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     

毛细管电泳分析核酸的方法及应用

毛细管电泳是一门速发展的分离技术。根据分离同制不同,毛细管电泳分离核酸的方法有:胶束电动力学毛细管色谱(MEKC)、毛细管区带电泳(CZE)和毛细管凝胶电泳(CGE)。毛细管电泳主要用于核酸纯度的鉴定、定量分析、测序,片段大小多态性分析、构象多态性分析和蛋白与核酸相互作用的研究。     

应用非变性毛细管电泳检测乙肝病毒基因型B和基因型C

目的 建立非变性毛细管电泳分离乙肝病毒特异性巢式PCR产物方法,用于乙肝病毒B基因型和C基因型判断.方法 建立线性聚丙烯-聚乙二胺-TBE非变性毛细管电泳方法,分离母婴阻断失败患儿血清中乙肝病毒基因型特异性巢式PCR的扩增产物,采用表观分子量鉴定B、C基因型特异性和非特异性扩增产物.结果 毛细管电泳条件为30.2cm×50μm毛细管,分离缓冲液为2％(w/v)线性聚丙烯酰胺+0.4％(w/v)聚乙二醇+lxTBE缓冲液(pH8.3).在9kV电压下12分钟内可分离DNA分子量梯度标准品(50 bp至300 bp)及PCR扩增产物,表观分子量与电泳迁移时间的曲线适合度大于0.9999.B型扩增产物表观分子量范围为282bp至285bp,非特异性扩增产物为276bp至280bp.C型为ll9bp至120bp,当前扩增条件下不产生非特异性扩增产物.结论 非变性毛细管电泳能够方便和准确地判断非特异性的B型扩增产物.     

血红蛋白毛细管电泳和血液学指标在地贫筛查中的诊断价值

目的探讨毛细管电泳法检测血红蛋白和血液学指标在地中海贫血筛查中的诊断价值。方法收集中山大学附属第一医院东院在2016年1月至2017年7月期间进行地中海贫血筛查的患者6416例,其中血红蛋白电泳、血液学指标和基因检测数据齐全的病例152例,分析联合毛细管电泳法和血液学指标筛查地贫的效能。结果152例同时进行了毛细管电泳、血细胞分析、基因检测的病例中,疑似地贫患者的符合率为55.4%,异常血红蛋白者的符合率为80%,单纯MCV、MCH或电泳异常者的符合率为14.5%,筛查正常者的符合率达90.9%;筛查总符合率为37.6%。其中MCV、MCH具有较高的灵敏度和特异性,RBC有较高的灵敏度,但特异性低;电泳结果具有较高的灵敏度,对β-地贫患者的检出具有很高的诊断价值,并可以检测出异常血红蛋白带;毛细管电泳联合MCV、MCH筛查地中海贫血具有较高的灵敏度和特异性。结论单纯血细胞分析或单纯电泳结果在地贫筛查中诊断价值一般,联合血细胞分析和毛细管电泳结果在地贫筛查中诊断价值高。     

毛细管电泳在核酸及核苷酸分析中的应用

毛细管电泳为生物活性物质分析的有力方法。由于其高效、快速、微量、易定量和易自动化的特点，有可能在很在范围内取代传统电泳技术。本文介绍了毛细管电泳在ＤＮＡ测序，ＤＮＡ大片段，核苷酸分析中的应用，归纳了其特点，影响因素，应用优势。     

应用毛细管电泳筛选DNA点突变

毛细管电泳在最近几年得到了迅速发展。它提供了快速、高分辨、自动操作、数据自动获取的在线检测,并且应用于ＤＮＡ的突变分析。本文介绍了利用毛细管电泳来筛选ＤＮＡ点突变,并对未来的发展进行了展望。     

毛细管电泳与核酸分析

毛细管电泳技术以其快速、高效、高灵敏度、重复性好、易定量、非同位素操作、可实时监测及全自动化等优点在核酸研究的各个方面得到了广泛应用。本文着重介绍了毛细管电泳技术在4个领域中的应用:①未知和已知单核苷酸改变的寻找;②短串联重复序列的检测;③DNA测序;④基因及其表达产物的定量分析。毛细管电泳作为一项具有许多优点的分离分析技术正在并将继续在核酸分析领域发挥重要作用。     

应用毛细管电泳筛选DNA点突变

毛细管电泳在最近几年得到了迅速发展。它提供了快速，高分辨，自动操作，数据自动获取的在线检测，并且应用于ＤＮＡ的突变分析。本文介绍了利用毛细管电泳来筛选ＤＮＡ点突变。并对未来的发展进行了展望。     

