Rapid quantification of DNA methylation by high performance capillary electrophoresis

The actual methods to evaluate total DNA methylation based on high performance liquid chromatography (HPLC) are long and tedious due to the specific running buffers required. In this work, a new open-tube capillary electrophoresis system has been applied to the separation of acid hydrolyzed genomic DNA and so, to the evaluation of genomic DNA methylation. Several running conditions were tested but separation of cytosine and 5-methyl-cytosine was only possible by sodium dodecyl sulfate (SDS) micelle system. The importance of sample dissolution preparation has also been demonstrated. The results of this study open up the possibility of quantification of the relative methylation degree of rapid genomic DNA by a simple method based on high performance capillary electrophoresis (HPCE).     

高效毛细管区带电泳法快速测定尿液中的肌酐

建立了一种快速测定尿中肌酐浓度的高效毛细管电泳方法。利用非涂层石英毛细管(64.5 cm×50μm i.d),以pH 2.5,0.1 mmol/L H3PO4作为电泳缓冲液,检测波长191 nm,用0.05 Pa压力进样4 s,在电压16 kV快速分离尿液中的肌酐,采用外标法定量。肌酐的迁移时间约为5.5 min,肌酐浓度在34.5~8840μmol/L范围内呈良好的线性(r2=0.999)。平均日内精密度为2.5%,日间精密度为3.0%。回收率94.1%~99.0%。与全自动生化分析仪碱性苦味酸速率法相比有良好的相关性(r=0.990,n=56)。高效毛细管电泳法测定尿肌酐可应用于临床样品的检测。     

毛细管区带电泳法快速测定人血血清蛋白

人血血清蛋白电泳分析是临床上诊断多种疾病的常用生化指标 ,也是临床实验室检查的常规项目。目前采用的方法有醋酸纤维薄膜电泳和琼脂糖电泳 ,尤以前者为主。由于醋酸纤维薄膜电泳法操作繁琐 ,每一步骤均需手工完成 ,影响因素较多 ,初学者往往不易掌握 ,且测定的重复性较差。高效毛细管电泳是近几年来迅速发展起来的分离分析技术 [1,2 ]。用该技术分析人血清蛋白国内外虽有报道 ,但由于人血清中蛋白质组分甚为复杂 ,采用不同的分离方法和条件可出现不同数量的组分峰 ( 6、7个甚至 1 0个以上组分 ) ,与目前临床上常用的醋酸纤维薄膜电泳图谱…     

A practical approach to high performance capillary electrophoresis with biosynthetic human growth hormone as a model protein

Biosynthetic human Growth Hormone (B-hGH) is a protein comprising 191 amino acids. The molecular weight is 22,125 and the isoelectric point is close to pH 5. Due to the ready availability of closely related analogues B-hGH was used as a model protein thus allowing for the demonstration and evaluation of the high resolution capability of high performance capillary electrophoresis (HPCE). The same apparatus was used throughout the experiments and an optimum signal-to-noise ratio was found at 200 nm. Linearity was observed between peak area, retention time and the hGH concentration or sample introduction time. Baseline separation of hGH, desamido hGH and didesamido hGH was obtained. Examples showing analysis with 1 million theoretical plates per meter, high speed separation, simultaneous analysis of multiple samples, sample stacking, hGH tryptic digest, and hGH lysate are reported. The use of electrophoretic velocities instead of apparent velocities for peak identification is illustrated.     

Integrated platform for detection of DNA sequence variants using capillary array electrophoresis

We have developed a highly versatile platform that performs temperature gradient capillary electrophoresis (TGCE) for mutation/single-nucleotide polymorphism (SNP) detection, sequencing and mutation/SNP genotyping for identification of sequence variants on an automated 24-, 96- or 192-capillary array instrument. In the first mode, multiple DNA samples consisting of homoduplexes and heteroduplexes are separated by CE, during which a temperature gradient is applied that covers all possible temperatures of 50% melting equilibrium (Tms) for the samples. The differences in Tms result in separation of homoduplexes from heteroduplexes, thereby identifying the presence of DNA variants. The sequencing mode is then used to determine the exact location of the mutation/SNPs in the DNA variants. The first two modes allow the rapid identification of variants from the screening of a large number of samples. Only the variants need to be sequenced. The third mode utilizes multiplexed single-base extensions (SBEs) to survey mutations and SNPs at the known sites of DNA sequence. The TGCE approach combined with sequencing and SBE is fast and cost-effective for high-throughput mutation/SNP detection.     

