无胶筛分毛细管电泳分析几百个碱基对核酸的条件优化

通过正交设计实验综合分析了内充羟丙基甲基纤维素（ＨＰＭＣ）无胶筛分毛细管电泳中的分离场强、ＨＰＭＣ浓度、柱长度和柱内径对核酸分离的影响。结果表明,柱长度越长、柱内径越小、分离场强越小,分离效果越好。考虑实际情况,为能在短时间内使几百个碱基对的核酸得到有效分离,一般选择３７ｃｍ×７５μｍｉ．ｄ．的涂壁毛细管、柱内质量浓度为８ｇ／Ｌ的ＨＰＭＣ、场强为３２４Ｖ／ｃｍ的条件,并在此种条件下分析了ＡｐｏＢ１００基因的低浓度聚合酶链式反应（ＰＣＲ）扩增产物（７１０ｂｐ）。     

Rapid microwell polymerase chain reaction with subsequent ultrathin-layer gel electrophoresis of DNA

Large-scale genotyping, mapping and expression profiling require affordable, fully automated high-throughput devices enabling rapid, high-performance analysis using minute quantities of reagents. In this paper, we describe a new combination of microwell polymerase chain reaction (PCR) based DNA amplification technique with automated ultrathin-layer gel electrophoresis analysis of the resulting products. This technique decreases the reagent consumption (total reaction volume 0.75-1 microL), the time requirement of the PCR (15-20 min) and subsequent ultrathin-layer gel electrophoresis based fragment analysis (5 min) by automating the current manual procedure and reducing the human intervention using sample loading robots and computerized real time data analysis. Small aliquots (0.2 microL) of the submicroliter size PCR reaction were transferred onto loading membranes and analyzed by ultrathin-layer gel electrophoresis which is a novel, high-performance and automated microseparation technique. This system employs integrated scanning laser-induced fluorescence-avalanche photodiode detection and combines the advantages of conventional slab and capillary gel electrophoresis. Visualization of the DNA fragments was accomplished by "in migratio" complexation with ethidium bromide during the electrophoresis process also enabling real time imaging and data analysis.     

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.     

Determination of thiopental in human serum and plasma by high-performance capillary electrophoresis-micellar electrokinetic chromatography.

The quantitation of thiopental in human serum and plasma was investigated using high-performance capillary electrophoresis (HPCE) in a micellar configuration and the results were compared with reversed-phase high-performance liquid chromatography (HPLC). Thiopental and an internal standard (carbamazepine for HPCE and thiamylal for HPLC) were extracted from serum or plasma using pentane and a phosphate buffer (pH 6.4). HPCE analysis took place in a phosphate-borate buffer with 50 mM sodium dodecyl sulphate using an automated instrument and HPLC was performed with a C8 column and a mobile phase of phosphate buffer-acetonitrile (65:35, v/v). HPCE and HPLC data from 66 patient samples compared well based on linear regression analysis. However, estimates obtained with the inclusion of the internal standard were lower than those based on the sample peak only. This example allows the elucidation of the advantages of using HPCE as an assay methodology for the therapeutic monitoring of thiopental and other drugs.     

Automated DNA fragment collection by capillary array gel electrophoresis in search of differentially expressed genes

An automatic DNA fragment collector using capillary array gel electrophoresis has been developed. A sheath flow technique is used for not only detection but also collection of DNA fragments. In a sheath flow cell, the DNA fragments separated by 16 capillaries flow independently into corresponding sampling capillaries. The fraction collector consists of 16 sampling trays and each sampling tray is set beneath each end of the sampling capillaries to collect the flow-through DNA fragments. Certain DNA fragments are automatically sorted by controlling the movement of the sampling trays according to the signals from the system. The collector experimentally separated two mixtures of polymerase chain reaction (PCR) products: one prepared by using eight different sizes (base lengths from 161 to 562) of DNAs; and the other prepared by a differential display (DD) method with cDNA fragments. Collected DNA fragments are amplified by PCR and measured by electrophoresis. DNA fragments with base length differences of one (base lengths 363 and 364) were successfully separated. A separated DNA fragment from the DD sample was also successfully sequenced. In addition, differentially expressed DNA fragments were automatically sorted by comparative analysis, in which two similar cDNA fragment groups, labeled by two different fluorophores, respectively, were analyzed in the same gel-filled capillary. These results show that the automatic DNA fragment collector is useful for gene hunting in research fields such as drug discovery and DNA diagnostics.     