芯片毛细管电泳检测平台的建立及其应用

目的采用芯片毛细管电泳检测基因点突变,为β-地中海贫血产前基因诊断提供一种快速、灵敏的检测技术。方法通过多重引物延伸反应扩增β-地中海贫血常见的突变位点,并采用芯片毛细管电泳双通道检测扩增产物,以辨别样品的基因型。结果建立了β-地中海贫血常见基因型的芯片毛细管电泳检测方法,对39例β-地中海贫血患者和5例产前诊断病例的检测结果符合试剂盒检测结果。结论芯片毛细管电泳检测β-地中海贫血具有快速、样品量少等优点,为产前遗传病的检测提供了一种新的技术手段。     

应用毛细管无胶筛分电泳诊断缺失型杜氏肌营养不良基因

毛细管电泳是一项新兴的分离、分析技术,它以高效、快速、重复性好、自动化及微量等优点,在核酸分离分析的各方面均较以往的方法更具优势。本工作应用毛细管无胶筛分电泳与多重ＰＣＲ技术相结合对缺失型杜氏肌营养不良的病人进行了直接基因诊断。与传统方法相比,本技术在分离速度、分辨率及灵敏度方面均有较大改善。毛细管电泳技术将广泛用于遗传病基因诊断。     

Mechanistic Study of α‐Amino Acid N‐Carboxyanhydride (NCA) Polymerization by Capillary Electrophoresis

Polymerizations of N_ε‐trifluoroacetyl‐L ‐lysine N‐carboxyanhydride initiated by hexylamine were performed in N,N‐dimethylformamide with different monomer to initiator ratios and at different temperatures. Poly(N_ε‐trifluoroacetyl‐L ‐lysine) samples were characterized by size exclusion chromatography coupled with multi‐angle laser light detection (SEC‐MALLS) and capillary electrophoresis (CE). If SEC‐MALLS analysis emphasizes the double distribution in molecular weight, nonaqueous CE (NACE) brings additional quantitative information on the oligomeric distribution and on the distribution according to polymer functionality (end‐groups). At room temperature, two termination reactions were clearly identified for these polymerizations. The suppression of these side reactions is reported upon decreasing the reaction temperature to 0 °C. A strong reduction of the bimodal aspect of the molecular weight distribution was obtained with the addition of urea to the polymerization medium. The possibility of a living and controlled polymerization at 0 °C with urea was demonstrated and illustrated by the synthesis of two block copolypeptides.

     

Microfabrication Technology for the Production of Capillary Array Electrophoresis Chips

Improvements in the fabrication, sample handling and electrical addressing of capillary array electrophoresis (CAE) chips have permitted the development of high density, high-throughput devices capable of analyzing 48 samples in about 20 minutes. The fabrication of high density capillary arrays on 10 cm diameter substrates required the characterization of glasses that yield high quality etches and the development of improved sacrificial etch masks. Using these improved fabrication techniques, high-quality, deep channel etches are routinely obtained. Methods for bonding large area substrates and for drilling arrays of 100 or more access holes have also been developed. For easier sample introduction, we use an array of sample wells fabricated from an elastomeric sheet. The practicality of these technologies is demonstrated through the analysis of 12 DNA samples in parallel on a microfabricated CAE chip, the development of methods for injecting multiple samples onto a single capillary without cross contamination, and the operation of a microfabricated array of 12 capillaries with 4 sample injections per capillary that can analyze 48 samples.     

A Novel immunofluorescence capillary electrophoresis assay system for the determination of chloramphenicol in milk

An immunofluorescence capillary electrophoresis technique is described for the detection of chloramphenicol where the analyte was allowed to compete with a chloramphenicolfluorescein conjugate for binding to polyclonal anti‐chloramphenicol antibody. The bound and unbound forms of the chloramphenicol‐fluorescein conjugate were then analyzed by capillary zone electrophoresis with laser‐induced fluorescence detection, which permitted direct quantitation of the extent of immunocomplex formation. The amount of unbound conjugate in the reaction mixture was proportional to the quantity of chloramphenicol in the sample. Using this assay, it was possible to detect concentrations as low as 0.1–0.5 ng ml^‐1 of chloramphenicol in a standard buffer and at least 01 ng ml^‐1 of chloramphenicol in milk after extraction with ethyl acetate using a simple protocol. This highly sensitive procedure was rapid, requiring less than 30min to complete. The immunofluorescence capillary electrophoresis system should be similarly applicable to the assay of a wide variety of analytes.     

