毛细管无胶筛分电泳

围绕着毛细管无胶筛分电泳的介质和机理,概括介绍了近年来这种技术在各个方面的发展,及其在DNA片段的分离、PCR扩增产物的检测和蛋白质分子量的测定等方面的应用前景．     

无胶筛分毛细管电泳在分析苹果发育时期转变蛋白质变化规律中的应用

对无胶筛分毛细管电泳体系的筛分介质浓度、缓冲体系的离子强度、分离电压、进样量及温度等条件进行了优化, 最终确定最佳分离条件为7.5% Dextran, 7.5%丙三醇, 0.15 mol.L-1 TB, 0.1% SDS, 分离电压23.5 kV, 进样量1.5 ps i×90秒, 温度25°C。应用此方法对金冠×红玉苹果杂种后代韧皮部蛋白质进行测定, 得到了多个与阶段转变相关的蛋白质, 在韧皮部50节上, 有分子量为5.0 kDa的特异蛋白质出现, 分子量为24.5 kDa的蛋白质含量在阶段转变过程中明显升高。     

无胶筛分毛细管电泳在分析苹果发育时期转变蛋白质变化规律中的应用

对无胶筛分毛细管电泳体系的筛分介质浓度、缓冲体系的离子强度、分离电压、进样量及温度等条件进行了优化,最终确定最佳分离条件为7.5% Dextran,7.5%丙三醇,0.15mol·L^-1 TB,0.1%SDS,分离电压23.5kV,进样量1.5psi×90秒,温度25℃。应用此方法对金冠×红玉苹果杂种后代韧皮部蛋白质进行测定,得到了多个与阶段转变相关的蛋白质,在韧皮部50节上,有分子量为5.0kDa的特异蛋白质出现,分子量为24.5kDa的蛋白质含量在阶段转变过程中明显升高。     

DNA分析与扩增

931549利用毛细管系列电泳进~DNA.]II序[英3/Huang,X.C.…,Ahal.Chem.一1992,64(8)一.2149~2154[译自DBA,1992,11(23),92—12849] 一种DNA测序方法利用了毛细管系列电泳,双色荧光检测和双染料标记。在一系列毛细管上分离Sanger DNA测序片段,再用双色激光激发的confocal荧光扫描器进行桂上检测。在单个毛细管里分离出4套DNA测序片段,然后用双编码法进行区别,其中每套片段用两个有特定比率的染料标记的引物标记。这样就有可能筛选出电泳迁移率相同的染料l关键的是甩不同染料标记的DNA片段之间不应该出现电泳迁移的差异,而且染料具…     

微流控芯片电泳及其法医学应用

在光学性能良好的Brofloat玻璃电泳芯片上,利用自行搭建的共聚焦激光诱导荧光检测系统,通过对芯片管道表面修饰、筛分介质、分离电场强度、进样方式、电泳温度、进样时间等条件的优化,对含15个STR基因座的法医DNA样品进行电泳分离测试实验.通过对芯片电泳条件优化获得了本电泳系统的最佳条件,成功实现8 min内完成DNA样品片段的分离,表明该微流控芯片电泳系统在法医DNA快速分析方面具有良好的应用前景.     

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。     

DNA链断裂检测技术的进展

DNA链损伤特别是DNA双链段裂(dsb)的检测方法是研究DNA辐射损伤的一个关键因素.已发展的检测DNA dsb的方法很多,但各种检测法均有其一定的优越性和适用范围,近年来应用较多并日益受到重视的新方法有原位杂交法,彗星试验(单细胞电泳法)以及高效毛细管电泳法等等.     