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.     

On-line pre-concentration and UV determination of DNA fragments by dynamic coating capillary electrophoresis and its application to detection of genetically modified oilseed rape based on PCR

In this work, it was demonstrated that on-line pre-concentration and separation of DNA fragments within bared silica column by dynamic coating capillary electrophoresis and UV detection. The DNA fragments were pre-concentrated with long electrokinetic injecting time (99 s), peak height increased dramatically as a function of injection time, especially for shorter length DNA. The concentration sensitivity of DNA fragments can be improved from 20- to 100-fold relative to a normal injection (5 s). The electro-osmotic flow (EOF) and DNA-wall interactions within the capillary were eliminated effectively by dynamic coating method. Employing 0.5% poly(ethylene oxide) (PEO) in Tris-phosphate-EDTA (TBE) buffer as sieving matrix, DNA fragments, ranging from 11 to 657 bp, were separated within 20 min. The linear coefficient of linear relation between the migration and DNA length is 0.999. The DNA fragments amplified from transgenic oilseed rape by polymerase chain reaction (PCR) were separated and detected by this method, demonstrating the potential use of this method for effective DNA analysis and detection of genetically modified organisms (GMO).     

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.     

Quantitation of PCR Products by Capillary Electrophoresis in a Single Run

IntroductionItisnecessarytoevaluatetheamountofDNAfragmentinsuchcasesasgeneexpressioninvestigati0n.HybridizationwithradioactivitytaggedoligonucleotidesfollowedbyphotograPhyanddensitometricscanisoftenemployedtomeettheneedsofsuchrequirementS,whichislaborintensive,timec0nsumingandunautomated-CaPillaryelectrophoresis(CE)tendst0beastrongtoolintheanalysis0fDNAfragmentSwiththeadvantagesofhighsensitivity,highres0luti0nandautomation.QuantitativeanalysisofDNAfragmentshasbeendonebysomeauthorsusingC…     

On-channel base stacking in microchip capillary gel electrophoresis for high-sensitivity DNA fragment analysis

We evaluated a novel strategy for high-sensitivity DNA fragment analysis in a conventional glass double-T microfluidic chip. The microchip allows for a DNA on-channel concentration based on base stacking (BS) with a microchip capillary gel electrophoretic (MCGE) separation step in a poly(vinylpyrrolidone) (PVP) sieving matrix. Depending if low conductivity caused a neutralization reaction between the hydroxide ions and the run buffer component Tris+, the stacking of DNA fragments were processed in the microchip. Compared to a conventional MCGE separation with a normal electrokinetic injection, the peak heights of 50-2650-base pair (bp) DNA fragments on the MCGE-BS separation were increased 3.9-8.0-fold. When we applied the MCGE-BS method to the analysis of a clinical sample of bovine theileria after PCR reaction, the peak height intensity of the amplified 816-bp DNA fragment from the 18S rRNA of T. buffeli was enhanced 7.0-fold compared to that of the normal injection method.     

