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1.
We have developed an affinity capillary electrophoresis (ACE) method for detection of gene point mutations using a DNA-polyacrylamide conjugate as a pseudostationary affinity phase. In this study, the target DNA was prepared by mixing two PCR products: the wild type of K-ras gene and its codon 12 point mutant. The ligand DNA was designed to be complementary to codons 11 and 12 of the wild type. The target DNA was denatured by the addition of formamide and by heating at 95 degrees C for 5 min, and then electrophoretically separated by difference in affinity to the pseudoimmobilized ligand DNA. The method successfully separated a mixture of the wild-type DNA and each of six codon 12 point mutants by the same ligand DNA. The limit of mutation detection was determined by mixing the wild-type DNA with decreasing concentrations of the mutant DNA. The lowest level of detection was 10% mutant DNA in a background of the wild type. The practicability of this method has been confirmed using a colorectal carcinoma cell line. This study is the first demonstration of detection of gene point mutation in polymerase chain reaction (PCR) products using ACE, and opens up a new possibility of CE-based gene diagnosis. 相似文献
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通过对PCR扩增的76例结直肠癌组织及癌旁正常组织DNA基因组共152个样本纯化变性后,采用毛细管电泳-激光诱导荧光检测(CE-LIF)结合单链构象多态性(SSCP)分析方法检测了人结直肠癌组织及癌旁正常组织中K-ras基因第12/13位密码子突变。所检测的76例结直肠癌患者中有30例患者存在基因突变,并对异常片段进行测序验证,测序证实以碱基G→A点突变为主。结果表明所建立的CE-LIF技术结合SSCP分析检测K-ras基因突变的方法高效、快速、灵敏、准确,适合于临床上大样本结直肠癌中K-ras基因突变分析,对选择抗结直肠癌药物有一定的指导作用。 相似文献
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Analyses of germline and somatic single-nucleotide DNA variations are important in both population genetics research and clinical practice. Reliable and inexpensive methods that are flexible and designed for automation are required for these analyses. Present day DNA sequencing technology is too expensive for testing all 22-25 000 human genes in populations genetics studies or in scanning large numbers of tumors for novel mutations. Denaturant capillary electrophoresis (DCE) has the potential to meet the need for large-scale analysis of DNA variants. Several different analyses can be performed by DCE, including mutation analysis, single-nucleotide polymorphism (SNP) discovery in individual and pooled samples, detection of allelic imbalance, and determination of microhaplotypes. Here we review the theoretical background of the method, its sensitivity, specificity, detection limit, throughput, and repeatability in the light of current literature in the field. 相似文献
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Denaturing CE (DCE) is a powerful tool for analysis of DNA variation. The development of commercial multi-CE instruments allows large-scale studies of DNA variation (many samples and many fragments). However, the cost of consumables like capillary arrays and sieving matrix might limit the use of DCE in such studies. Thus, we have tested 72 different in-house formulated sieving matrices' ability to suppress EOF and separate PCR-amplified alleles with the DCE variant, cycling temperature CE (CTCE). The data herein demonstrate that alleles can be baseline-separated by use of PVP and poly(N,N-dimethyl acrylamide) polymers at various percentages and pH. Allele separation by CTCE is matrix-independent and consequently applicable to any capillary instrument used for DNA separation. Formulation of sieving matrix for CTCE was done by dissolving appropriate amount of polymer powder into the running buffers. Allele separation was observed at different pH (7.5-8.5), concentrations and molecular size of the polymer, without compromising the separation and reproducibility. Finally, the cost reduction of homemade matrices is more than 1000-fold as compared to commercial sieving matrices. 相似文献
5.
