毛细管电泳检测肺癌基因突变的方法学研究

建立了一种毛细管电泳快速高效检测聚合酶链反应(PCR)扩增产物以及限制性内切酶酶切产物的方法,使其更好地用于基因诊断.以聚环氧乙烷(poly(ethylene oxide),PEO)为筛分介质,用涂层的毛细管柱(37 cm×75 μm,有效长度27 cm)分离pUC19 DNA/MspⅠ(HpaⅡ) Marker标准DNA片段.考察了筛分介质的质量浓度、pH值、毛细管柱的温度和运行电压.在1×TBE (pH 8.2)电泳液、电压15 kV、温度15 ℃,于10 min内成功分离了Marker标准DNA片段.该方法快速、灵敏、准确,用于临床76例肺癌患者正常组织和肿瘤组织p53基因和ras基因点突变情况的检测,结果满意.     

羟乙基纤维素中毛细管电泳DNA分离特性研究

本实验以羟乙基纤维素(HEC)为筛分介质,以100～1500 bp DNA ladder为分离对象,系统地研究了直流电场下毛细管电泳时DNA分离特性.论文考察了DNA迁移淌度及分离度随HEC溶液浓度和分子量、毛细管两端电场强度(E)、毛细管有效长度(le)及其内径形状、背景电解液(BGE)温度等因素变化规律.研究发现:(1)当筛分介质HEC浓度高于其阈值浓度c*时,HEC分子量越大,相邻DNA片段之间淌度差越大,HEC浓度越高,其迁移淌度越低;(2)对于相邻的DNA片段,le在一定范围内,其分离度随le增大而线性升高;(3)毛细管有效长度一定时,DNA淌度随毛细管侧面积与截面积之比R增大而升高,分离效率提高;(4)BGE温度升高,DNA在筛分介质中扩散效应增强,迁移淌度变大,相邻DNA片段间分离度减小.根据以上结论,在直流电场下毛细管电泳φ×174-Hirc II限制性酶切片段,并实现了其高分离度、快速分离.     

限制性内切酶指纹-高效毛细管电泳激光诱导荧光法测定原核/真核质粒

采用线性聚丙烯酰胺修饰石英毛细管的高效毛细管电泳无胶筛分技术,对原核/真核质粒用不同限制性内切酶酶切,运用限制性内切酶指纹-高效毛细管电泳激光诱导荧光(REF-HPCE-LIF)检测法同时对内切酶酶切后多个和较长DNA片段进行了检测,电泳缓冲液为1×TBE(pH8.3),阴极电压进样(10kV,5s),分离电压13kV,25℃,激光诱导荧光检测器检测(λex=520nm)。结果表明,所建立的REF-HPCE-LIF方法可对原核/真核酶切后多个和较长DNA片段进行检测,获得了满意的限制性内切酶指纹图谱,能够检测片段大小相差不超过10bp。所建立的方法较琼脂糖电泳分辨率高,可应用在检测多个和较长DNA片段的突变,在诊断肿瘤方面有一定应用前景。     

含有金纳米粒子的筛分介质对不同长度DNA片段的毛细管电泳分离

探讨了含有金纳米粒子(GNPs)的筛分介质在毛细管电泳(CE)中对不同长度DNA片段的分离.以聚环氧乙烷(PEO)-金纳米粒子(GNPs)-TBE为CE筛分介质,用涂层的毛细管柱(37 cm×75 μm,有效长度27 cm)分离DNA Marker D和1 kbp DNA Ladder Marker标准DNA片段,考察了CE过程中各参数(如筛分介质质量浓度、分离电压、温度和筛分介质pH值)对不同长度DNA片段分离的影响.对比了新型筛分介质与不含GNPs的PEO-TBE筛分介质的分离效果,并将新型筛分介质用于实际样品的检测.结果表明,在筛分介质中添加GNPs后能够改进CE的分离效果,且分离时间短.方法较适于分离较宽范围的DNA片段.     

毛细管电泳无胶筛分介质分离DNA的机理

快速、高效而灵敏的分离技术对于DNA的分析是至关重要的.使用无胶筛分介质的毛细管电泳是最重要的DNA分离技术之一,通常使用无交联的高分子溶液作为无胶筛分介质.本文在介绍高分子溶液理论的基础上,综述了DNA在毛细管电泳无胶筛分介质(缠结溶液和稀溶液)中的分离机理,主要包括Ogston筛分模型、各种修正的爬行模型、瞬态缠结偶合机理及其改进机理等.     

