Fast and easy coating for capillary electrophoresis based on a physically adsorbed cationic copolymer

In this work, a new copolymer synthesized in our laboratory is used as physically adsorbed coating for capillary electrophoresis (CE). The copolymer is composed of ethylpyrrolidine methacrylate (EPyM) and methylmethacrylate (MMA). The capillary coating is easily obtained by simply flushing into the tubing an EPyM/MMA solution. It is demonstrated that the composition of the EPyM/MMA copolymer together with the selection of the background electrolyte (BGE) and pH allow tailoring the direction and magnitude of the electroosmotic flow (EOF) in CE. It is also shown that the EOF obtained for the EPyM/MMA-coated capillaries was reproducible in all cases independently on pH or polymer composition. Thus, RSD values lower than 1.9% (n=5) for the same capillary and day were obtained for the migration time, while the repeatability interdays (n=5) was observed to provide RSD values lower than 0.5%. The stability of the coating procedure was also tested between capillaries (n=3) obtaining RSD values lower than 0.6%. It is demonstrated with several examples that the use of EPyM/MMA coatings in CE can drastically reduce the analysis time and/or to improve the resolution of the separations. It is shown that EPyM/MMA-coated capillaries allow the separation of basic proteins by reducing their adsorption onto the capillary wall. Also, EPyM/MMA-coated capillaries provide a faster separation of samples containing simultaneously positive and negative analytes. Moreover, it is demonstrated that the use of EPyM/MMA-coated capillaries can incorporate an additional chromatographic-like interaction with nucleosides that highly improves the separation of this group of solutes.     

New physically adsorbed polymer coating for reproducible separations of basic and acidic proteins by capillary electrophoresis

In this work, a new physically adsorbed coating for capillary electrophoresis (CE) is presented. The coating is based on a N,N-dimethylacrylamide-ethylpyrrolidine methacrylate (DMA-EPyM) copolymer synthesized in our laboratory. The capillary coating is simple and easy to obtain as only requires flushing the capillary with a polymer aqueous solution for 2 min. It is shown that by using these coated capillaries the electrostatic adsorption of a group of basic proteins onto the capillary wall is significantly reduced allowing their analysis by CE. Moreover, the DMA-EPyM coating provides reproducible separations of the basic proteins with RSD values for migration times lower than 0.75% for the same day (n = 5) and lower than 3.90% for three different days (n = 15). Interestingly, the electrical charge of the coated capillary wall can be modulated by varying the pH of the running buffer which makes possible the analysis of basic and acidic proteins in the same capillary. The usefulness of this coating is further demonstrated via the reproducible separation of whey (i.e. acidic) proteins from raw milk. The coating protocol should be compatible with both CE in microchips and CE-MS of different types of proteins.     

Comparison of three modifications of fused‐silica capillaries and untreated capillaries for protein profiling of maize extracts by capillary electrophoresis

In this work, capillary electrophoresis was applied to protein profiling of fractionated extracts of maize. A comparative study on the application of uncoated fused‐silica capillaries and capillaries modified with hydroxypropylmethylcellulose, ω‐iodoalkylammonium salt and a commercially available neutral capillary covalently coated with polyacrylamide is presented. The coating stability, background electrolyte composition, and separation efficiency were investigated. It was found that for zeins separation, the most stable and efficient was the capillary coated with polyacrylamide. Finally, the usefulness of these methods was studied for the differentiation of zein fraction in transgenic and nontransgenic maize. Zeins extracted from maize standards containing 0 and 5% m/m genetic modification were successfully separated, but slight differences were observed in terms of the zein content. Albumin and globulin fractions were analyzed with the use of unmodified fused‐silica capillary with borate buffer pH 9 and the capillary coated with polyacrylamide with phosphate buffer pH 3. In the albumin fraction, additional peaks were found in genetically modified samples.     

Determination of Genetically Modified Soybean by Multiplex PCR and CGE with LIF Detection