Analysis of large-volume DNA markers and polymerase chain reaction products by capillary electrophoresis in the presence of electroosmotic flow

We have demonstrated on-line concentration and separation of DNA in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO) solutions. After injecting large-volumes DNA samples, PEO solutions entered a capillary filled with 400 mM Tris-borate (TB) buffers by EOF and acted as sieving matrices. DNA fragments stacked between the sample zone and PEO solutions. Because sample matrixes affected PEO adsorption on the capillary wall, leading to changes in EOF, migration time, concentration, and resolving power varied with the injection length. When injecting phiX174 RF DNA-HaeIII digest prepared in 5 mM Tris-HCl buffer, pH 7.0, at 250 V/cm, peak height increased linearly as a function of injection volume up to 0.9 microl (injection time 150 s). The sensitivity improvement was 100-fold compare to that injected at 25 V/cm for 10 s (0.006 microl). When injecting 1.54 microl of GeneScan 1000 ROX, the sensitivity improvement was 265-fold. The sensitivity improvement was 40-fold when injecting 0.17 microl DNA sample containing pBR 322/HaeIII, pBR 328/BglI, and pBR 328/HinfI digests prepared in phosphate-buffered saline. This method allows the analysis of polymerase chain reaction (PCR) products amplified after 17 cycles when injecting 0.32 microl (at 30 cm height for 300 s). The total analysis time was shorter (91.6 min) than that (119.6 min) obtained from injecting PCR products after 32 cycles for 10 s.     

聚环氧乙烷无胶筛分毛细管电泳分离宽分子量范围DNA片段

在无胶筛分毛细管电泳中,以聚环氧乙烷为筛分介质,用硅烷化处理的毛细管柱（31.2 cm×75 μm有效长度21.0 cm）分离DL5000 DNA Marker（DNA长度为100~5000 bp）,研究筛分介质浓度、缓冲液pH、分离电压和溴化乙锭浓度对分离双链DNA片段的影响,优化出分离100~5000 bp DNA片段的最佳条件。毛细管电泳的最佳条件为PEO浓度0.5%、缓冲液pH值8.0、电压12 kV、溴化乙锭浓度3.0 μg/mL。此条件下,对山梨醇脱氢酶基因（SDH）和乙烯受体基因（ETR1）的聚合酶链式反应（PCR）扩增产物同时检测,分离、鉴定效果良好。     

多重聚合酶链反应-毛细管电泳-激光诱导荧光法检测三种食源性致病菌

建立了食品中常见致病菌大肠杆菌O157:H7的uidA基因、沙门菌的invA基因和志贺菌的ipaH基因的多重聚合酶链反应（PCR）产物的毛细管电泳快速检测方法。根据这3种致病菌的特异性基因序列设计多重PCR引物,优化PCR扩增反应体系,采用7.0 g/L 甲基纤维素为筛分介质,毛细管电泳-激光诱导荧光检测法同时检测了3种常见致病菌的PCR扩增产物。在优化的多重PCR反应和毛细管筛分电泳条件下,该方法可以同时检测沙门菌、志贺菌和大肠杆菌O157:H7基因的多重PCR扩增产物,22 min内即可完成3种常见致病菌的毛细管电泳检测。迁移时间的相对标准偏差为1.47%~2.07%。与凝胶电泳法比较,该法简便快速,灵敏度高,可用于多种致病菌脱氧核糖核酸的检测,为食品安全提供了一种可靠的快速检测方法。     

High-throughput polymerase chain reaction analysis of clinical samples by capillary electrophoresis with UV detection

Routine genetic analysis of large numbers of individuals by polymerase chain reaction (PCR) using capillary electrophoresis is often restricted by the low throughput of standard protocols and the tedious sample preparation process. Here, we demonstrate that capillary electrophoresis with UV detection can be used in PCR-based DNA analysis starting from clinical samples without purification or complicated sample manipulation. After PCR reaction using cheek cells, blood, or HIV-1 gag DNA, the reaction mixtures were injected into a capillary array either on-line or off-line by base stacking. The use of multiplexed absorption detection and the elimination of any purification steps both before and after PCR reaction can potentially provide significant benefits compared to current methods for DNA analysis with regard to time, cost, and labor.     

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.     

High-performance capillary electrophoretic analysis of chloramphenicol acetyl transferase activity.

