or cloning methods suffer from many disadvantages. They are unable to give a complete qualitative and quantitative view of the total amount of microorganisms themselves, their interactions among each other and with their environment. Obviously, the determination of static or dynamic balances among microorganisms is of fast growing interest. The generation of species specific and fluorescently labeled 16S ribosomal DNA (rDNA) fragments by the terminal restriction fragment length polymorphism (T-RFLP) technique is a suitable tool to overcome the problems other methods have. For the separation of these fragments polyacrylamide gel sequencers are preferred as compared to capillary sequencers using linear polymers until now because of their higher electrophoretic resolution and therefore sizing accuracy. But modern capillary sequencers, especially multicapillary sequencers, offer an advanced grade of automation and an increased throughput necessary for the investigation of complex communities in long-time studies. Therefore, we adapted a T-RFLP technique to an automated high-throughput multicapillary electrophoresis device (ABI 3100 Genetic Analysis) with regard to a precise qualitative and quantitative characterization of microbial communities.     

Capillary electrophoresis for the characterization of the complex dendrimeric contrast agent Gadomer

A CE method for the characterization of the complex dendrimeric contrast agent Gadomer has been developed and validated. The method was capable of separating the target substance Gadomer 24 from related dendrimers containing amino or carboxyl functions and from impurities of lower molecular weight. The compounds were separated in a fused-silica capillary. The optimized BGE consisted of 15 mM sodium phosphate, pH 6.3, containing 0.5 mM hexadecyltrimethylammonium bromide. The assay was validated with regard to linearity, specificity, accuracy, LOD and LOQ as well as robustness according to the guidelines of the International Conference on Harmonization. The method allows the determination of the purity and stability of the drug substance Gadomer as well as its injectable formulation. On the basis of the present study, a strategy for the quality assurance and quality control of the complex dendrimeric drug candidate Gadomer may be devised. The method may therefore serve as a key component in a set of analytical methodologies designed to assure and control the reproducible quality and safety of this innovative product. To the best of our knowledge, this is the first work reporting a validated method for the characterization, impurity profiling, and stability testing of a dendrimeric agent designed for clinical use.     

Capillary electrophoresis for the determination of new markers of natural latex quality

Nowadays, increasing use of latex products in the healthcare area has also led to an increase in new scientific controls of raw material and manufactured products in order to maintain higher standards in quality control. Since field latex is a rich environment for microbial growth, in the absence of adequate preservation, the carbohydrates that it contains become microbiologically oxidised to the so-called volatile fatty acids (VFAs). Samples of natural rubber latex coming from different countries and processed in different ways have been tested by applying a capillary electrophoresis method for the measurement of the short-chain organic acids present in sera. The separation was performed with phosphate buffer at pH 6.25 and cetyltrimethylammonium bromide (CTAB) as background electrolyte in an uncoated fused-silica capillary with -10 kV of applied potential. The sample needs no other pre-treatment more than coagulation to obtain the serum. In the assayed samples, it has been shown that poorly-preserved latex presented higher amounts of succinic acid and lower amounts of malic acid. The succinic to malic acid ratio may be an important parameter because it cannot be altered by dilution or similar processes such as the traditional VFA index, used to determine the quality of latex. Ratios for succinic to malic acid <0.6 have been found for well-preserved latex and >0.6 for poorly-preserved latex.     

Capillary electrophoresis with laser-induced fluorescence detection as a tool for enzyme characterization and inhibitor screening.

An effective, rapid and economical CE/LIF (capillary electrophoresis/laser-induced fluorescence) method was developed and applied to the characterization of signal peptidase (SPase) enzyme, which is a target for the screening of new drug candidates. In this method, CE separates the product from the substrate and LIF selectively detects the fluorescence-labeled product and substrate. By measuring the increase of the product as a function of time, one can monitor the progression of the enzyme reaction. The progression curves were also used for screening inhibitors for this enzyme. The effects of various reaction conditions were also studied and discussed. In addition, this CE/LIF method was applied to the determination of the enzyme activity, the quality control of the substrate and/or enzymes, and the cross-reactivity of inhibitors to the enzyme. It can be concluded that this method is suitable for high throughput screening (HTS) assays because it can deliver fast, sensitive, quantitative, and reliable results.     