A method for rapid screening, identification and detection of genetically modified soybean by multiplex polymerase chain reaction (PCR) and capillary gel electrophoresis with laser-induced fluorescence (CGE–LIF) was developed and applied to actual food samples. A triplex PCR procedure was used to amplify the parts of nopaline synthase (NOS) terminator, and the junction between cauliflower mosaic virus 35S promoter and chloroplast transit peptide CTP4 trait gene, as well as the lectin gene to allow the screening and identification of specific transgenic soybean line (glyphosate-tolerant soybean). The multiplex PCR parameters and conditions of capillary gel electrophoresis were optimized. The amplified DNA fragments were analyzed by CGE–LIF. The amplified PCR products were analyzed by CGE–LIF within about 20 min. The method developed is highly sensitive and allows the detection of a percentage of genetically modified soybean as low as 0.025%. The percentage is low enough to fulfill the requirement of the EU Regulation for transgenic food labeling of 1.0%. The sequences of the multiple PCR products were identical with those published in Genbank. The proposed method has been used in identification and detection of genetically modified soybean in various food samples. Compared with agarose gel electrophoresis (AGE), the proposed method is more rapid, accurate and requires a smaller amount of samples. Thus an efficient alternative method is provided for monitoring genetically modified soybean in order to meet the increasing demand of implementation of the genetically modified food labeling policy.     

转基因玉米的多重PCR-毛细管电泳-激光诱导荧光检测方法研究

采用三重PCR反应, 同时扩增CaMV 35S启动子、 hsp70 intron1和CryIA(b)基因之间序列以及Invertase基因, 扩增产物用无胶筛分毛细管电泳-激光诱导荧光检测, 从而建立了多重PCR-毛细管电泳-激光诱导荧光快速检测转基因玉米的新方法. 对影响多重PCR扩增和毛细管电泳的因素进行了优化. 在优化的条件下, 本方法可以同时检测转基因玉米样品中3种外源基因. 经序列测试证实, 三重PCR 扩增产物的序列与原基因完全一致, 表明扩增结果可靠. 该方法能检出0.05% MON810转基因玉米成分, 远低于欧盟对转基因食品规定标识的质量分数阈值(1％). 该方法对玉米及其制品的检测结果与实时荧光PCR方法的检测结果一致, 与传统的琼脂糖凝胶电泳法相比, 具有特异性高\, 快速及灵敏等优点, 适用于玉米中转基因成分以及转基因玉米MON810品系的快速筛选、 鉴定和检测, 能满足我国实施转基因食品标签法规的要求.     

Gold nanoparticle-coated capillaries for protein and peptide analysis on open-tubular capillary electrochromatography

We report a new method of immobilization of gold nanoparticles (AuNPs) on a fused-silica capillary through covalent binding. The resulting modified capillary was applied to electrophoretic systems to improve the efficiency of separation and the selectivity of selected solutes. The immobilization of AuNPs on the capillary wall was performed in a very simple and fast way without requiring heating. The surface features of an AuNP-coated capillary column were determined using the scanning electron microscopy. The chromatographic properties of AuNP-coated capillaries were investigated through variation of the buffer pH and separation voltage. Effective separations of synthetic peptides mixture were obtained on the AuNP-coated capillaries. The method shows a remarkable stability since it was reused about 900 times. The capacity factor was duplicated. Therefore, this modification is stable and can be applied to different separation purposes. A complex mixture of tryptic peptide fragments of HSA was analyzed in both the bare- and the AuNP-coated capillaries. Better electrophoretic peptide profile was observed when using the AuNP-coated capillary.     

Analysis of DNA restriction fragments and polymerase chain reaction products by capillary electrophoresis

Capillary electrophoresis (CE) is a new, high-resolution tool for the analysis of DNA restriction fragments and DNA amplified by the polymerase chain reaction (PCR). By combining many of the principles of traditional slab gel methods in a capillary format, it is possible to perform molecular size determinations of human and plant PCR amplification products and DNA restriction fragments. DNA restriction fragments and PCR products were analyzed by dynamic sieving electrophoresis (DSE) and capillary gel electrophoresis (CGE). As part of this study, sample preparation procedures, injection modes, and the use of molecular mass markers were evaluated. Optimum separations were performed using the uPage-3 (3% T, 3% C) CGE columns with UV detection at 260 nm. Membrane dialysis and ultrafiltration/centrifugation proved to be nearly equivalent methods of sample preparation. Reproducibility studies demonstrated that blunt-ended, non-phosphorylated markers (specifically allele generated markers) provide the most accurate calibration for PCR product analysis. This study demonstrates that CE offers a high-speed, high-resolution analytical method for accurately determining molecular size and/or allelic type as compared with traditional methodologies.     

