On-line nanoliter cycle sequencing reaction with capillary zone electrophoresis purification for DNA sequencing

An integrated system for DNA sequencing based on a nanoreactor for cycle-sequencing reaction coupled with on-line capillary zone electrophoresis (CZE) for purification and capillary gel electrophoresis (CGE) for separation is presented. Less than 100 nl of premixed reagent solution, which includes dye-labeled terminator pre-mix, bovine serum albumin and template, was hydrodynamically injected into a fused-silica capillary (75 microm I.D.) inside a laboratory-made microthermocycler for cycle sequencing reaction. In the same capillary, the reaction products were purified by CZE followed by on-line injection of the DNA fragments into another capillary for CGE. Over 540 base pairs (bp) of DNA can be separated and the bases called for single-standed DNA with 0.9% error rate. The total time was about 3.5 h, or a cycle time of 2 h with staggered operation. For double-stranded DNA, a longer reaction time was required and base calling up to 490 bp with 1.2% error rate was achieved. The whole system is readily adaptable to automated multiplex operation for DNA sequencing or polymerase chain reaction analysis.     

Capillary-based fully integrated and automated system for nanoliter polymerase chain reaction analysis directly from cheek cells

A miniaturized, integrated and automated system based on capillary fluidics has been developed for nanoliter DNA analysis directly from cheek cells. All steps for DNA analysis, including injecting aqueous reagents and DNA samples, mixing the solutions together, thermal cell lysis, polymerase chain reaction (PCR), transfer and injection of PCR product, separation, sizing and detection of those products are performed in a capillary-based integrated system. A small amount of cheek cells collected by a plastic toothpick is directly dissolved in the PCR cocktail in a plastic vial or mixed on-line with a small volume of PCR cocktail (125 nl) in the capillary. After thermal cell lysis and PCR in a microthermal cycler, the DNA fragments are mixed with DNA size standards and transferred to a micro-cross for injection and separation by capillary gel electrophoresis. Programmable syringe pumps, switching valves, multiposition and freeze-thaw valves are used for microfluidic control in the entire system. This work establishes the feasibility of performing all the steps of DNA analysis from real samples in a capillary-based nanoliter integrated system.     

Optimizing separation conditions for riboflavin,flavin mononucleotide and flavin adenine dinucleotide in capillary zone electrophoresis with laser-induced fluorescence detection

DNA sequencing is performed in a multiplexed capillary electrophoresis system by UV absorption detection. Four individual electropherograms are obtained by simultaneously running the unlabeled DNA products of the four ddNTP-terminated reactions in the capillary array. The sequence of the template used in the cycle-sequencing reaction can be determined by overlaying the four electropherograms. Two internal standards are employed to adjust for the variance in migration times among the capillaries. After applying the correction algorithm, base calling can be done at a high level of confidence.     

Characterization of acridone dyes for use in four-decay detection in DNA sequencing

Four acridone dyes and dye-labeled primers were characterized for use in four-decay DNA sequencing. In the four-decay scheme, fluorescence lifetime replaces spectral ("color") selectivity for distinguishing between four base-specific labels in a single-lane capillary electrophoresis (CE) separation of the DNA fragments. Prior to the introduction of the acridone dyes, a major obstacle to four-decay detection was the lack of four suitable dyes with resolvable lifetimes. The four acridone dyes, whether free in solution or tethered to DNA primer, exhibit significant differences among their lifetimes and are well-suited to use together in four-decay sequencing. The lifetimes of the four dye-labeled DNA primers that were sequentially injected and detected on-the-fly in a 2% POP6 sequencing gel were 4, 6, 11 and 14 ns. A 405 nm violet laser diode provides optimal excitation of the four dyes.     