Fluorescence-based approach for detecting and characterizing antibiotic-induced conformational changes in ribosomal RNA: comparing aminoglycoside binding to prokaryotic and eukaryotic ribosomal RNA sequences

Aminoglycoside antibiotics bind specifically to a conserved sequence of the 16S ribosomal RNA (rRNA) A site and interfere with protein synthesis. One model for the mechanism underlying the deleterious effects of aminoglycosides on protein synthesis invokes a drug-induced conformational change in the rRNA that involves the destacking of two adenine residues (A1492 and A1493 in Escherichia coli) at the A site. We describe here a fluorescence-based approach for detecting and characterizing this drug-induced conformational change in the target rRNA. In this approach, we insert the fluorescent base analogue 2-aminopurine in place of A1492 in an E. coli 16S rRNA A-site model oligonucleotide (EcWT) as well as in a mutant form of this oligomer (A1408G) in which A1408 has been replaced with a guanine. The presence of guanine at 1408 instead of adenine represents one of the major sequence differences between prokaryotic and eukaryotic A sites, with the latter A sites being resistant to the deleterious effects of aminoglycosides. Binding of the aminoglycoside paromomycin to the 2AP-substituted forms of EcWT and A1408G induced changes in fluorescence quantum yield consistent with drug-induced base destacking in EcWT but not A1408G. Isothermal titration calorimetry studies reveal that paromomycin binds to the EcWT duplex with a 31-fold higher affinity than the A1408G duplex, with this differential affinity being enthalpic in origin. In the aggregate, these observations are consistent with both rRNA binding affinity and drug-induced base destacking being important determinants in the prokaryotic specificity of aminoglycosides. Combining fluorescence quantum yield and lifetime data allows for quantification of the extent of drug-induced base destacking, thereby providing a convenient tool for evaluating the relative impacts of both novel and existing A-site targeting ligands on rRNA conformation and potentially for predicting relative antibiotic activities and specificities.     

Capillary electrophoresis of double-stranded DNA fragments using a new fluorescence intercalating dye EvaGreen

EvaGreen is a new DNA intercalating dye successfully used in quantitative real-time PCR. In the present work, we firstly apply EvaGreen to the analysis of dsDNA by CE with LIF detection. Comparisons of EvaGreen dye with the commonly used dyes SYBR Green I and SYBR Gold were preformed in dsDNA analysis by CE. The linear range of dsDNA using EvaGreen was slightly wider than that using SYBR Gold and SYBR Green I, and the detection limits of dsDNA were not significantly different for the three dyes. Good separations of dsDNA fragments were obtained using the three dyes. Reproducibility of migration time and the peak area of dsDNA fragments with EvaGreen were better than those for SYBR Green I and SYBR Gold. The RSD values were 0.24-0.27% for migration time and 3.45-7.59% for peak area within the same day, 1.35-1.63% for migration time and 6.72-12.05% for peak area for three days. Our data demonstrated that EvaGreen is well suited for the dsDNA analysis by CE with LIF detection.     

Single-strand conformation polymorphism analysis of candidate genes for reliable identification of alleles by capillary array electrophoresis

