Functionalized gold nanoparticles as additive to form polymer/metal composite matrix for improved DNA sequencing by capillary electrophoresis

A new matrix additive, poly (N,N-dimethylacrylamide)-functionalized gold nanoparticle (GNP-PDMA), was prepared by “grafting-to” approach, and then incorporated into quasi-interpenetrating network (quasi-IPN) composed of linear polyacrylamide (LPA, 3.3 MDa) and PDMA to form novel polymer/metal composite sieving matrix (quasi-IPN/GNP-PDMA) for DNA sequencing by capillary electrophoresis. Without complete optimization, quasi-IPN/GNP-PDMA yielded a readlength of 801 bases at 98% accuracy in about 64 min by using the ABI 310 Genetic Analyzer at 50 °C and 150 V/cm. Compared with previous quasi-IPN/GNPs, quasi-IPN/GNP-PDMA can further improve DNA sequencing performances. This is because the presence of GNP-PDMA can improve the compatibility of GNPs with the whole sequencing system, enhance the entanglement degree of networks, and increase the GNP concentration in system, which consequently lead to higher restriction and stability, higher apparent molecular weight (MW), and smaller pore size of the total sieving networks. Furthermore, the composite matrix was also compared with quasi-IPN containing higher-MW LPA and commercial POP-6. The results indicate that the composite matrix is a promising one for DNA sequencing to achieve full automation due to the separation provided with high resolution, speediness, excellent reproducibility, and easy loading in the presence of GNP-PDMA.     

Protein separation by capillary gel electrophoresis: A review

Capillary gel electrophoresis (CGE) has been used for protein separation for more than two decades. Due to the technology advancement, current CGE methods are becoming more and more robust and reliable for protein analysis, and some of the methods have been routinely used for the analysis of protein-based pharmaceuticals and quality controls. In light of this progress, we survey 147 papers related to CGE separations of proteins and present an overview of this technology. We first introduce briefly the early development of CGE. We then review the methodology, in which we specifically describe the matrices, coatings, and detection strategies used in CGE. CGE using microfabricated channels and incorporation of CGE with two-dimensional protein separations are also discussed in this section. We finally present a few representative applications of CGE for separating proteins in real-world samples.     

Low viscosity DNA sieving matrices for capillary electrophoresis

The rapid development of DNA capillary electrophoresis (CE) technology has increased the demand of new low viscosity sieving matrices with high separation capacity. The high throughput, resolution and automatic operation of CE systems have stimulated the application of the technique to different kinds of DNA analysis, including DNA sequencing, separation of restriction fragments, PCR products and synthetic oligonucleotides. In addition specific methods for PCR-based mutation assays for the study of known and unknown point mutations have been developed for use in CE. The key component for a large scale application of CE to DNA analysis is the availability of appropriate sieving matrices. This article gives an overview of the linear polymers used as DNA separation matrices with particular emphasis on the polymers that combine high sieving capacity, low viscosity and chemical resistance.     

High-speed DNA sequencing by tube-based capillary electrophoresis

We assessed the feasibility of high-speed DNA sequencing by tube-based capillary electrophoresis (TCE) with electrokinetic sample injections. We developed a water-circulated TCE system to control the capillary temperature precisely. Using this system and a ready-made sieving matrix at 50 degrees C, single-stranded DNA size marker fragments were separated at various pairs of the electric field strength, E, of 128-480 V/cm and the capillary effective length, L, of 100-360 mm. Assuming the read length (RL) is the fragment size at which the peak width equals the peak interval per base in obtained electropherograms, we estimated the values of RL (E, L), the RL at the pair (E, L). The points in ELz-space, (E, L, RL(E, L)), form a curved surface expressed by z = RL(E, L). Analyzing the contour lines of this curved surface, we determined the pairs of E and L providing target RLs of 300-500 bases within a minimum time. At a pair optimized for a 500-base RL (330 V/cm, 200 mm), one-color sequencing fragments were successfully separated up to 529 bases within 9.6 min. These results demonstrate that high-speed DNA sequencing comparable with that obtained by microfabricated chip-based capillary electrophoresis (MCE) can be achieved with TCE, which is more suitable in automation than MCE.     

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.     