北京油鸡MSAP毛细管电泳荧光检测技术的建立

采用毛细管电泳荧光检测技术, 对北京油鸡肌肉组织基因组进行甲基化敏感扩增片段多态性(Methylation sensitive amplified polymorphism, MSAP)检测。通过对基因组DNA用量、预扩产物稀释倍数、选择性引物浓度、Mg2+浓度、dNTPs浓度和电泳内标量等6个主要参数进行分析和优化, 建立了适合北京油鸡基因组DNA甲基化分析的MSAP毛细管电泳荧光检测技术。重复实验表明, 建立的毛细管电泳荧光标记的MSAP检测技术能够自动地、高通量地分析北京油鸡基因组的DNA甲基化状态, 也适用于其他动植物基因组的DNA甲基化研究。     

琼脂糖凝胶电泳技术

九、碱性琼脂糖凝胶 科学家们认为,琼脂糖凝胶电泳是分离、鉴定和纯化DNA片段的一种极好的方法。这种技术操作简单,快速,并且还能解决在密度梯度离心中所存在的DNA片段不能充分分离的难题。此外,还能直接确定凝胶中DNA的位置:其方法是先用低浓度的能发荧光的具有插入本领的染料(溴乙锭),对胶中DNA条带进行染色;然后,把电泳胶置于紫外光下进行直接检测,其灵敏度可达1毫微克(ng)。     

枯草芽孢杆菌基因片段的琼脂糖凝胶电泳分离I．色氨酸C基因片段的分离

限制性核酸内切酶EcoRl酶切枯草芽孢杆菌(Bactllus subtlis) SR 22的 DNA,用琼脂糖凝胶电泳分离了色氨酸C基因(trpC)的片段,位于凝肢柱的10—11厘米处。冷冻挤压回收该段的DNA,荧电光分光光度计直接测定回收液中DNA的含量。电泳后比电泳前trpC 转化活性提高了37.7倍。这一段DNA的平均分子量为5.1×106,trpC片段的纯度为9.3％。     

Capillary electrophoresis of DNA in uncrosslinked polymer solutions: evidence for a new mechanism of DNA separation

The electrophoretic separation of DNA molecules is usually performed in thin slabs of agarose or polyacrylamide gel. However, DNA separations can be achieved more rapidly and efficiently within a microbore fused silica capillary filled with an uncrosslinked polymer solution. An early assumption was that the mechanism of DNA separation in polymer solution(SINGLEBOND)capillary electrophoresis (PS(SINGLEBOND)CE) is the same as that postulated to occur in slab gel electrophoresis, i.e., that entangled polymer chains form a network of "pores" through which the DNA migrates. However, we have demonstrated that large DNA restriction fragments (2.0(SINGLEBOND)23.1 kbp) can be separated by CE in extremely dilute polymer solutions, which contain as little as 6 parts per million [0.0006% (w/w)] of uncrosslinked hydroxyethyl cellulose (HEC) polymers. In such extremely dilute HEC solutions, far below the measured polymer entanglement threshold concentration, pore-based models of DNA electrophoresis do not apply. We propose a transient entanglement coupling mechanism for the electrophoretic separation of DNA in uncrosslinked polymer solutions, which is based on physical polymer/DNA interactions. (c) 1996 John Wiley & Sons, Inc.     

Poly(ethyleneoxide) for high resolution and high-speed separation of DNA by capillary electrophoresis

Capillary electrophoresis (CE) with polyacrylamide gels has already been demonstrated to allow single-base resolution of single-stranded DNA. However, linear polyacrylamide is not an ideal matrix because of a high viscosity and difficulties in preparing the polymer with well defined pore sizes. Alternatively, poly(ethyleneoxide) (PEO) with a large range of molecular masses from 300 000 to 8 000 000 is available commercially. In addition, it is easy to prepare homogeneous solutions to provide highly reproducible separation performance with sufficient resolution. Single-base resolution of double-stranded DNA between 123 and 124 base pairs can be achieved by the use of homogeneous matrices prepared from PEO (2.5% M_r 8 000 000), and even better resolution is achieved by using mixed polymer matrices. With further work, it should be possible to change the fractions and the total amounts of polymers to achieve even higher resolution for different samples with different size ranges of fragments. Another advantage of mixed polymer matrices is that relatively high resolution can be obtained while maintaining a relatively low viscosity compared to linear polyacrylamide with identical contents of formamide and urea, which makes it easier to fill these matrices into small capillaries.     