Star-shaped polymers for DNA sequencing by capillary electrophoresis

The separation and sequencing of DNA are the main objectives of the Human Genome Project, and this project has also been very useful for gene analysis and disease diagnosis. Capillary electrophoresis (CE) is one of the most common techniques for the separation and analysis of DNA. DNA separations are usually achieved using capillary gel electrophoresis (CGE) mode, in which polymer gel is packed into the capillary. Compared with a traditional CGE matrix, a hydrophilic polymer matrix, which can be adsorb by the capillary wall has numerous advantages, including stability, reproducibility and ease of automation. Various water-soluble additives, such as linear poly(acrylamide) (PAA) and poly(N,N-dimethylacrylamide) (PDMA), have been employed as media. In this study, different star-shaped PDMA polymers were designed and synthesized to achieve lower polymer solution viscosity. DNA separations with these polymers avoid the disadvantages of high viscosity and long separation time while maintaining high resolution (10 bp between 271 bp and 281 bp). The influences of the polymer concentration and structure on DNA separation were also determined in this study; higher polymer concentration yielded better separation performance, and star-like polymers were superior to linear polymers. This work indicates that modification of the polymer structure is a potential strategy for optimizing DNA separation.     

Integrated electroosmotically-driven on-line sample purification system for nanoliter DNA sequencing by capillary electrophoresis

An integrated on-line system is developed for DNA sequencing at the nanoliter scale. The technique involves the use of a nanoreactor for small-volume cycle-sequencing reaction, capillary zone electrophoresis (CZE) for purification of the sequencing fragments, and capillary gel electrophoresis (CGE) for separation of the purified DNA fragments. The nanoreactor and CZE are integrated into one capillary, where a 100-nl dye-labeled terminator cycle-sequencing reaction is carried out followed by CZE to separate excess dye-labeled terminators from the sequencing fragments. On-line electrokinetic injection of the purified DNA fragments into the CGE system is accomplished at a small-volume tee connector by which the CZE capillary is interfaced to the CGE system. The utility of the system is demonstrated in sequencing nanoliter volumes of single-stranded DNA (M13mp18) and double-stranded DNA (pGEM). The use of voltage to drive both CZE and CGE makes it feasible for automation and future adaptation of the whole system to a microchip.     

Determination of PCR products by CE with contactless conductivity detection

The use of CE with contactless conductivity detection for the determination of PCR products is demonstrated for the first time. The separation of specific length PCR products according to their size could be achieved using 5% PVP as a sieving medium in a separation buffer consisting of 20 mM Tris and 20 mM 2‐(cyclohexylamino)ethansulphonic acid (pH 8.5). A fused silica capillary of 60 cm length and 50 μm id and an applied separation voltage of –15 kV were employed and separations could be completed within 20–50 min. PCR amplified DNA fragments of different sizes obtained from different bacterial plasmid templates as well as a fragment from genomic DNA of genetically modified soybeans could be successfully identified.     

Fast and sensitive detection of genetically modified yeasts in wine

In this work, a novel screening methodology based on the combined use of multiplex polymerase chain reaction (PCR) and capillary gel electrophoresis with laser induced fluorescence (CGE-LIF) is developed for the fast and sensitive detection of genetically modified yeasts in wine. As model, a recombinant EKD-13 Saccaromyces cerevisiae strain was selected and different wines were prepared using either recombinant or conventional yeasts. Special emphasis is put on the yeast DNA extraction step, exploring different commercial and non-commercial methods, in order to overcome the important difficulty of obtaining amplifiable DNA from wine samples. To unequivocally detect the transgenic yeast, two specific segments of the transgenic construction were amplified. In addition, a third primer pair was used as amplification control to confirm the quality of the yeast DNA obtained from the extraction step. CGE-LIF provides high sensitivity, good analysis speed and impressive resolution of DNA fragments, making this technique very convenient to optimize multiplex PCR parameters and to analyze the amplified DNA fragments. Thus, the CGE-LIF method provided %RSD values for DNA migration times lower than 0.82% (n=10) with the same capillary and lower than 1.92% (n=15) with three different capillaries, allowing the adequate size determination of the PCR products with an error lower than 4% compared to the theoretically expected. The whole method developed in this work requires less than one working day and grants the sensitive detection of transgenic yeasts in wine samples.     

IUPAC collaborative trial study of a method to detect genetically modified soy beans and maize in dried powder.

