毛细管电泳技术在微生物学研究中的应用

毛细管电泳(capillary electrophoresis,CE)又称高效毛细管电泳(HPCE),是当前发展最快的分析技术之一.近年来,CE已被广泛应用于核酸、蛋白质、多肽、药物等大分子物质的分析,核酸序列测定等生命科学的各个领域.目前,在微生物学领域,CE除了在微生物基因测序等方面得到广泛应用外,在微生物学检测方面,CE也得到了广泛的应用.本文对近年来CE在细菌、真菌、病毒等微生物颗粒及基因检测方面的应用加以综述.     

毛细管电泳在DNA分析中的应用

简要介绍了CE技术原理,综述毛细管电泳在DNA分子微量检测、片段分离、基因突变及高通量DNA分析与测序中的应用和进展。     

毛细管电泳芯片及其应用

毛细管电泳芯片是近十年发展起来的一项新技术。与普通毛细管电泳相比,毛细管电泳芯片具有体积小、分离速度快、效果好、所用样品量少等优点。章介绍了毛细管电泳芯片的芯片结构及其应用等的研究进展。     

毛细管电泳的最新进展

毛细管电泳是近年发展最快的分离分析技术之一。它具有高灵敏度、高分辨率、高速度等优点,广泛应用于各个领域。随着毛细管电泳技术的不断发展,逐渐出现了非水毛细管电泳,毛细管阵列电泳,毛细管电泳免疫分析,毛细管电色谱手性拆分等分支。     

蛋白质和多肽研究的毛细管电泳技术

毛细管沈阳东大迪克化工药业有限公司电泳（capillary　electrophoresis，CE）也常称高效毛细管电泳（high performance capillary electrophoresis．HPCE）．是以内径20-200μm的柔性毛细管柱作为分离通道、以高压直流电场为驱动力对各种小分子、大分子以至细胞等进行高效分离、检测或微量制备等有关技术的总称（参见图1）。     

高效毛细管电泳在蛋白质分析上的应用

高效毛细管电泳（ＨＰＣＥ）是继高效液相色谱技术之后的又一新型分析及分离技术。本文应用高效毛细管电泳技术对基因工程干扰素、疫苗、动物脏器提取物等蛋白质产品进行了分离分析和纯度鉴定,并与凝胶电泳的分析结果进行对比。实验结果表明,ＨＰＣＥ可以用于生物产品分离、生物遗传研究和医学临床等领域的蛋白质定量分析、组分测定和纯度鉴定,是一种很有应用前途的新技术。     

毛细管电泳及其在蛋白质折叠研究中的应用

简要介绍了毛细管电泳的常用分离模式及其原理,并对毛细管电泳在蛋白质化学领域中的新应用——研究蛋白质折叠和发展前景作了评述。     

平板通道毛细管电泳

平板通道毛细管电泳又称微芯片毛细管电泳,是一门新兴的分析技术,具有小型化、自动化、快速、高效等优点。本文简要介绍了平板制作、进样、电泳分离、检测及其在生物方面的应用等。     

人工神经原网络处理PCR数据在细菌鉴定中的应用

目的16SrRNA和16S-23SrRNA间区片段是常用细菌分类鉴定靶点,本研究探讨人工神经原网络（ANN）对上述位点PCR扩增产物数据分析在细菌快速鉴定方面的价值。方法2对15SrRNA基因荧光引物和1对16S-23SrRNA区间基因引物用于扩增血液标本中分离出的317株细菌。相关毛细管电泳（CE）限制性片段长度多态性（RFLP）和单链构象多态性（SSCP）数据进行人工神经原网络分析。结果16S-23SrRNA基因的RFLP数据对未知菌鉴定的准确率高于16SrRNA基因的SSCP数据,分别为98．0％和79．6％。结论实验证明了人工神经原网络作为一种模式识别方法对于简化细菌鉴定十分有价值。     

平板通道毛细管电泳

平板通道毛细管电泳又称微芯片毛细管电泳,是一门新兴的分析技术,具有小型化、自动化、快速、高效等优点。本文简要介绍了平板制作、进样、电泳分离、检测及其在生物方面的应用等。     

Automated DNA mutation analysis by single-strand conformation polymorphism using capillary electrophoresis with laser-induced fluorescence detection

Automation is essential for rapid genetic-based mutation analysis in clinical laboratory to screen a large number of DNA samples. We propose in this report an automatic process using Beckman Coulter P/ACE™ capillary electrophoresis (CE) with laser-induced fluorescence (LIF) system to detect a single-point mutation in the codon 12 of human K-ras gene. Polymerase chain reaction (PCR) using a fluorescently labeled reverse primer and a plain forward primer to specifically amplify a selected 50 bp DNA fragment in human K-ras gene. The amplified DNA is placed on the sample tray of the CE system with a pre-programmed step for single-strand conformation polymorphism (SSCP) analysis. Sample injection and denaturation processes are performed online along with separation and real-time data analysis. The concept of automation for rapid DNA mutation analysis using CE-LIF system for SSCP is presented.     