Comblike, monodisperse polypeptoid drag-tags for DNA separations by end-labeled free-solution electrophoresis (ELFSE)

The development of innovative technologies designed to reduce the cost and increase the throughput of DNA separations continues to be important for large-scale sequencing and genotyping efforts. We report research aimed at the further development of a free-solution bioconjugate method of DNA size separation by capillary electrophoresis (CE), in particular, the determination of an optimal molecular architecture for polyamide-based "drag-tags". We synthesized several branched poly(N-methoxyethyl glycine)s (poly(NMEG)s, a class of polypeptoids) as novel friction-generating entities for end-on attachment to DNA molecules. A 30-mer poly(NMEG) "backbone," comprising five evenly spaced reactive epsilon-amino groups, was synthesized on solid phase, cleaved, and purified to monodispersity by RP-HPLC. Three different comblike derivatives of this backbone molecule were created by (1) acetylating the epsilon-amino groups or (2) appending small, monodisperse NMEG oligomers (a tetramer and an octamer). Grafting of the oligo(NMEG)s was done using solution-phase amide bond formation chemistry. Once purified to total monodispersity, the three different drag-tags were studied by free-solution electrophoresis to observe the effect of branching on their hydrodynamic drag or "alpha" and hence their ability to separate DNA. Drag was found to scale linearly with total molecular weight, regardless of branch length. The octamer-branched drag-tag-DNA conjugate was used to separate ssDNA products of 50, 75, 100, and 150 bases in length by free-solution CE in less than 10 min. Hence, the use of branched or comblike drag-tags is both a feasible and an effective way to achieve high frictional drag, allowing the high-resolution separation of relatively large DNA molecules by free-solution CE without the need to synthesize very long polymers.     

Use of nanomaterials in capillary and microchip electrophoresis

This review gives an overview of different separation strategies with nanomaterials and their use in capillary electrophoresis (CE) and capillary electrochromatography, as well as in microchip electrophoresis, including metal and metal oxide nanoparticles, carbon nanotubes, fullerene and polymer nanoparticles, as well as silica nanoparticles. The paper highlights the new developments and innovative applications of nanoparticles as pseudostationary phases or immobilized on the capillary surface for CE separation. The separation and characterization of target nanoparticles with different sizes by CE are reviewed likewise.     

Use of nanomaterials in capillary and microchip electrophoresis

This review gives an overview of different separation strategies with nanomaterials and their use in capillary electrophoresis (CE) and capillary electrochromatography, as well as in microchip electrophoresis, including metal and metal oxide nanoparticles, carbon nanotubes, fullerene and polymer nanoparticles, as well as silica nanoparticles. The paper highlights the new developments and innovative applications of nanoparticles as pseudostationary phases or immobilized on the capillary surface for CE separation. The separation and characterization of target nanoparticles with different sizes by CE are reviewed likewise.     

Effect of temperature on the separation of DNA fragments by high-performance liquid chromatography and capillary electrophoresis: a comparative study

This study investigates the effect of experimental temperature on the separation of DNA fragments, 21–587 bp, by both high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). The results show that the temperature plays an important role in the HPLC separation of DNA fragments. The optimum temperature was found to be between 40 and 50°C for HPLC, while 25°C was the optimum temperature for the CE separation. Also, although CE migration times became shorter, efficiency and resolution decreased with an increase in temperature from 25 to 50°C, but the separation was not significantly affected. Also, the optimum HPLC temperature might be different depending on the fragment sizes to be resolved.     