Recent developments in DNA sequencing by capillary and microdevice electrophoresis

The present review covers papers published in the years 1997 and 1998 on DNA sequencing by capillary and microdevice electrophoresis. The article does not include other electrophoretic DNA applications such as analysis of oligonucleotides, genotyping, and mutational analysis. Capillary gel electrophoresis (CGE) is starting to become a viable competitor to slab gel electrophoresis for DNA sequencing. Commercially available multicapillary array sequencers are now entering sequencing facilities which to date have totally relied on traditional slab gel technology. CGE research on DNA sequencing therefore becomes increasingly concerned with the critical task of fine-tuning the operational parameters to create robust sequencing systems. Electrophoretic microdevices are being considered the next technological step in DNA sequencing by electrophoresis.     

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.     

Control of the cultivation process of antithrombin III and its characterization by capillary electrophoresis

The production by baby hamster kidney cells of recombinant antithrombin III (r-AT III), the main inhibitor of thrombin, factor Xa and other proteases of the clotting cascade, was monitored by capillary isotachophoresis using mixtures of continuous spacers. The results were compared with those obtained by capillary zone electrophoresis (CZE). The downstream process, which incorporated anion-exchange and heparin affinity chromatography, was monitored by CZE under acidic conditions and voltage ramping. The purified product was characterized by its isoelectric point and molecular mass. Isoelectric points of the three major and three minor isoforms of AT III were evaluated by capillary isoelectric focusing using a pH range of 4–6 and various mobilization procedures. The molecular mass of AT III was investigated by capillary gel electrophoresis (CGE), applying removable dextran gels. Both parameters could be determined within 30 min using only one coated capillary. The results showed an excellent correspondence with those achieved with conventional slab gels. The affinity complex between AT III and thrombin could also be detected by CGE and the heparin dependence of the affinity reaction could be investigated.     

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.     

毛细管电泳的原理和应用(第六讲)CE在DNA、糖和离子分析中的应用

简要介绍了ＣＥ在ＤＮＡ、糖和离子分析中的应用。ＤＮＡ分析包括分离原理、方法及检测手段，碱基、核苷和核苷酸分析，纯度检测和微量制备，ｓｓＤＮＡ和ｄｓＤＮＡ分析，基因突变检测及ＤＮＡ序列分析等。糖分析包括衍生化技术、检测方法、所用模式及糖复合物的分析等。离子分析包括阴离子分析原理、检测方法、影响因素、应用及阳离子分析等。     

毛细管电泳的原理和应用(第六讲)CE在DNA、糖和离子分析中的应用

 简要介绍了ＣＥ在ＤＮＡ、糖和离子分析中的应用。ＤＮＡ分析包括分离原理、方法及检测手段，碱基、核苷和核苷酸分析，纯度检测和微量制备，ｓｓＤＮＡ和ｄｓＤＮＡ分析，基因突变检测及ＤＮＡ序列分析等。糖分析包括衍生化技术、检测方法、所用模式及糖复合物的分析等。离子分析包括阴离子分析原理、检测方法、影响因素、应用及阳离子分析等。     

Protein mapping of peanut extract with capillary electrophoresis

Protein separation can be achieved with different modes of capillary electrophoresis, such as with capillary gel electroporesis (CGE) or with capillary zone electrophoresis (CZE). CZE protein mapping of peanut extract was approached in four different ways, combining neutral-coated or multilayer-coated capillaries with pHs well over or under the isoelectric point range of the proteins of interest. At acidic pHs, the mobility ranges of the major peanut allergens Ara h1, Ara h2, Ara h3, and Ara h6 were identified. Although the pH is a major factor in CZE separation, buffers with different compositions but with the same pH and ionic strength showed significantly different resolutions. Different components of the electrolyte were studied in a multifactorial design of experiment. CE-SDS and CZE proved to be suitable for protein mapping and we were able to distinguish different batches of peanut extract and burned peanut extract.     

Nanoliter-scale sample preparation methods directly coupled to polymethylmethacrylate-based microchips and gel-filled capillaries for the analysis of oligonucleotides.