Capillary electrophoresis for pharmacokinetic studies

Different analytical techniques involving capillary electrophoresis for the determination of drugs and metabolites in biological fluids are described. Pharmacokinetic studies carried out using capillary electrophoresis are presented, as well as the in vitro metabolism investigations. The advantages and the limitations of capillary electrophoresis for pharmacokinetic studies are discussed.     

荧光标记复合PCR扩增微卫星位点的构建与优化

本研究采用毛细管电泳技术,构建并优化了荧光标记复合PCR同时扩增多个微卫星位点。主要过程为：首先根据设计所扩增微卫星位点的期望长度,将9个微卫星位点分成两组,5个位点用FAM（蓝色）标记,4个位点用HEX（绿色）标记;两种荧光类型分组优化,用琼脂糖胶电泳检测。其次,荧光标记的复合PCR扩增8个中华绒螯蟹样品的9个微卫星位点,采用ABI3730xl毛细管电泳检测,以ROX500（红色）为长度标准物,结果经Genemapper3.5软件 分析,检测结果表明毛细管电泳检测荧光标记复合PCR产物不仅精确读取微卫星位点的长度（分辨率高达1bp）,还能区分微卫星位点复制时滑链所引起的“回声斑”;调整各微卫星位点引物比列使所有位点扩增强弱均匀。最后,逐一检测复合PCR基本参数（dNTP浓度、 PCR程序和模版DNA用量）对复合PCR产物的影响,优化PCR。结果表明通过毛细管电泳检测荧光标记复合PCR产物来读取微卫星位点的基因型具有精确性、高效性和稳定性。     

Validation of fluorescence-labeled artificial nonhuman sequences for single-strand conformation polymorphism mutation detection in familial hypercholesterolemia

We developed a two-in-one, polymerase chain reaction (PCR)-based method with a specific amplification step and a universal amplification step in one tube to screen for the presence of DNA variations. The method relies on fluorescence-labeled artificial nonhuman sequences for mutation detection. To document utility, we applied this method as a high-throughput capillary single-strand conformation polymorphism screening system to identify 30 mutations in the low-density lipoprotein receptor gene. The sensitivity of mutant allele detection compared to wild-type allele detection was 93%. We conclude that the "two-in-one PCR" is sensitive, simple, and cost effective.     

Evaluation of ADA Gene Expression and Transduction Efficiency in ADA/SCID Patients Undergoing Gene Therapy

A capillary electrophoresis (CE) method was developed for ADA/SCID diagnosis and monitoring of enzyme replacement therapy, as well as for exploring the transfection efficiency for different retroviral vectors in gene therapy.     

Direct quantification of gene expression using capillary electrophoresis with laser-induced fluorescence

Quantification of gene expression provides valuable information regarding the response of cells or tissue to stimuli and often is accomplished by monitoring the level of messenger RNA (mRNA) being transcribed for a particular protein. Although numerous methods are commonly used to monitor gene expression, including Northern blotting, real-time polymerase chain reaction, and RNase protection assay, each method has its own drawbacks and limitations. Capillary electrophoresis with laser-induced fluorescence (CE-LIF) can reduce protocol time, eliminate the need for radioactivity, and provide superior sensitivity and dynamic range for quantification of RNA. In addition, CE-LIF can be used to directly determine the amount of an RNA species present, something that is difficult and not normally accomplished using current methods. Gene expression is detected using a fluorescently labeled riboprobe specific for a given RNA species. This direct approach was validated by analyzing levels of 28S RNA and also used to determine the amount of discoidin domain receptor 2 mRNA in cardiac tissue.     