Scanning the beta-globin gene for mutations in large populations by denaturing capillary and gel electrophoresis 总被引:1,自引:0,他引:1
Separation of mutant from nonmutant DNA sequences of 100 bp may be accomplished by using defined denaturing conditions of chemical denaturant and/or elevated temperature during electrophoresis on either polyacrylamide slab gels (denaturing gradient gel electrophoresis, DGGE) or capillary gels (constant denaturant capillary electrophoresis, CDCE). In analysis of mutant directly from a polymerase chain reaction (PCR) product mixture, both have detection sensitivities of approximately 1%. CDCE that facilitates an intermediate mutant enrichment step permits detection of mutants at fractions as low as 2 x 10(-6). Here we report the successful application of both approaches to scan for mutations of the human beta-globin gene (HBB) in two human population samples of approximately 5000 persons in the HBB. Using DGGE, the coding region and flanking intronic splice sites of HBB were scanned in a population of 4949 Han Chinese individuals in pool sizes of 48 individual DNA samples. Four point mutations ranging in mutant frequency from 0.5 to 0.0002 were identified. Using CDCE with a mutant enrichment step, these same sequences were scanned in a population of 5028, predominantly African-American juveniles (<9 years) as a single pooled DNA sample. Three point mutations were identified ranging in mutant frequency from 0.13 to 0.0005. This study shows that both the DGGE/small pool and the CDCE/large pool approaches offer the means to define the fine structure map of genetic variation in large population samples, and with appropriately engineered facilities to provide high throughput, should be useful in pangenomic scans to discover genes carrying casual mutations for common diseases. 相似文献
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高效毛细管电泳分离及检测蛋氨酸对映体 总被引:1,自引:0,他引:1
蛋氨酸(Methionine)学名甲硫基丁氨酸,分子式C5H11NO2S,相对分子质量149.2。在蛋白质合成中,蛋氨酸是信息核糖核酸“翻译”成蛋白质过程中的第一步。无蛋氨酸的存在,蛋白质生物的合成就无法开始。适用于防治肝脏疾病和砷中毒,也用作食品的添加剂。蛋氨酸有D,L两种构型。目前尚未见用Cu^2+做手性选择剂毛细管电泳电化学拆分D,L-蛋氨酸的报道。电化学检测(主要是安培检测和电导检测)具有操作简单、维护方便、无须衍生化处理等优点, 相似文献
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平衡混合物的非平衡毛细管电泳(NECEEM)可用于测定复合物的解离常数(K_d)。该文以单链脱氧核糖核苷酸结合蛋白(SSB)和单链脱氧核糖核苷酸(ssDNA)相互作用为模型,分析了相互作用强弱的电泳图谱特征,测定了不同条件下复合物的K_d,建立了基于积分偏差的误差分析方法。结果表明,SSB与ssDNA相互作用强弱所致的电泳图差异、SSB与ssDNA的浓度比、毛细管分离温度均影响K_d的计算。此外,基于25℃时SSB与5′-GGTTGGTGTGGTTGG-3′(15mer)人凝血酶适配体作用模型的数值分析结果表明,在现有实验条件下,手动积分峰面积时产生的面积偏差对K_d计算结果有影响,但在10%的偏差区间内,所求K_d值的最大相对偏差小于7%,所求K_d值的误差可忽略。该文证实了基于NECEEM求解动力学相关参数的方法可靠。 相似文献
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非水介质毛细管电泳电导检测罗红霉素及其制剂 总被引:5,自引:0,他引:5
采用甲醇为分离介质,三(羟甲基)氨基甲烷-硼酸(Tris—H3BO3)为支持电解质,采用负高压,使用电导检测,对罗红霉素及其制剂进行了毛细管电泳分离检测,对电泳介质的种类、浓度、表观pH、以及操作电压和进样时问对分离的影响进行了研讨,在选定的条件下,罗红霉素的线性范围为19.0—142.0mg/L,检出限为0.8mg/L(S/N≥3),峰面积的相对标准偏差RSD(n=6)为4.3%。3种供试品中罗红霉素的平均加标回收率分别为97.7%、94.8%、93.6%。 相似文献
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We assessed the feasibility of high-speed DNA sequencing by tube-based capillary electrophoresis (TCE) with electrokinetic sample injections. We developed a water-circulated TCE system to control the capillary temperature precisely. Using this system and a ready-made sieving matrix at 50 degrees C, single-stranded DNA size marker fragments were separated at various pairs of the electric field strength, E, of 128-480 V/cm and the capillary effective length, L, of 100-360 mm. Assuming the read length (RL) is the fragment size at which the peak width equals the peak interval per base in obtained electropherograms, we estimated the values of RL (E, L), the RL at the pair (E, L). The points in ELz-space, (E, L, RL(E, L)), form a curved surface expressed by z = RL(E, L). Analyzing the contour lines of this curved surface, we determined the pairs of E and L providing target RLs of 300-500 bases within a minimum time. At a pair optimized for a 500-base RL (330 V/cm, 200 mm), one-color sequencing fragments were successfully separated up to 529 bases within 9.6 min. These results demonstrate that high-speed DNA sequencing comparable with that obtained by microfabricated chip-based capillary electrophoresis (MCE) can be achieved with TCE, which is more suitable in automation than MCE. 相似文献
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The feasibility of using the affinity CE methodologies pre-equilibrium CZE and CE frontal analysis was tested on interaction systems exhibiting rapid on-and-off kinetics. Experimentally, the methodologies differ only with respect to the volume of sample introduced into the capillary. Pre-equilibrium CZE has been considered amendable to interactions with slow on-and-off kinetics only; however, it has recently been applied in studies of interactions with fast on-and-off kinetics. The effect of varying the sample volume introduced hydrodynamically into the capillary on the apparent degree of complexation was studied. For two different binding systems, the fraction of free analyte was found to be overestimated using pre-equilibrium CZE as compared to volumes providing plateau peak conditions as used with frontal analysis. Results indicate that frontal analysis conditions lead to more robust binding assays and thus more reliable data. The validity of data obtained by pre-equilibrium CZE may be low, thus the use of an experimental setup providing plateau peaks is highly recommended. It is suggested that the effect of altering the sample volume on the degree of binding should be investigated as part of method development and validation. 相似文献