We are currently developing miniaturized, chip-based electrophoresis devices fabricated in plastics for the high-speed separation of oligonucleotides. One of the principal advantages associated with these devices is their small sample requirements, typically in the nanoliter to sub-nanoliter range. Unfortunately, most standard sample preparation protocols, especially for oligonucleotides, are done off-chip on a microliter-scale. Our work has focused on the development of capillary nanoreactors coupled to micro-separation platforms, such as micro-electrophoresis chips, for the preparation of sequencing ladders and also polymerase chain reactions (PCRs). These nanoreactors consist of fused-silica capillary tubes (10-20 cm x 20-50 microns I.D.) with fluid pumping accomplished using the electroosmotic flow generated by the tubes. These reactors were situated in fast thermal cyclers to perform cycle sequencing or PCR amplification of the DNAs. The reactors could be interfaced to either a micro-electrophoresis chips via capillary connectors micromachined in polymethylmethacrylate (PMMA) using deep X-ray etching (width 50 microns; depth 50 microns) or conventional capillary gel tubes using zero-dead volume glass unions. For our chips, they also contained an injector, separation channel (length 6 cm; width 30 microns; depth 50 microns) and a dual fiber optic, near-infrared fluorescence detector. The sequencing nanoreactor used surface immobilized templates attached to the wall via a biotin-streptavidin-biotin linkage. Sequencing tracks could be directly injected into gel-filled capillary tubes with minimal degradation in the efficiency of the separation process. The nanoreactor could also be configured to perform PCR reactions by filling the capillary tube with the PCR reagents and template. After thermal cycling, the PCR cocktail could be pooled from multiple reactors and loaded onto a slab gel or injected into a capillary tube or microchip device for fractionation.     

亲和毛细管电泳技术及其应用

对近几年新发展起来的亲和毛细管电泳技术（ACE）的原理、分类及方法作了简要介绍,着重介绍了亲和毛细管区带电泳、毛细管亲和凝胶电泳、胶束电动色谱中的亲和电泳、亲和毛细管等电聚焦、亲和探针毛细管电泳等过程和方法。对ACE在分子生物学、生物化学中的应用及该技术在亲和常数测定、核酸片段识别、竞争免疫分析、药物先导化合物的筛选等方面的应用也作了介绍。     

Application of capillary electrophoresis to monitor populations of Cellulomonas cartae KYM-7 and Agrobacterium tumefaciens KYM-8 in mixed culture.

A bacterial cell mixture ot Cellulomonas cartae KYM-7 and Agrobacterium tumefaciens KYM-8 was analyzed by capillary zone electrophoresis (CZE) and capillary gel electrophoresis (CGE). Both pherograms showed two discrete peaks. The cells in the peaks were collected, Gram stained, and examined with a microscope. The cells of the two strains were well separated by OGE, and each OGE peak consisted almost entirely of cells of one strain (greater than 98% purity), whereas each CZE peak contained cells of both strains (about 90% purity). In the concentration range of 1.0 x 10(10) to 1.0 x 10(12) cells/mL, the area of CGE peaks was proportional to the amount of cells. The growth of the two strains in mixed culture was measured by OGE. The OGE quantification data were in good agreement with those obtained using fluorescence in situ hybridization. The CGE analyses were accomplished in 1 h, using a relatively uncomplicated procedure. Thus, OGE exhibited great advantages in accuracy, rapidity, and simplicity.     

Recent progress in chiral separation principles in capillary electrophoresis

This review summarizes recent developments in the field of chiral separations by electromigration techniques including capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), isotachophoresis (ITP), electrokinetic chromatography (EKC), and capillary electrochromatography (CEC). This overview focuses on the development of new chiral selectors and the introduction of new techniques rather than applications of already established selectors and methods. The mechanisms of the different chiral separation principles are discussed.     