Capillary electrophoresis-based single strand DNA conformation analysis in high-throughput mutation screening

The generation of the draft human genome sequence has created new possibilities for diagnosis, prevention, and treatment of human disease. One consequence of these new possibilities is an increasing need for methods and technology that can be used for high-throughput screening for mutations in large DNA sample materials. In recent years, a number of mutation screening methods have emerged that are based on the analysis of sequence-dependent changes in the conformation of single- and double-stranded DNA using capillary electrophoresis. Common features of these methods are high sensitivity and reproducibility as well as the possibility for automation and massive parallelization. Thus, at present they are among the most attractive technologies for high-throughput mutation screening. This review describes the recent advances in capillary electrophoresis-based single strand conformation polymorphism (CE-SSCP) for detection of unknown mutations, and assesses its practical usability for high-throughput mutation screening based on the available literature. In addition, future prospects are outlined in light of the recent advances in microchip-based capillary electrophoresis.     

基于毛细管电泳的单基因病扩展性携带者筛查技术的建立及临床评估

目的 建立并评估一种基于毛细管电泳技术的单基因病扩展性携带者筛查方法。 方法 采用多个基于毛细管电泳的技术检测 1 099 例样本中 24 个基因相关的 20 种疾病中的 448 个致病变异并进行验证。 结果 毛细管电泳检测结果与其他方法验证的结果完全一致。 在 1 099 例受检者中, 检出 190 例携带者, 总体携带率为17. 3 % (190 / 1 099), 检出变异个数最多的是 GJB2 基因。 在检测的1 075 例女性样本中, 共 检出 8 例为 X 连锁遗传病致病基因的携带者, 携带率为 7. 4 ‰ (8 / 1 075)。 结论 成功建立了一种基于毛 细管电泳的扩展性携带者筛查技术。     

High performance microfluidic capillary electrophoresis devices

This paper presents a novel microfluidic capillary electrophoresis (CE) device featuring a double-T-form injection system and an expansion chamber located at the inlet of the separation channel. This study addresses the principal material transport mechanisms depending on parameters such as the expansion ratio, the expansion length, the fluid flow. Its design utilizes a double-L injection technique and combines the expansion chamber to minimize the sample leakage effect and to deliver a high-quality sample plug into the separation channel so that the detection performance of the device is enhanced. Experimental and numerical testing of the proposed microfluidic device that integrates an expansion chamber located at the inlet of the separation channel confirms its ability to increase the separation efficiency by improving the sample plug shape and orientation. The novel microfluidic capillary electrophoresis device presented in this paper has demonstrated a sound potential for future use in high-quality, high-throughput chemical analysis applications and throughout the micro-total-analysis systems field.     

优化PCR体系结合毛细管电泳技术检测FMR1基因前突变与全突变

目的 通过优化PCR并结合毛细管电泳,建立高扩增效率、高分辨率的FMR1基因CGG重复序列异常扩增检测体系.方法 选择标准样本和经Southern印迹技术确定(CGG)n的正常、前突变、全突变男性和女性样本15例,进行PCR检测体系的优化.优化的PCR扩增产物经琼脂糖凝胶电泳、聚丙烯酰胺凝胶电泳和毛细管电泳等多种方法进行结果比较.结果 经优化的PCR体系可以检测出(CGG)n大于260个拷贝的全突变男性和(CGG)n达到183个拷贝的前突变女性.毛细管电泳能够清晰分辨出相差1个CGG的两个等位基因,结果具有良好的可重复性.结论 该PCR检测体系大幅度提高了普通PCR方法的扩增效率和分辨率,明显降低了对于Southern印迹技术的依赖,可以作为临床筛查FMR1基因突变的首选方法.     

Accurate determination of the number of CAG repeats in the Huntington disease gene using a sequence-specific internal DNA standard

We have developed a sequence-specific internal DNA size standard for the accurate determination of the number of CAG repeats in the Huntington disease (HD) gene by cloning key fragments (between 15 and 64 CAG repeats) of the HD gene. These fragments, pooled to produce a sequence-specific DNA ladder, enabled us to observe the true number of CAG repeats directly, with no need for calculations. Comparison of the calculated numbers of CAG repeats in the HD gene using this sequence-specific DNA standard with a commercially available standard (GENESCAN-500 TAMRA) showed that the latter underestimated the number of CAG repeats by three when analyzed by capillary electrophoresis on the ABI 310 Genetic Analyzer (POP4 polymer). In contrast, the use of the same standard overestimated the number of CAG repeats by one when the samples were analyzed by denaturing polyacrylamide electrophoresis on ABI 377 DNA Sequencer (6% denaturing polyacrylamide gel). This suggests that our sequence-specific standard provides greater accuracy for the determination of the true number of CAG repeats in the HD gene than commercially available standards. The sequence-specific standard can be radioactively labeled and successfully replace conventional DNA size standards when analyzing polymerase chain reaction (PCR)-amplified HD alleles by denaturing polyacrylamide electrophoresis.