毛细管电泳-激光诱导荧光法分离检测DNA片段及基因扩增产物

以羟丙甲基纤维素和非交联聚丙烯酰胺浴液为筛分介质,将毛细管电泳-激光诱导荧光法用于DNA片段及基因扩增产物的分离检测。探讨了非胶筛分介质中高分子化合物的浓度、电解质的浓度、内插试剂用量等对DNA片段分离检测的影响;考察了DNA片段迁移时间和峰面积的重现性及DNA片段定量检测的关系。建立了一种快速、灵敏的DNA片段及基因扩增产物分离检测方法。     

毛细管电泳无胶筛分介质分离DNA的机理

快速、高效而灵敏的分离技术对于DNA的分析是至关重要的。使用无胶筛分介质的毛细管电泳是最重要的DNA分离技术之一,通常使用无交联的高分子溶液作为无胶筛分介质。本文在介绍高分子溶液理论的基础上,综述了DNA在毛细管电泳无胶筛分介质(缠结溶液和稀溶液)中的分离机理,主要包括Ogston筛分模型、各种修正的爬行模型、瞬态缠结偶合机理及其改进机理等。     

毛细管电泳聚乙烯吡咯烷酮与羟乙基纤维素混配无胶筛分介质分离DNA片段

本文报道了毛细管电泳聚乙烯吡咯烷酮与羟乙基纤维素混配无胶筛分介质分离较短的 p GEM7Zf(+) Hae DNA片段 (DNA长度为 1 8～ 675bp)。研究表明 ,在 1 %的羟乙基纤维素无胶筛分介质中 ,加入 2 %的聚乙烯吡咯烷酮能显著提高 DNA片段的分辨率和分离效率。在混配无胶筛分介质中 ,聚乙烯吡咯烷酮有两种作用 ,一是动态涂渍 ,降低毛细管内壁对 DNA片段与 DNA荧光插入试剂的吸附 ,改善分离效率 ;二是两种不同长度、性质的线性高分子能形成更为致密的“缠绕网络”,有利于较短的 DNA片段电泳分离。     

添加剂在毛细管电泳分离DNA中的作用研究进展

使用无胶筛分介质的毛细管电泳（包括毛细管阵列电泳和微芯片电泳）是最重要的DNA分离技术之一。在低粘度的无胶筛分介质中加入某种添加剂是一种有效且简单的克服填充困难和提高DNA分离性能的方法。本文就各种添加剂（如多羟基化合物、粘土、金纳米粒子、乳胶粒等）对提高无胶筛分介质中DNA的分离性能的作用进行了综述。     

毛细管电泳检测神经细胞凋亡

建立了毛细管电泳检测凋亡细胞DNA片段的方法.以DNA相对分子质量标准品为溶质,考察了分离条件(电压、进样时间、温度、聚合物浓度)对分离的影响.在优化条件下,利用毛细管无胶筛分电泳对缺氧缺血过程中不同时间点神经PC12细胞DNA片段进行了分析,并与流式细胞仪结果比较,研究缺氧缺血时胶质细胞凋亡过程.     

Rapid separation and laser-induced fluorescence detection of mutated DNA by capillary electrophoresis in a self-coating, low-viscosity polymer matrix

The detection of point and other simple mutations in DNA is important for cancer research and diagnosis and other biological studies. Capillary electrophoresis has been successfully used for separating DNA fragments. However, a low-viscosity polymer sieving buffer for DNA separation with on-line coating has never been reported. In this paper, a new method using capillary electrophoresis with on-line coating and laser-induced fluorescence detection (CE-LIF) for screening for point or simple DNA mutations has been demonstrated. The method uses an on-line dynamic coating technique that increases capillary lifetime and analysis reproducibility, and employs a low-viscosity polymer solution, which allows the user to rinse the capillary rapidly and refill with polymer solution easily. Experiments proved that the additives in the separation buffer for on-line capillary coating do not affect the separation efficiency of the running buffer, and do not interfere with the formation of hydrogen-bonded network between boric acid, mannitol and hydroxypropylmethylcellulose polymers. The stability of the dynamically coated capillary was quantitatively studied; the capillary lifetime was increased 6- to 7-fold compared with that of permanently coated CE columns. Standard DNA fragments containing mutations, with sizes of 209, 219, and 338 bps, were successfully separated and detected with this system, after the mutated DNA fragments were cleaved by CEL-I endonuclease. The technique is very sensitive for the size-separation of low-range, middle-range, and high-range DNA fragments. Results were compared with the HPLC methods developed by Transgenomic, Inc. and were in good agreement. The method should be applicable to mutation detection for all relevant biological and clinical studies. The factors influencing separations and the stability of dynamic capillary coatings are also discussed in the paper.     