This paper presents results of a collaborative trial study (IUPAC project No. 650/93/97) involving 29 laboratories in 13 countries applying a method for detecting genetically modified organisms (GMOs) in food. The method is based on using the polymerase chain reaction to determine the 35S promotor and the NOS terminator for detection of GMOs. reference materials were produced that were derived from genetically modified soy beans and maize. Correct identification of samples containing 2% GMOs is achievable for both soy beans and maize. For samples containing 0.5% genetically modified soy beans, analysis of the 35S promotor resulted also in a 100% correct classification. However, 3 false-negative results (out of 105 samples analyzed) were reported for analysis of the NOS terminator, which is due to the lower sensitivity of this method. Because of the bigger genomic DNA of maize, the probability of encountering false-negative results for samples containing 0.5% GMOs is greater for maize than for soy beans. For blank samples (0% GMO), only 2 false-positive results for soy beans and one for maize were reported. These results appeared as very weak signals and were most probably due to contamination of laboratory equipment.     

DNA separation by capillary electrophoresis with hydrophilic substituted celluloses as coating and sieving polymers. Application to the analysis of genetically modified meals

A coating procedure based on the physical adsorption of hydroxypropyl cellulose onto the wall of a capillary column has been successfully used for the separation of DNA fragments up to 500 bp. The method uses a running Tris-phosphate-EDTA buffer containing 2-hydroxyethyl cellulose as sieving polymer. The separation procedure shows good reproducibility (measured as RSD%) for consecutive runs (<0.64), for different days (< 1.15) and capillaries (<2.15), short analysis times, and a long coating lifetime. Good reproducibility and efficiency are even achieved by performing the separation in the presence of additives such as ethidium bromide and mannitol. The method is applied to the detection of GMOs in soybean and maize meals with an accurate evaluation of the length of DNA sequences, previously amplified by polymerase chain reaction.     

Detection and identification of multiple genetically modified events using DNA insert fingerprinting

Current screening and event-specific polymerase chain reaction (PCR) assays for the detection and identification of genetically modified organisms (GMOs) in samples of unknown composition or for the detection of non-regulated GMOs have limitations, and alternative approaches are required. A transgenic DNA fingerprinting methodology using restriction enzyme digestion, adaptor ligation, and nested PCR was developed where individual GMOs are distinguished by the characteristic fingerprint pattern of the fragments generated. The inter-laboratory reproducibility of the amplified fragment sizes using different capillary electrophoresis platforms was compared, and reproducible patterns were obtained with an average difference in fragment size of 2.4 bp. DNA insert fingerprints for 12 different maize events, including two maize hybrids and one soy event, were generated that reflected the composition of the transgenic DNA constructs. Once produced, the fingerprint profiles were added to a database which can be readily exchanged and shared between laboratories. This approach should facilitate the process of GMO identification and characterization.     

Improved separation of double-stranded DNA fragments by capillary electrophoresis using poly(ethylene oxide) solution containing colloids

The analysis of double-stranded (ds) DNA fragments by capillary electrophoresis (CE) using poly(ethylene oxide) (PEO) solution containing gold nanoparticles (GNPs) is presented, focusing on evaluating size dependence of the GNPs and PEO on resolution and speed. To prevent the interaction of the capillary wall with DNA, the capillary was dynamically coated with polyvinylpyrrolidone. Using different PEO solutions containing GNPs ranging in diameter from 3.5 to 56 nm, we have achieved reproducible, rapid, and high-resolution DNA separations. The results indicate that the sizes of PEO and GNPs as well as the concentration of PEO affect resolution. The separation of DNA ranging in size from 8 to 2176 base pairs (bp) was accomplished in 5 min using 0.2% PEO (8 MDa) containing 56 nm GNPs. We have also demonstrated the separations of the DNA fragments ranging from 5 to 40 kbp using 0.05% PEO (2 MDa) containing 13 nm GNPs or 0.05% PEO (4 MDa) containing 32 nm GNPs. With very low viscosity (< 15 cP), automatic replacement of the sieving matrices is easy, indicating a great potential for high-throughput DNA analysis using capillary array electrophoresis systems.     

Effect of ionic strength, pH and polymer concentration on the separation of DNA fragments in the presence of electroosmotic flow

DNA separations in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO) solutions have been demonstrated. During the separations, PEO entered capillaries filled with Tris-borate (TB) free buffers by EOF and acted as sieving matrices. We have found that ionic strength and pH of polymer and free solutions affect the bulk EOF and resolution differently from that in capillary zone electrophoresis. The EOF coefficient increases with increasing ionic strength of the free TB buffers as a result of decreases in the adsorption of PEO molecules. In contrast, the bulk EOF decreases with increasing the ionic strength of polymer solutions using capillaries filled with high concentrations of free TB buffers. Although resolution values are high due to larger differential migration times between any two DNA fragments in a small bulk EOF using 10 mM TB buffers, use of a capillary filled with at least 100 mM TB free buffers is suggested for high-speed separations. On the side of PEO solutions, 1.5% PEO solutions prepared in 100 to 200 mM TB buffers are more proper in terms of resolution and speed. The separation of DNA markers V and VI was accomplished less than 29 min in 1.5% PEO solutions prepared in 100 mM TB buffers, pH 7.0 at 500 V/cm using a capillary filled with 10 mM free TB buffers, pH 7.0.     