Band broadening of DNA fragments isolated by polyacrylamide gel electrophoresis in capillary electrophoresis

Polyacrylamide gel electrophoresis (PAGE) is used frequently for isolation and purification of DNA fragments. In the present study, DNA fragments extracted from polyacrylamide gels showed significant band broadening in capillary electrophoresis (CE). A pHY300PLK (a shuttle vector functioning in Escherichia coli and Bacillus subtilis) marker, which contained nine fragments ranging from 80 to 4870 bp, was separated by PAGE, and each fragment was isolated by phenol/chloroform extraction and ethanol precipitation. After extraction from the polyacrylamide gel, the peaks of the isolated DNA fragments exhibited band broadening in CE, where a linear poly(ethylene oxide) was used as a sieving matrix. The theoretical plate numbers of the DNA fragments contained in the pHY300PLK marker were >10⁶ for all the fragments before extraction. However, the DNA fragments extracted from the polyacrylamide gel showed decreased theoretical plate numbers (5–20 times smaller). The degradation of the theoretical plate number was significant for middle sizes of the DNA fragments ranging from 489 to 1360 bp, whereas the largest and smallest fragments (80 and 4870 bp) had no obvious influence. The band broadening was attributed to contamination of the DNA fragments by polyacrylamide fibers during the separation and extraction process.     

Mechanism of sequence-based separation of single-stranded DNA in capillary zone electrophoresis

Separation of DNA by length using CGE is a mature field. Separation of DNA by sequence, in contrast, is a more difficult problem. Existing techniques generally rely upon changes in intrinsic or induced differences in conformation. Previous work in our group showed that sets of ssDNA of the same length differing in sequence by as little as a single base could be separated by CZE using simple buffers at high ionic strength. Here, we explore the basis of the separation using circular dichroism spectroscopy, fluorescence anisotropy, and small angle X-ray scattering. The results reveal sequence-dependent differences among the same length strands, but the trends in the differences are not correlated to the migration order of the strands in the CZE separation. They also indicate that the separation is based on intrinsic differences among the strands that do not change with increasing ionic strength; rather, increasing ionic strength has a greater effect on electroosmotic mobility in the normal direction than on electrophoretic mobility of the strands in the reverse direction. This increases the migration time of the strands in the normal direction, allowing more time for the same-length strands to be teased apart based on very small differences in the intrinsic properties of the strands of different sequence. Regression analysis was used to model the intrinsic differences among DNA strands in order to gain insight into the relationship between mobility and sequence that underlies the separation.     

Matrix conditioning for lengthened capillary DNA sequencing

Capillary electrophoresis (CE) is currently the preferred format for both DNA sequencing and small DNA fragment analysis. The present study provides a simple revision of the procedure used for CE of DNA with a commercial DNA sequencing apparatus from Applied Biosystems. The revision is electrophoretic conditioning of the sieving matrix (typically POP-6) before sample injection. The effects of this preconditioning are revealed during subsequent analyses performed without replenishing the sieving matrix. The primary effect of preconditioning is to increase peak separations during a subsequent CE. The preconditioning has the following characteristics: (i) The effect on peak separation progressively increases as the preconditioning time increases to at least 6 h. (ii) The effect on peak separation scales approximately as the product of the preconditioning time and the magnitude of the electrical field (162 - 320 V/cm) during preconditioning. (iii) The preconditioning persists for more than 72 h at zero field. Preconditioning of the matrix substantially improves resolution of fragment analysis in the range of 700-2000 nucleotides. For DNA sequencing, the primary impact of preconditioning is, thus far, extension of the range of low-quality base calls at the end of sequence reading. Matrix preconditioning is a new factor to consider when interpreting data obtained by CE in polymer solutions. The mechanism of preconditioning is not yet known.     

Integrated circuit microchip system with multiplex capillary electrophoresis module for DNA analysis

In this paper, we describe the use of an integrated circuit (IC) microchip system as a detector in multiplex capillary electrophoresis (CE). This combination of multiplex capillary gel electrophoresis and the IC microchip technology represents a novel approach to DNA analysis on the microchip platform. Separation of DNA ladders using a multiplex CE microsystem of four capillaries was monitored simultaneously using the IC microchip system. The IC microchip-CE system has advantages such as low cost, rapid analysis, compactness, and multiplex capability, and has great potential as an alternative system to conventional capillary array gel electrophoresis systems based on charge-coupled device (CCD) detection.     