Separation methods for pharmacologically active xanthones

Xanthones, as a kind of polyphenolic natural products with many strong bioactivities, are attractive for separation scientists due to the similarity and diversity of their structures resulting in difficult separation by chromatographic methods. High performance liquid chromatography (HPLC) and thin layer chromatography (TLC) are traditional methods to separate xanthones. Recently, capillary electrophoresis (CE), as a micro-column technique driven by electroosmotic flow (EOF), with its high efficiency and high-speed separation, has been employed to separate xanthones and determine their physicochemical properties such as binding constants with cyclodextrin (CD) and ionization constants. Since xanthones have been used in clinic treatment, the development of chromatographic and CE methods for the separation and determination of xanthones plays an essential role in the quality control of some herbal medicines containing xanthones. This article reviewed the separation of xanthones by HPLC, TLC and CE, citing 72 literatures. This review focused on the CE separation for xanthones due to its unique advantages compared to chromatographic methods. The comparison of separation selectivity of different CE modes including capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC), microemulsion electrokinetic capillary chromatography (MEEKC) and capillary electrochromatography (CEC) was discussed. Compared with traditional chromatographic methods such as HPLC and TLC, CE has higher separation efficiency, faster separation, lower cost and more flexible modes. However, because of low sensitivity of UV detector and low contents of xanthones in herbal medicines, CE methods have seldom been applied to the analysis of real samples although CE showed great potential for xanthone separation. The determination of xanthones in herbal medicines has been often achieved by HPLC. Hence, how to enhance CE detection sensitivity for real sample analysis, e.g. by on-line preconcentration and CE-MS, would be a key to achieve the quantitation of xanthones.     

HIV-1 Conserved Elements p24CE DNA Vaccine Induces Humoral Immune Responses with Broad Epitope Recognition in Macaques

To target immune responses towards invariable regions of the virus, we engineered DNA-based immunogens encoding conserved elements (CE) of HIV-1 p24^gag. This conserved element vaccine is designed to avoid decoy epitopes by focusing responses to critical viral elements. We previously reported that vaccination of macaques with p24CE DNA induced robust cellular immune responses to CE that were not elicited upon wild type p55^gag DNA vaccination. p24CE DNA priming followed by p55^gag DNA boost provided a novel strategy to increase the magnitude and breadth of the cellular immune responses to HIV-1 Gag, including the induction of strong, multifunctional T-cell responses targeting epitopes within CE. Here, we examined the humoral responses induced upon p24CE DNA or p55^gag DNA vaccination in macaques and found that although both vaccines induced robust p24^gag binding antibody responses, the responses induced by p24CE DNA showed a unique broad range of linear epitope recognition. In contrast, antibodies elicited by p55^gag DNA vaccine failed to recognize p24CE protein and did not recognize linear epitopes spanning the CE. Interestingly, boosting of p24CE DNA primed animals with p55^gag DNA resulted in augmentation of antibodies able to recognize p24^gag as well as the p24CE proteins, thereby inducing broadest immunity. Our results indicate that an effectively directed vaccine strategy that includes priming with the conserved element vaccine followed by boost with the complete immunogen induces broad cellular and humoral immunity focused on the conserved regions of the virus. This novel and effective strategy to broaden responses could be applied against other antigens of highly diverse pathogens.     

Determination of illicit and/or abused drugs and compounds of forensic interest in biosamples by capillary electrophoretic/electrokinetic methods

The application of capillary electrophoresis (CE) methods in forensic toxicology for the determination of illicit and/or misused drugs in biological samples is reviewed in the present paper. Sample pretreatments and direct injection modes used in CE for analysis of drugs in biological fluids are briefly described. Besides, applications of separation methods based on capillary zone electrophoresis or micellar electrokinetic chromatography with UV absorbance detection to (i) analysis of drugs of abuse, (ii) analysis of other drugs and toxicants of potential forensic interest and (iii) for metabolism studies are reviewed. Also, alternative CE methods are briefly discussed, including capillary isotachophoresis and separation on mixed polymer networks. High sensitivity detection methods used for forensic drug analysis in biological samples are then presented, particularly those based on laser induced fluorescence. A glimpse of the first examples of application of CE–mass spectrometry in forensic toxicology is finally given.     

Capillary electrophoresis of subcellular-sized particles

Utilization of capillary electrophoresis (CE) for characterization and analytical separation of submicron- and micron-sized organic and inorganic particles as well as biological vesicles is reviewed. CE has been applied to charged polystyrene size standards, inorganic and organic colloidal particles, lipoprotein particles, liposomes, microsomes and viruses. These particle separations generally occur in a size-dependent manner and provide values of electrophoretic mobility which are in good agreement with those obtained by other electrophoretic techniques.