A simple capillary gel electrophoresis approach for efficient and reproducible DNA separations. Analysis of genetically modified soy and maize

It is generally assumed that in order to achieve suitable separations of DNA fragments, capillary gel electrophoresis (CGE)-coated capillaries should be used. In this work, a new method is presented that allows to obtain reproducible CGE separations of DNA fragments using bare fused-silica capillaries without any previous coating step. The proposed method only requires: (i) a capillary washing with 0.1 M hydrochloric acid between injections and (ii) a running buffer composed of Tris-phosphate-ethylenediamine tetraacetic acid (EDTA) and 4.5% of 2-hydroxyethyl cellulose (HEC) as sieving polymer. The use of this new CGE procedure gives highly resolved and reproducible separations of DNA fragments ranging from 50 to 750 bp. The separation of these DNA fragments is accomplished in less than 30 min with efficiencies up to 1.7 x 10(6) plates/m. Reproducibility values of migration times (given as %RSD) for the analyzed DNA fragments are better than 1.0% (n = 4) for the same day, 2.2% (n = 16) for four different days, and 2.3% (n = 16) for four different capillaries. The usefulness of this separation method is demonstrated by detecting genetically modified maize and genetically modified soy after DNA amplification by PCR. This new CGE procedure together with LIF as detector provides sensitive analysis of 0.9% of Bt11 maize, Mon810 maize, and Roundup Ready soy in flours with S/ N up to 542. These results demonstrate the usefulness of this procedure to fulfill the European regulation on detection of genetically modified organisms in foods.     

DNA fragment analysis by an affordable multiple-channel capillary electrophoresis system

We are demonstrating a cost-effective multichannel capillary electrophoresis system for a high-efficiency double-stranded DNA (dsDNA) fragments analysis. This bench-type high-performance DNA analysis (HDA) system uses fluorescence-type detection with inexpensive solid-state light sources and nonmoving integrated emission collection micro-optics. DNA samples are analyzed simultaneously by using a multiple usage and disposable multicapillary cartridge, which contains integrated capillary channels, optical fibers and an integrated sieving gel reservoir. Using commercially available dsDNA size markers as indicators, the HDA system provides high resolving power in 7 min separations. The system can hold a total of 192 samples in two 96-well polymerase chain reaction (PCR) plates, which can be automatically analyzed within 2.5 h. This affordable system can be used in laboratories to replace slab gel electrophoresis for routine and high-throughput dsDNA analysis.     

DNA fragment separations by on‐line combination of capillary isotachophoresis–capillary zone electrophoresis with UV detection

A high‐speed DNA fragment separation system based on an on‐line combination of capillary ITP with CZE (CITP‐CZE) and using UV detection at 260 nm was developed. A novel CITP‐CZE buffer system of pH 6.1 was designed for the separation of ten DNA fragments with sizes ranging from 100 to 1000 bp. An effect of underivatized α‐, β‐ and γ‐cyclodextrins on the resolution of DNA fragments in the CZE step of the CITP‐CZE combination was systematically investigated. Methylhydroxyethylcellulose present in the BGE was used to eliminate the EOF. DNA ladder fragments were separated within 10 min with LODs in the range of 1–5 ng/μL (S/N = 3). The RSDs of the migration time and peak area of individual DNA fragments were in the range of 1–3 and 3–9%, respectively. The developed CITP‐CZE system was further applied to the analysis of digest plasmid DNA samples.     

High-throughput DNA analysis by microchip electrophoresis

DNA analysis plays a great role in genetic and medical research, and clinical diagnosis of inherited diseases and particular cancers. Development of new methods for high throughput DNA analysis is necessitated with incoming of post human genome era. A new powerful analytical technology, called microchip capillary electrophoresis (MCE), can be integrated with some experimental units and is characterized by high-speed, small sample and reagent requirements and high-throughput. This new technology, which has been applied successfully to the separation of DNA fragments, analysis of polymerase chain reaction (PCR) products, DNA sequencing, and mutation detection, for example, will become an attractive alternative to conventional methods such as slab gel electrophoresis, Southern blotting and Northern blotting for DNA analysis. This review is focused on some basic issues about DNA analysis by MCE, such as fabrication methods for microchips, detection system and separation schemes, and several key applications are summarized.