Methyl malondialdehyde as an internal standard for the determination of malondialdehyde

Methyl malondialdehyde (Me-MDA) is suggested as an internal standard for the determination of the lipid peroxidation product, malondialdehyde (MDA). A procedure for synthesising the Me-MDA sodium salt is described in detail. The purity and identity of the synthesised Me-MDA have been confirmed using nuclear magnetic resonance and UV spectroscopy, and by micellar electrokinetic chromatography. The applicability of Me-MDA as an internal standard has been demonstrated for rat brain homogenate samples. These samples were purified solely through ultrafiltration. The preferred analytical technique was capillary zone electrophoresis (CZE) with UV detection at 267 nm. The limits of detection (3 S/N) for the CZE separations of Me-MDA and MDA were 0.5 and 0.2 μM, respectively, and the total analysis time was approximately 10 min. Details of separations are also presented using high-performance liquid chromatography (HPLC) with UV detection at 245 nm, and gas chromatography, together with either electron capture or mass spectrometric detection. The GC separations require derivatisation of MDA and Me-MDA with pentafluorophenylhydrazine while the CZE and HPLC separations can be performed on the native molecules.     

Polymerase chain reaction method to identify Down syndrome model segmentally trisomic mice

The Ts65Dn segmentally trisomic mouse possesses an extra copy of a segment of chromosome 16 translocated to chromosome 17. This segment includes the mouse homolog of the Down syndrome critical region of human chromosome 21. The Ts65Dn mouse serves as a useful model to study the developmental regulation of the Down syndrome phenotype. To identify mice bearing the extra chromosome 16 segment, we developed a polymerase chain reaction (PCR) method as an alternative to karyotyping. Conditions under which segments of genes on chromosome 16 (App and Dyrk1a) could be coamplified with a control gene on chromosome 8 (Acta1) so that the yield of each PCR product was proportional to the amount of its template were determined. The amplification products were resolved and quantified by two methods. In the first method, the DNA segments were separated by agarose gel electrophoresis and stained with ethidium bromide. The fluorescence yields were quantified by photodensitometry. In the second method, the fragments were resolved and quantified by the high-performance DNA analysis system, a high-throughput, multichannel, microcapillary electrophoresis instrument. The results of both methods were within 10% of the expected ratio of 1.5. Application of these methods has allowed the maintenance of a Ts65Dn breeding colony through six generations and should permit the precise and efficient identification of trisomic and disomic animals at any developmental stage with minimally invasive procedures.     

Capillary electrophoresis with noncovalently bilayer-coated capillaries for stability study of allergenic proteins in simulated gastrointestinal fluids

A novel noncovalently bilayer-coated capillary using cationic polymer polybrene (PB) and anionic polymer (sodium 4-styrenesulfonate) (PSS) as coatings was prepared. This PB–PSS coating showed good migration-time reproducibility for proteins and high stability in the range of pH 2–10 and in the presence of 1 M NaOH, acetonitrile and methanol. Capillary electrophoresis with PB–PSS coated capillaries was successfully applied to quantitatively investigate the stability of bovine serum albumin, ovomucoid, β-lactoglobulin and lysozyme in simulated gastrointestinal fluids. β-lactoglobulin A and β-lactoglobulin B were both stable in simulated gastric fluid with degradation percentages of 34.3% and 17.2% after 60 min of incubation, respectively. Bovine serum albumin, ovomucoid and lysozyme were stable in simulated intestinal fluid with degradation percentages of 17.7%, 23.4% and 22.8% after 60 min of incubation, respectively. The superiority of the proposed method over sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and capillary electrophoresis with untreated fused silica capillaries was demonstrated and emphasized.     

Capillary isoelectric focusing of a difficult-to-denature tetrameric enzyme using alkylurea–urea mixtures

Capillary isoelectric focusing (cIEF) is normally run under denaturing conditions using urea to expose any buried protein residues that may contribute to the overall charge. However, urea does not completely denature some proteins, such as the tetrameric enzyme Erwinia chrysanthemil-asparaginase (ErA), in which case electrophoresis-compatible alternative denaturants are required. Here, we show that alkylureas such as N-ethylurea provide increased denaturation during cIEF. The cIEF analysis of ErA in 8 M urea alone resulted in a cluster of ill-resolved peaks with isoelectric points (pI values) in the range 7.4 to 8.5. A combination of 2.0 to 2.2 M N-ethylurea and 8 M urea provided sufficient denaturation of ErA, resulting in a main peak with a pI of 7.35 and an acidic species minor peak at 7.0, both comparing well with predicted pI values based on the sum of protein residue pK_a values. Recombinant deamidated ErA mutants were also demonstrated to migrate to pI values consistent with predictions (pI 7.0 for one deamidation). The quantitation of ErA acidic species in samples from full-scale manufacturing (1.0–3.5% of total peak area) was found to be reproducible and linear. Use of alkylureas as denaturing agents in capillary electrophoresis and cIEF should be considered during biopharmaceutical assay development.