DNA capillary electrophoresis using poly(vinyl alcohol). I. Inner capillary coating

Two new methods of inner capillary coating with poly(vinyl alcohol) (PVAL) have been investigated and evaluated by performing DNA capillary electrophoresis (CE) using PVAL as a separation medium and by measuring the electroosmotic flow (EOF) mobility. The treatment of capillaries with a silanol-group modified PVAL (PVAL-Si) has been found to give good coating effects for improving the resolution of DNA CE and for reducing the EOF. This coating must be effectively achieved by combining the adsorptive property of PVAL chains onto silica with the reaction between the silanol groups of PVAL-Si and the silica surface. The adsorption of PVAL onto silica has been observed by using atomic force microscopy (AFM) for PVAL-Si as well as for a nonmodified PVAL as a control. The coating with PVAL that links to the capillary wall surface with more hydrolytically stable bonding, -Si-C-, has been formed by performing the Grignard reaction, followed by in-capillary polymerization of vinyl acetate (VAc) and hydrolysis. This coating has been found to be effective for improving the resolution of DNA CE and for reducing the EOF.     

Portable capillary electrophoresis system with potential gradient detection for separation of DNA fragments

A portable capillary electrophoresis (CE) system with a novel potential gradient detection (PGD) was utilized to separate DNA fragments. For the first time it was demonstrated that separation of DNA fragments in polymer solution could be detected by a portable CE system integrated with PGD, with a limit of detection (LOD) comparable to that of the CE-ultraviolet (UV) method. Effects of buffer solution, sieving medium, and applied voltage were also investigated. The portable CE-PGD system shows several potential advantages, such as simplicity, cost effectiveness, and miniaturization.     

Nonaqueous and aqueous capillary electrophoresis of synthetic polymers

In this work, the use of capillary electrophoresis (CE) to analyze synthetic polymers is reviewed including works published till February 2004. The revised works have been classified depending on the CE mode (e.g., free solution capillary electrophoresis, capillary gel electrophoresis, etc.) and type of buffer (i.e., nonaqueous, aqueous and hydro-organic background electrolytes) employed to separate synthetic macromolecules. Advantages and drawbacks of these different separation procedures for polymer analysis are discussed. Also, physicochemical studies of complex polymer systems by CE are reviewed, including drug release studies, synthetic polyampholytes, dendrimers, fullerenes, carbon nanotubes and associative copolymers.     

A technique for capillary joining in capillary electrophoresis

Summary Aspects of cracking and joining capillaries have been investigated. Capillary coupling was achieved using various methods. The most successful used hydrofluoric acid-etched capillaries to form male and female ends which were then joined together. This type of joint was used to connect sections of capillary of similar and different internal diameters with minimal loss in resolution, peak width and number of theoretical plates. (Uridine and hypoxanthine was used as a test mixture). For hypoxanthine on a 50 m/50 m etched joined capillary 10 cm from the detector window the number of theoretical plates was 96.6% of that for hypoxanthine on an unbroken capillary. Following the relative success of capillary joining, a coupled capillary flowcell (50 m/200 m) was produced and evaluated.     

Aptamer-based label-free method for hemin recognition and DNA assay by capillary electrophoresis with chemiluminescence detection

An aptamer-based label-free approach to hemin recognition and DNA assay using capillary electrophoresis with chemiluminescence detection is introduced here. Two guanine-rich DNA aptamers were used as the recognition element and target DNA, respectively. In the presence of potassium ions, the two aptamers folded into the G-quartet structures, binding hemin with high specificity and affinity. Based on the G-quartet–hemin interactions, the ligand molecule was specifically recognized with a K _d ≈ 73 nM, and the target DNA could be detected at 0.1 μM. In phosphate buffer of pH 11.0, hemin catalyzed the H₂O₂-mediated oxidation of luminol to generate strong chemiluminescence signal; thus the target molecule itself served as an indicator for the molecule–aptamer interaction, which made the labeling and/or modification of aptamers or target molecules unnecessary. This label-free method for molecular recognition and DNA detection is therefore simple, easy, and effective. Figure A label-free approach to aptamer-based hemin recognition and DNA detection is introduced, which gives great potential for using a small molecule itself as the indicator for molecular recognition and DNA detection thereby avoiding any labeling or modification step     