This study highlights the potential utility of high-performance capillary electrophoresis (HPCE) for monitoring enzyme activity. Free-zone capillary electrophoresis is used to rapidly and reproducibly analyze the activity of the bacterial enzyme chloramphenicol acetyl transferase (CAT) which converts the substrates acetyl coenzyme A (CoA) and chloramphenicol to acetyl chloramphenicol and CoA. The results of this study indicate that HPCE may be an excellent tool for studying enzyme activities since it has several advantages over standard single parameters assays, most notably, the ability to monitor both loss of substrate and appearance of products simultaneously. Conditions have been identified for optimal separation of the substrate (chloramphenicol) from the products (acetylated derivatives). This presents a unique potential of HPCE for the analysis of enzymatic reactions that may be applied to areas of analytical research presently utilizing enzymatic reactions. One such analytical method is the CAT assay used for analysis of gene promoter activity. In this study, HPCE is shown to yield similar quantitative results with nonradiolabelled substrate in a fraction of the time. HPCE has several advantages over standard techniques including speed of analysis, no need for radiolabelled substrate, small sample volumes, high sensitivity/resolution and excellent quantitative capabilities.     

二氢叶酸还原酶抑制剂的高效毛细管电泳法筛选模型的建立与应用

建立了一种采用毛细管电泳法(CE)测定二氢叶酸还原酶(DHFR)反应动力学参数的新方法。以含0.002%Brij-35的50 mmol/L 的硼砂缓冲溶液（pH 9.18）作为电泳介质,检测波长280 nm,于19 min内实现了体系中各组分的基线分离。根据酶反应过程中反应物和反应产物的浓度变化计算有关反应动力学参数。将已知的氨甲喋呤、甲氧苄胺嘧啶和叶酸3种抑制剂作用于所建立的二氢叶酸还原酶体系,测得抑制剂的半数抑制浓度与文献值相接近,证明本体系可用于二氢叶酸还原酶抑制剂(DHFRI)的筛选。     

单链构象多态性-毛细管电泳分析中样品纯化研究

聚合酶链反应(PCR)样品中的Cl^-、引物等小分子对单链构象多态性一毛细管电泳分析有重要影响。本文用毛细管电泳一间接紫外法测定了PCR样品中的Cl^-浓度,并对乙醇沉淀法和试剂盒纯化法用于PCR样品的纯化效果进行比较,在此基础上研究了Cl^-浓度、引物及其二聚体在SSCP-CE分析中的影响。结果表明,它们主要影响DNA进样量而对分离效率影响不大。     

多响应曲面优化-毛细管电泳-激光诱导荧光快速检测食源性致病菌

建立了食品中常见致病菌: 沙门菌的invA基因、大肠杆菌O157∶H7的rfbO157基因、志贺菌的ipaH基因及副溶血性弧菌Vpara(16S-23S rDNA IGS)基因的多重PCR产物-毛细管电泳快速检测方法. 根据沙门菌、大肠杆菌O157∶H7、志贺菌及副溶血性弧菌的特异性基因保守序列设计出多重PCR引物, 优化PCR 扩增反应体系. 采用多响应曲面法优化毛细管电泳的分离条件, 以含有DNA荧光染料SYBR Green Ⅰ的1.0％甲基纤维素为筛分介质, 通过毛细管电泳-激光诱导荧光同时检测4种常见致病菌的PCR 扩增产物. 在优化的多重PCR反应体系和毛细管筛分电泳条件下, 此方法可以同时检测出沙门菌的invA基因、大肠杆菌O157∶H7的rfbO157基因、志贺菌的ipaH基因及副溶血性弧菌Vpara(16S-23S rDNA IGS)基因的多重PCR扩增产物, 25 min内即可完成检测. 迁移时间的日内相对标准偏差为0.92%~1.58%. 通过多响应曲面的优化, 有效改善了毛细管电泳对DNA分子的分离能力.     

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.