A single-strand conformation polymorphism method by capillary electrophoresis with laser-induced fluorescence for detection of the T1151A mutation in hMLH1 gene

Mutation of hMLH1 gene plays an important role in human tumorigenesis. A highly sensitive single-strand conformation polymorphism (SSCP) method for detection of the T1151A mutation in exon 12 of the hMLH1 gene was for the first time developed employing laser-induced fluorescence capillary electrophoresis (LIF-CE). Effects of the concentration of linear polyacrylamide solution, running temperature, running voltage and the addition of glycerol on SSCP analysis were investigated, and the optimum separation conditions were defined. Thirty colorectal cancer patients and eight lung cancer patients were screened and the T1151A mutation was found in four of them. Based on CE-sequencing the mutation was further confirmed. To our knowledge, this is for the first time that the T1151A mutation is found in lung cancer. Our method is simple, rapid, and highly sensitive and is well suited to the analysis of large numbers of clinical samples.     

CE of Small DNA Fragments Using Linear Polyacrylamide Matrices

Mutations at codons 248 and 249 of p53 gene showed a relatively high incidence in gastric cancer patients. Development of novel methods for the detection of codon mutations is of great importance for gastric cancer research. Studies have showed that the separation matrix can significantly influence the separation efficiency and resolution of small DNA fragments in CE. In order to achieve baseline separation of PCR-amplified products of small DNA fragments from gastric cancer tissue, linear polyacrylamides (LPA I and LPAII) were designed and synthesized in the current study. LPAI and LPAII were used as separation matrixes to separate small size fragments (less than 70 bp) of pBR322/BsuRI DNA Marker and the separation conditions were optimized. Optimum separations were performed at 25 kV in reversed-polarity mode with capillary temperature set at 15 °C. The signal of DNA fragments was detected using laser-induced fluorescence detector, with an argon ion laser as the excitation source that emits at 488 nm. A 520 nm bandpass filter was used as an emission cut-off filter. The resolution of small DNA fragments was higher when LPAI was used as separation matrix compared to LPAII, accompanied with longer migration time. The results indicated that LPAI as separation matrix was more efficient for the separation of small DNA fragments (less than 70 bp) than other LPAs. A rapid and sensitive analysis method for the separation and detection of small DNA fragments (less than 70 bp) was established in this study. The method was successfully applied to detect the mutations at codons 248 and 249 in p53 gene from gastric cancer tissues.     

Genetic mutation analysis by CE with LIF detection using inverse-flow derivatization of DNA fragments

Inverse-flow derivatization is a novel approach to obtain fluorescent DNA derivatives in DNA analysis based on CE with LIF detection. In the present work, we want to explore the feasibility of the application of this method into the mutation detection based on constant denaturant capillary electrophoresis (CDCE) and SSCP analysis. The DNA fragments were first amplified by PCR using a pair of common primers without fluorescent label, and then the mutations were determined by CDCE or SSCP analysis based on CE-LIF with inverse-flow derivatization of DNA fragments. The experimental conditions were investigated systematically, and different labeling modes including inverse-flow derivatization, on-column derivatization and fluorescent labeled primer technique were compared. The inverse-flow derivatization was successfully used in the detection of C677T mutation in the methylenetetrahydrofolate reductase gene by CDCE or SSCP analysis. Our preliminary results demonstrate that inverse-flow derivatization is very simple, inexpensive and sensitive and well suitable for the genetic analysis in clinical diagnosis.     

BRCA1 mutation screening using restriction endonuclease fingerprinting-single-strand conformation polymorphism in an automated capillary electrophoresis system