We investigated the reliability of capillary array electrophoresis-single strand conformation polymorphism (CAE-SSCP) to determine if it can be used to identify novel alleles of candidate genes in a germplasm collection. Both strands of three different size fragments (160, 245 and 437 bp) that differed by one or more nucleotides in sequence were analyzed at four different temperatures (18 degrees C, 25 degrees C, 30 degrees C, and 35 degrees C). Mixtures of amplified fragments of either the intron interrupting the C-terminal WRKY domain of the Tc10 locus or the NBS domain of the TcRGH1 locus of Theobroma cacao were electroinjected into all 16 capillaries of an ABI 3100 Genetic Analyzer and analyzed three times at each temperature. Multiplexing of samples of different size range is possible, as intermediate and large fragments were analyzed simultaneously in these experiments. A statistical analysis of the means of the fragment mobilities demonstrated that single-stranded conformers of the fragments could be reliably identified by their mobility at all temperatures and size classes. The order of elution of fragments was not consistent over strands or temperatures for the intermediate and large fragments. If samples are only run once at a single temperature, small fragments could be identified from a single strand at a single temperature. A combination of data from both strands of a single run was needed to identify correctly all four of the intermediate fragments and no combination of data from strands or temperatures would allow the correct identification of two large fragments that differed by only a single single-nucleotide polymorphism (SNP) from a single run. Thus, to adequately assess alleles at a candidate gene locus using SSCP on a capillary array, fragments should be < or =250 bp, samples should be analyzed at two different temperatures between 18 degrees C and 30 degrees C to reduce the variability introduced by the capillaries, data should be combined from both strands and both temperatures, and undenatured double-stranded (ds)DNA molecular weight standards, such as ROX 2500, should be included as internal standards.     

Capillary electrophoresis for capture and concentrating of target nucleic acids by affinity gels modified to contain single-stranded nucleic acid probes

Selective capture and pre-concentration of target nucleic acids from relatively complicated samples may provide a method to facilitate introduction to a microfluidic-based detection system to improve detection limits. An acrylamide polymer gel modified with Acrydite^™ that contained 20mer oligonucleotide probe was prepared and loaded into a capillary column. The results indicated that the amount of probe DNA that was captured into the acrylamide was about 40% of the starting monomer, and different quantities of probe could therefore be coupled into the gel. The gel was passivated by pre-treatment with non-complementary DNA oligonucleotide to block non-selective adsorption sites, and the gel was determined to be stable for multiple cycles of use. The probe could hybridize with target sequences that were introduced by electrokinetic injection from a sample solution. The target could be freed from the polymer gel by use of a combination of heating, chaotropic salt and voltage conditions. Target capture efficiency was up to 90% when using samples that did not saturate probe sites in the columns, and recovery of target from the gel could be as high as 95%.     

Capillary electrophoresis: a useful tool for the management of plant genetic resources

Capillary electrophoresis (CE) is a powerful analytical tool that is widely applied to the analysis of biological samples. Proteins, peptides, nonprotein amino acids, phenolic compounds, and ions can be analysed using this electrophoretic methodology. This review summarises some applications of CE to the evaluation and characterisation of plant genetic resources of both Triticum and legume species, as carried out at the Istituto di Genetica Vegetale, National Research Council (IGV-CNR) in Bari (Italy). Different protein fractions as well as nonprotein amino acids were investigated by capillary zone electrophoresis (CZE), the most user-friendly mode of CE application. The described case studies show that CZE can be applied to some institutional activities of gene banks such as the evaluation of genetic diversity within stored collections, the acquisition of new samples, the differentiation of species belonging to the same genus, the identification of misclassified accessions, and the measurement of compounds relevant to nutrition or health.     

Single-strand conformation polymorphism for p53 mutation by a combination of neutral pH buffer and temperature gradient in capillary electrophoresis

A large number of point mutations in the p53 gene have been detected by capillary zone electrophoresis via single-strand conformation polymorphism (SSCP) analysis. A much improved detection sensitivity was obtained via the following modifications in running conditions: use of low-viscosity 3% hydroxyethylcellulose (HEC), a neutral pH (pH 6.8) buffer, in which the standard Tris moiety was substituted with a 2-(N-morpholino)ethanesulfonic acid (MES)/Tris mixture, use of SYBR Green II for improved fluorescent signal at the lower pH adopted; and, finally, the use of a temperature gradient in the 15-25 degrees C interval, for favoring the conformational transitions in the mutated samples. The typical temperature gradient activated had a slope of 2 degrees C/min and were induced externally. A total of 24 samples from affected patients, both in the homo- and heterozygous state, were analyzed. All the mutations could be detected by this improved protocol, raising the sensitivity from the standard ca. 80% of conventional SSCP to essentially 100% with the present methodology. All the mutations were confirmed by sequence analysis of the affected samples.     