毛细管电泳分析中手性化合物的定性检测

毛细管电泳(CE)作为一种新型分离分析技术,具有分离效率高、分析速度快、样品用量少、分离模式灵活多样等众多优势,在手性物质分离等领域应用广泛。在以往的工作中,手性化合物的CE分离模式、手性拆分剂选择及提高分离度等研究已作了详尽报道,而成功分离后的手性物质定性、对映体出峰顺序确认等问题也至关重要。该文以CE手性化合物分离分析中是否依赖标准品分类,及其定性检测方法进行了总结。利用CE分离分析手性样品,若待测物有手性对映体标准品时,其定性通常通过比较标准品迁移时间或标准加入法完成。基于检测器的不同,依赖标准品的CE分析主要分为光学、质谱和电化学3类检测模式。其中光学检测又包含紫外-可见(UV)、激光诱导荧光(LIF)、化学发光(CL)等多种检测方式。不同的检测方式决定了样品前处理方式的差异,随之形成的谱图及手性化合物定性方式也大相径庭。当缺少手性对映体标准品时,可用的CE手性分离分析方法主要有酶消解和抗体添加法、计算法等。前两种方法主要依据对映体与特定消解酶或抗体间的相互作用,完成手性物质的选择定性,适用范围均较局限。相比较,借助理论计算,使CE结合圆二色光谱(CD)的计算法操作简单,定性准...     

高效毛细管电泳法测定二氢叶酸还原酶活力的研究

通过胶束电动毛细管电泳法研究分离二氢叶酸还原酶体系中二氢叶酸、四氢叶酸、 NADP、 NADPH和酶5种组分,在含0.002%Brij-35的pH 9.18 50 mmol/L 的硼砂缓冲溶液中,5种组分在18min内得到基线分离.通过对其产物四氢叶酸峰面积的定量测定,计算出二氢叶酸还原酶的米氏常数,建立了毛细管电泳法对二氢叶酸还原酶活力的测定方法.     

DNA capillary electrophoresis using poly(vinyl alcohol). I. Inner capillary coating

Two new methods of inner capillary coating with poly(vinyl alcohol) (PVAL) have been investigated and evaluated by performing DNA capillary electrophoresis (CE) using PVAL as a separation medium and by measuring the electroosmotic flow (EOF) mobility. The treatment of capillaries with a silanol-group modified PVAL (PVAL-Si) has been found to give good coating effects for improving the resolution of DNA CE and for reducing the EOF. This coating must be effectively achieved by combining the adsorptive property of PVAL chains onto silica with the reaction between the silanol groups of PVAL-Si and the silica surface. The adsorption of PVAL onto silica has been observed by using atomic force microscopy (AFM) for PVAL-Si as well as for a nonmodified PVAL as a control. The coating with PVAL that links to the capillary wall surface with more hydrolytically stable bonding, -Si-C-, has been formed by performing the Grignard reaction, followed by in-capillary polymerization of vinyl acetate (VAc) and hydrolysis. This coating has been found to be effective for improving the resolution of DNA CE and for reducing the EOF.     

High-throughput gender determination using automated denaturant gel capillary electrophoresis

Sex determination of anonymous samples is a requirement before analysis of DNA variation on X or Y chromosomes. Based on this, we designed a method for screening samples on different DNA capillary sequencing instruments with a sensitivity that is able to quantify sex chromosome abnormalities. The two different amelogenin alleles sited on the X and Y chromosomes were polymerase chain reaction amplified with the same set of primers and separated by denaturant capillary electrophoresis (DCE). Sex chromosome ratios could be reproducibly determined with a relative standard deviation of 8.7%, which is sufficient to distinguish a normal XY karyotype from an XYY karyotype associated with Klinefelter syndrome. Reconstruction experiments demonstrated sensitivity down to a simulated Y:X allelic ratio of 1:127 in all three instruments, enabling the prediction of sex chromosomal aneuploidies. When tested on anonymous pooled and single samples, DCE gave a good prediction of the male to female ratio in pools of 1000 blood donors. In conclusion, DCE is a simple and robust method for sex determination that can be readily performed on commercially available CE systems.     