Efficient mutation scanning techniques are needed for the rapid detection of novel disease-associated mutations and rare-sequence variants of putative importance. The large size of the breast cancer 1 gene (BRCA1) and the many mutations found throughout its entire coding sequence make screening for mutations in this gene particularly challenging. We have developed a method for screening exon 11 of the BRCA1 gene based on restriction enzyme digestion of fluorescence-labeled polymerase chain reaction (PCR) products followed by single-strand conformation polymorphism (SSCP) using an automated capillary electrophoresis system, denoted capillary restriction endonuclease fingerprinting (REF)-SSCP electrophoresis. Using this strategy on a control set of samples, we were able to detect 17 of 18 known sequence alterations. The method was then applied to screen 73 Norwegian females with family histories of breast and/or ovarian cancer. A total of 172 sequence alterations were detected, including substitutions, insertions, and deletions. One novel substitution of unknown function was identified. Sequencing of all samples negative in the capillary REF-SSCP system gave no additional mutations confirming the high sensitivity of the described methodology. Capillary REF-SSCP electrophoresis appeared as a technically convenient technique, requiring amplification of fewer PCR fragments than traditional SSCP. The novel strategy allows high-throughput mutation scanning without radioactive labeling and polyacrylamide gel electrophoresis (PAGE).     

Study on detection of mutation DNA fragment in gastric cancer by restriction endonuclease fingerprinting with capillary electrophoresis

The DNA fragment detection focusing technique has further enhanced the sensitivity and information of DNA targets. The DNA fragment detection method was established by capillary electrophoresis with laser‐induced fluorescence detection and restriction endonuclease chromatographic fingerprinting (CE‐LIF‐REF) in our experiment. The silica capillary column was coated with short linear polyarclarylamide (SLPA) using nongel sieving technology. The excision product of various restricted enzymes of DNA fragments was obtained by REF with the molecular biology software Primer Premier 5. The PBR322/BsuRI DNA marker was used to establish the optimization method. The markers were focused electrophoretically and detected by CE‐LIF. The results demonstrate that the CE‐LIF‐REF with SLPA can improve separation, sensitivity and speed of analysis. This technique may be applied to analysis of the excision product of various restricted enzymes of prokaryotic plasmid (pIRES2), eukaryote plasmid (pcDNA3.1) and the PCR product of codon 248 region of gastric cancer tissue. The results suggest that this method could very sensitively separate the excision products of various restricted enzymes at a much better resolution than the traditional agarose electrophoresis. Copyright © 2011 John Wiley & Sons, Ltd.     

Determination of global DNA methylation level by capillary electrophoresis using octyl-modified quaternized cellulose as an electrolyte additive

A new cellulose derivative, octyl-modified quaternized cellulose (OMQC), was synthesized and used as electrolyte additive for the analysis of 5-methylcytosine by capillary electrophoresis with UV detection. While added in the background electrolyte, OMQC carrying octyl groups and quaternary ammonium groups exhibited dynamic coating ability. Capillary coated with OMQC was able to generate a stable anodal electro-osmotic flow even at pH 12.0. After several running conditions were optimized, a new method for quantification of genomic methylation level was developed on the basis of hydrolysis of DNA by formic acid and separation of nucleic acid bases by capillary electrophoresis. Cytosine and 5-methylcytosine were separated with a resolution near 4.0 in less than 10 min. The detection limits (S/N?=?3) were 1.1 and 1.5 μg/mL for cytosine and 5-methylcytosine, respectively.     

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.     

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。     

A simple capillary gel electrophoresis approach for efficient and reproducible DNA separations. Analysis of genetically modified soy and maize

It is generally assumed that in order to achieve suitable separations of DNA fragments, capillary gel electrophoresis (CGE)-coated capillaries should be used. In this work, a new method is presented that allows to obtain reproducible CGE separations of DNA fragments using bare fused-silica capillaries without any previous coating step. The proposed method only requires: (i) a capillary washing with 0.1 M hydrochloric acid between injections and (ii) a running buffer composed of Tris-phosphate-ethylenediamine tetraacetic acid (EDTA) and 4.5% of 2-hydroxyethyl cellulose (HEC) as sieving polymer. The use of this new CGE procedure gives highly resolved and reproducible separations of DNA fragments ranging from 50 to 750 bp. The separation of these DNA fragments is accomplished in less than 30 min with efficiencies up to 1.7 x 10(6) plates/m. Reproducibility values of migration times (given as %RSD) for the analyzed DNA fragments are better than 1.0% (n = 4) for the same day, 2.2% (n = 16) for four different days, and 2.3% (n = 16) for four different capillaries. The usefulness of this separation method is demonstrated by detecting genetically modified maize and genetically modified soy after DNA amplification by PCR. This new CGE procedure together with LIF as detector provides sensitive analysis of 0.9% of Bt11 maize, Mon810 maize, and Roundup Ready soy in flours with S/ N up to 542. These results demonstrate the usefulness of this procedure to fulfill the European regulation on detection of genetically modified organisms in foods.