A single-strand conformation polymorphism method for the large-scale analysis of mutations/polymorphisms using capillary array electrophoresis

We present a high-throughput single-strand conformation polymorphism (SSCP) method, performed on a commercially available capillary array DNA sequencer. We tested various sieving matrices and electrophoretic conditions, using 51 DNA fragments which included 45 fragments carrying only one single nucleotide polymorphism (SNP), 4 fragments having two SNPs and 2 fragments with insertion or deletion. Resolution of alleles was improved by increasing concentrations of both sieving matrices and buffers, and all examined polymorphisms of DNA fragments were detected, most of them (45 fragments) as clearly split allele peaks in heterozygotes. Allele frequencies of SNPs can be estimated accurately by determining the relative amounts of alleles in pooled DNA. In this method, the turn-around time for the analysis of 96 samples is less than 3 h. These results demonstrate that capillary array-based SSCP is an efficient and accurate technique for the large-scale quantitative analysis of mutations/polymorphisms.     

Effect of temperature gradients on single-strand conformation polymorphism analysis in a capillary electrophoresis system using Pluronic polymer matrix

Capillary electrophoresis-single strand conformation polymorphism (CE-SSCP) analysis is a prominent bioseparation method based on the mobility diversity caused by sequence-induced conformational differences of single-stranded DNA. The use of Pluronic polymer matrix has opened up new opportunities for CE-SSCP, because it improved the resolution for various genetic analyses. However, there still exists a challenge in optimizing Pluronic-based CE-SSCP, because the physical properties of Pluronic solutions are sensitive to temperature, particularly near the gelation temperature, where the viscoelasticity of Pluronic F108 solutions sharply changes from that of a Newtonian fluid to a hydrogel upon heating. We have focused on a set of experiments to control the ambient temperature of the CE system with the aim of enhancing the reliability of the CE-SSCP analysis by using the Applied Biosystems ABI 3130xl genetic analyzer with Pluronic F108 solution matrix. The ambient temperature control allowed us to vary the inlet and outlet portion of the capillary column, while the temperature of the column was kept at 35 °C. The resolution to separate 2 single-base-pair-differing DNA fragments was significantly enhanced by changing the temperature from 19 to 30 °C. The viscoelastic properties of the F108 solution matrix upon heating were also investigated by ex situ rheological experiments with an effort to reveal how the development of gels in Pluronic solutions affects the resolution of CE-SSCP. We found that the column inlet and outlet temperatures of the capillary column have to be controlled to optimize the resolution in CE-SSCP by using the Pluronic matrix.     

Capillary electrophoresis as a tool for optimization of multiplex PCR reactions

Copying multiple regions of a DNA molecule is routinely performed today using the polymerase chain reaction (PCR) in a process commonly referred to as multiplex PCR. The development of a multiplex PCR reaction involves designing primer sets and examining various combinations of those primer sets and different reaction components and/or thermal cycling conditions. The process of optimizing a multiplex PCR reaction in order to obtain a well-balanced set of amplicons can be time-consuming and labor-intensive. The rapid separation and quantitation capabilities of capillary electrophoresis make it an efficient technique to help in the multiplex PCR optimization process.     

Capillary electrophoresis as a tool for optimization of multiplex PCR reactions

Copying multiple regions of a DNA molecule is routinely performed today using the polymerase chain reaction (PCR) in a process commonly referred to as multiplex PCR. The development of a multiplex PCR reaction involves designing primer sets and examining various combinations of those primer sets and different reaction components and/or thermal cycling conditions. The process of optimizing a multiplex PCR reaction in order to obtain a well-balanced set of amplicons can be time-consuming and labor-intensive. The rapid separation and quantitation capabilities of capillary electrophoresis make it an efficient technique to help in the multiplex PCR optimization process.