Separation and determination of enkephalin-related peptides using capillary electrophoresis

CZE with UV-absorption detection has been used for the separation and determination of enkephalin-related peptides. The experimental conditions, such as pH and concentration of running buffer, applied voltage, injection method, and time, were investigated in detail. Excellent separation efficiency could be obtained for ten enkephalin-related peptides with a 50 microm (ID) x 58 cm capillary using sodium dihydrogen phosphate as the running buffer (pH 3.11) when 20 kV of applied voltage was used. The concentration detection limits were found to be in the range of 0.31-1.94 microg/mL (defined as S/N = 3). The proposed method has been applied to analyze the spiked cerebrospinal fluid (CSF) sample, and the results showed that CZE is a powerful technique for separation and detection of the above biological peptides.     

Simultaneous determination of paraquat and diquat in serum using capillary electrophoresis.

The use of capillary electrophoresis for simultaneous separation and detection of the two bipyridylium herbicides, paraquat and diquat, was investigated. Both herbicides were extracted from fortified sera with disposable ODS-silica cartridges. Separation was carried out using a capillary tube (50 microns i.d., 750 mm) of fused silica containing 10 mM glycine-HCl buffer (pH 3.0), 40 mM NaCl and 20% methanol as the carrier. Paraquat and diquat were completely separated in 10 min at an applied potential of 20 kV. On-column UV monitoring allowed detection of both herbicides simultaneously. The assay sensitivity was 0.05 micrograms/mL (signal-to-noise ratio, 2:1), which probably increases with increase in the sample volume of serum. Analytical recovery of both herbicides added to serum was about 97% at concentrations of 0.5-2.0 micrograms/mL.     

高效毛细管电泳同时分离测定栀子苷、芍药苷及丹皮酚的研究

建立了同时分离测定栀子苷、芍药苷和丹皮酚的高效毛细管电泳新方法.以十六烷基三甲基溴化铵为表面活性剂形成胶束、甲醇作为有机改性剂、 NaOH溶液为背景电解质,在234 nm 波长下紫外检测.对十六烷基三甲基溴化铵、 NaOH的浓度以及有机改性剂、分离电压等因素对分离的影响做了系统的研究, 确立了栀子苷、芍药苷和丹皮酚的最佳分离条件.栀子苷、芍药苷、丹皮酚分别在20～100 μg/mL,5～80 μg/mL,2～20 μg/mL范围内呈较好的线性关系,回收率分别为99.00%～100.27%,99.49%～101.83%,97.11%～99.06%.方法已用于加味逍遥丸中3种成分的检测.     

A new approach to dynamic deactivation in capillary zone electrophoresis

This paper describes a simple means of obtaining high resolution separations of basic proteins at a pH below their pl. Small amounts of a cationic fluorosurfactant are added to the running buffer. A positively charged wall is thereby obtained which will repel positively charged proteins. The particular chemistry of fluorosurfactants is believed to enhance the efficiency of the deactivation. Examples are presented of the separation of some model proteins, including a human insulin-like growth factor (IGF-I), and a misfolded by-product of the growth factor.     

End-column amperometric detection of melatonin by high performance capillary electrophoresis

A rapid and sensitive method is described for the detection of melatonin in rat pineal gland and melatonin tablets. Capillary electrophoresis (CE) with electrochemical detection (EC) was used. This method had high sensitivity and good reproducibility. The detection limit of melatonin was as low as 1.3x10(-9) mol/l (0.30 fg). There was a linear range of concentration from 3.9x10(-7) to 3.25x10(-5) mol/l with a correlation coefficient of 0.999. The linear equation was Y=8.27+0.0042X with a slope of 0.0042 pA/nM. The relative standard deviations of the peak current response and the migration time for 16 continuous injections of 6.5 muM melatonin were 4.5 and 0.48%, respectively. The proposed method was applied to detect melatonin in the rat pineal gland and the melatonin tablets. Good results were obtained compared with previous reports.     