Quasi-interpenetrating network formed by polyacrylamide and poly(N,N-dimethylacrylamide) used in high-performance DNA sequencing analysis by capillary electrophoresis

Quasi-interpenetrating network (IPN) formed by polyacrylamide and poly(N,N-dimethylacrylamide) was designed, synthesized, and tested as a high-performance DNA separation medium by capillary electrophoresis. The performance of quasi-IPN on DNA sequencing was determined by the acrylamide to dimethylacrylamide molar ratio, polyacrylamide molecular weight, and its size distribution. Under optimal operating conditions, quasi-IPN was able to achieve one-color DNA sequencing up to 1000 bases in 39 min, or 1200 bases in 60 min. Its performance was compared with some of the existing commercialized products, such as POP6 from Applied Biosystems and MegaBACE matrix from Amersham Biosciences. By using the ABI 310 Genetic Analyzer, even without optimized base-calling software, quasi-IPN yielded a read length of up to 700 bases of contiguous sequence (50-750 bases) in 35 min with 99.6% accuracy, or 750 bases of contiguous sequence (50-800 bases) in 37 min with 98.0% accuracy.     

Inverse-flow derivatization for capillary electrophoresis of DNA fragments with laser-induced fluorescence detection

A novel method is presented to detect DNA fragments separated by capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection using inverse-flow derivatization. In electrophoresis, the intercalating dye, thiazol orange was only added to the separation buffer at the positive polarity. The negatively charged DNA fragments migrated from the negative polarity to the positive polarity, while the positively charged dye migrated in the opposite direction. When DNA fragments met with dye ions, the DNA–dye complexes were formed. The complexes continued migrating to the positive end, due to their net negative charges. When the complexes passed through the detection window, the fluorescent signals were generated. Importantly, DNA fragments migrated as their native state before DNA–dye complexes were formed. This procedure was used to detect double stranded DNA (dsDNA) and single stranded DNA (ssDNA) fragments, and polymerase chain reaction (PCR) products. The excellent resolution and good reproducibility of DNA fragments were achieved in non-gel sieving medium. This procedure may be useful in genetic mutation/polymorphism detections.     

Fluorescein thiocarbamyl amino acids as internal standards for migration time correction in capillary sieving electrophoresis

A number of algorithms have been developed to correct for migration time drift in capillary electrophoresis. Those algorithms require identification of common components in each run. However, not all components may be present or resolved in separations of complex samples, which can confound attempts for alignment. This paper reports the use of fluorescein thiocarbamyl derivatives of amino acids as internal standards for alignment of 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ)-labeled proteins in capillary sieving electrophoresis. The fluorescein thiocarbamyl derivative of aspartic acid migrates before FQ-labeled proteins and the fluorescein thiocarbamyl derivative of arginine migrates after the FQ-labeled proteins. These compounds were used as internal standards to correct for variations in migration time over a two-week period in the separation of a cellular homogenate. The experimental conditions were deliberately manipulated by varying electric field and sample preparation conditions. Three components of the homogenate were used to evaluate the alignment efficiency. Before alignment, the average relative standard deviation in migration time for these components was 13.3%. After alignment, the average relative standard deviation in migration time for these components was reduced to 0.5%.     

A new quantitative method for circulating DNA level in human serum by capillary zone electrophoresis with laser-induced fluorescence detection

We present a method for the quantification of circulating DNA in serum by capillary zone electrophoresis (CZE) with laser-induced fluorescence detection (LIF). The serum was digested by proteinase to release free DNA. SYBR Gold was utilized as DNA intercalating dye, fluorescein as internal standard (ISTD). CZE-LIF was applied for the separation and quantification of total circulating DNA. Good linearity (R = 0.9992) in the low range of DNA concentrations (0.5-40 ng/mL) and a detection limit of 0.5 ng/mL for DNA (S/N = 3) were obtained. Our data demonstrated that CZE-LIF system has a good linearity with excellent sensitivity and satisfactory reproducibility in the quantification of circulating DNA in serum. This method was successfully used for the quantification of circulating DNA levels in serum. We observed that the circulating DNA levels in certain cancer patients were significantly higher than that in healthy individuals. Compared to current methods, our protocol does not need the extraction of DNA from serum. Our preliminary results have illustrated that CZE-LIF system is simple, rapid, and sensitive, and it is well suitable for large-scale quantification of circulating DNA levels in clinical diagnosis.