Capillary electrophoresis using copolymers of different composition as physical coatings: a comparative study

In this work, a comparative study on the use of different polymers as physically adsorbed coatings for CE is presented. It is demonstrated that the use of ad hoc synthesized polymers as coatings allows tailoring the EOF in CE increasing the flexibility of this analytical technique. Namely, different polymers were synthesized at our laboratory using different percentages of ethylpyrrolidine methacrylate (EpyM) and N,N-dimethylacrylamide (DMA). Thus, by modifying the percentage of EpyM and DMA monomers it is possible to manipulate the positive charge of the copolymer, varying the global electrical charge on the capillary wall and with that the EOF. These coated capillaries are obtained by simply flushing a given EpyM-DMA aqueous solution into bare silica capillaries. It is shown that by using these coated capillaries at adequate pHs, faster or more resolved CE separations can be achieved depending on the requirements of each analysis. Moreover, it is demonstrated that these coated capillaries reduce the electrostatic adsorption of basic proteins onto the capillary wall. Furthermore, EpyM-DMA coatings allow the reproducible chiral separation of enantiomers through the partial filling technique (PFT). The EpyM-DMA coated capillaries are demonstrated to provide reproducible EOF values independently of the pH and polymer composition with%RSD values lower than 2% for the same day. It is also demonstrated that the coating procedure is reproducible between capillaries. The compatibility of this coating protocol with CE in microchips is discussed.     

Separating DNA sequencing fragments without a sieving matrix.

The possibility of separating appropriately labeled DNA fragments using free-flow capillary electrophoresis was predicted a few years ago based on simple theoretical arguments. Free-flow separation of double-stranded DNA (dsDNA) fragments in the 100-1000 base range was later demonstrated using a streptavidin label. In this article, we now report that end-labeled free-flow electrophoresis (ELFSE) can also be used to sequence single-stranded DNA (ssDNA). The first 100 bases of a DNA sequencing reaction were read without any sieving matrix when fractionated streptavidin was added to the 5'-end of the ssDNA fragments. These separations required only 18 min and did not require coated capillaries. An analysis of the results indicates that sample injection, analyte-wall interactions and thermal diffusion are the limiting factors at this time. Extrapolating from our data, we predict that several hundred bases could be sequenced in less than 30 min with the proper conditions. ELFSE thus offers an attractive potential alternative to polymer solutions for DNA sequencing in capillaries and microchips.     

Capillary electrophoresis of DNA damage after irradiation: apoptosis and necrosis

Apoptosis is a type of cellular death but also directly regulates tumorigenesis through different gene expression. This phenomenon is often used as end-point in studies of radio- and chemosensitivity of cancer cells. Restriction DNA fragments have been separated quickly, efficiently and successfully by capillary gel electrophoresis (CGE). In this study CGE has been applied to distinguish between the discrete pattern of degraded DNA produced by apoptosis and randomized DNA breaks produced by ionizing radiation. The influence of different variables has been discussed and an example of fast separation by CGE of the apoptotic fragments produced by UV light treatment is shown.     

Separation of DNA restriction fragments by high performance capillary electrophoresis with low and zero crosslinked polyacrylamide using continuous and pulsed electric fields

This paper presents results on the separation of DNA restriction fragments by high performance capillary electrophoresis (HPCE). Capillaries containing polyacrylamide with low amounts of crosslinking agent (i.e. 0.5% C) were first studied. The greater molecular accessibility offered with columns of low crosslinking, relative to higher crosslinked gels (e.g. 5% C), permitted high efficiency separations of double stranded DNA fragments up to 12,000 base pairs in length. Capillaries containing no crosslinking agent, i.e. linear polyacrylamide, were then examined. Ferguson plots (i.e. log mobility vs. %T) were used to assess the size selectivity of linear polyacrylamide capillaries. In another study, it was determined that the relative migration of DNA species was a strong function of applied electric field and molecular size. Lower fields yielded better resolution than higher fields for DNA molecules larger than about 1000 base pairs, albeit at the expense of longer separation time. Based on these results, we have examined pulsed field HPCE and have demonstrated the use of this approach to enhance separation.