Application of high performance capillary electrophoresis on toxic alkaloids analysis

We employed CE to identify mixtures of the toxic alkaloids lappaconitine, bullatine A, atropine sulfate, atropine methobromide, scopolamine hydrobromide, anisodamine hydrobromide, brucine, strychnine, quinine sulfate, and chloroquine in human blood and urine, using procaine hydrochloride as an internal standard. The separation employed a fused-silica capillary of 75 microm id x 60 cm length (effective length: 50.2 cm) and a buffer containing 100 mM phosphate and 5% ACN (pH 4.0). The sample was injected in a pressure mode and the separation was performed at a voltage of 16 kV and a temperature of 25 degrees C. The compounds were detected by UV absorbance at wavelengths of 195 and 235 nm. All the ten alkaloids were separated within 16 min. The method was validated with regard to precision (RSD), accuracy, sensitivity, linear range, LOD, and LOQ. In blood and urine samples, the detection limits were 5-40 ng/mL and linear calibration curves were obtained over the range of 0.02-10 microg/mL. The precision of intra- and interday measurements was less than 15%. Electrophoretic peaks could be identified either by the relative migration time or by their UV spectrum.     

高效毛细管电泳法拆分芳香醇类化合物的研究

以磺化的环糊精为手性选择剂,采用毛细管电泳法对10种结构相近的芳香醇类化合物进行了手性拆分的研究.分离过程通过11次实验分别建立了10种手性醇对映体分离度的目标函数,考察了3个主要因素（溶液的pH、分离电压和手性选择剂浓度）对分离效率的影响.从分离度响应曲面图可得到最佳的分离条件,在选定的优化条件下,10种手性芳香醇在10 min内均得到了满意的拆分效果.     

In vitro evolution of functional DNA using capillary electrophoresis

Electrophoretic selection with capillary electrophoresis (CE) is used, for the first time, to isolate functional nucleic acid sequences using SELEX (systematic evolution of ligands by exponential enrichment). SELEX uses molecular evolution to select functional sequences (aptamers) from random RNA or DNA libraries. Conventional SELEX is usually performed with affinity chromatography, which may introduce significant bias into the selection step. Important biases include the slow kinetics involved in the elution of strongly bound sequences and performing the selection with the target molecule tethered to the stationary support, not in free solution. In this novel CE-SELEX approach, selection occurs in free solution. The nucleic acid sequences that bind the target undergo a mobility shift, migrating at a different rate, allowing them to be separated from the inactive sequences. Thus, there is no need to wash the active sequences off a column as in conventional SELEX, eliminating any kinetic bias. In this work, the viability of CE-SELEX was demonstrated by performing selections against immunoglobulin E (IgE). Anti-IgE aptamers with dissociation constants as low as 40 nM were obtained in only two rounds of selection.     

A new concept for sequencing DNA by capillary electrophoresis.

It is proposed that the scaling symmetry of constant charge density with increasing molecular weight, which prevents the separation by electrophoresis of DNA molecules in solution (with respect to molecular weight) be broken by the attachment of a perturbing entity (protein, virus or charged sphere) to one end of the molecule. An application of this idea to a concept for sequencing DNA by capillary electrophoresis is discussed, and the possibility of using the reattachment of the RecA protein to separate large segments of DNA in solution by electrophoresis following sequence-specific cleavage is mentioned.     

Enantiomeric purity determination of propranolol by capillary electrophoresis using dual cyclodextrins and a polyacrylamide-coated capillary.

The use of a chirally selective capillary electrophoresis method is reported for the enantioselective purity determination of propranolol drug substance. The method employed a combination of both charged and neutral cyclodextrin. An internally coated capillary was used to suppress electroosmotic flow and potential peak tailing. The method was capable of monitoring below 0.1% m/m of the undesired impurity. Acceptable validation data was also obtained for recovery, linearity, and for both short and long-term injection precision.