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Summary In isothermal CE the migration velocity of analytes and the number of theoretical plates delivered are expected to be proportional to the field strength. In reality ohmic heating of the capillary causes distortions: the migration velocity increases more rapidly while the plate count increases less rapidly, and may even fall at high values of the field. These distortions are worse the larger the bore of the capillary and the higher the concentration of buffer. A detailed investigation of these effects using capillaries cooled by natural convection has confirmed that self heating of the capillary is indeed largely responsible. The extent of self heating has been determined by three independent methods and to a first approximation is proportional to the power dissipation in the capillary. Decreasing viscosity with temperature is responsible for the nonlinearity of the dependence of velocity upon field strength while increase in the diffusion coefficient of analytes is responsible for the poorer than expected performance at high field strengths. 相似文献
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《Journal of separation science》2018,41(13):2819-2827
Malignant transformation in gliomas is frequently supplemented by somatic mutations in isocitrate dehydrogenase 1 and isocitrate dehydrogenase 2 genes. It has recently emerged that mutations in these genes are associated with prolonged survival and should be used as prognostic factor in management of brain cancer patients. There are several approaches in use for the detection of isocitrate dehydrogenase 1 and 2 mutations; however, these often exhibit shortcomings such as convoluted protocols with long processing time, complex (and costly) dedicated fluorescent probes, and/or demand on amounts of input DNA. Therefore, a simple and rapid method would be highly desired. Here, we present development and validation of simple and reliable isocitrate dehydrogenase 1 and 2 mutation detection assay using denaturing capillary electrophoresis. The detection sensitivity in terms of the limiting mutated allele fraction detectable estimated from a series of dilution runs was 2.9%. The method was validated by comparing to results obtained by a widely accepted detection technique, the multiplex ligation‐dependent probe amplification, on a set of 85 brain tumors. The concordance of both methods was 100%, but denaturing capillary electrophoresis assay required fivefold lower input of DNA (1 versus 5 μL of DNA at concentrations typically between 10 and 30 ng/μL). 相似文献
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An estimation method for determination of binding constants of receptors to ligands by affinity capillary electrophoresis
was evaluated. On the basis of the theories of pseudostationary phase or so-called dynamic stationary phase, the retention
factor (k) was used to represent the interaction between the receptor and ligand. k could be easily deduced from the migration times of the ligand and the receptor. Then, with the linear relationship of k versus the concentration of ligand in the running buffer, the binding constant K
b was calculated from the slope and intercept. In order to test its feasibility, the calculation method was demonstrated using
three model systems: the interactions between vancomycin and N-acetyl-d-Ala-d-Ala, ristocetin and N-acetyl-d-Ala-d-Ala, and carbonic anhydrase B and an arylsulfonamide. Estimated binding constants were compared with those determined by
other techniques. The results showed that this estimation method was reliable. This calculation method offers a simple and
easy approach to estimating binding constants of ligands to receptors. 相似文献
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Enzymes immobilized on the inner surface of an electrophoretic capillary were used to increase sensitivity and resolution in capillary zone electrophoresis (CZE). Sensitivity is enhanced by inserting a piece of capillary containing the immobilized enzyme into the main capillary, located before the detector, in order to transform the analyte into a product with a higher absorptivity. This approach was used to determine ethanol. In order to improve resolution, capillary pieces containing immobilized enzymes were inserted at various strategic positions along the electrophoretic capillary. On reaching the enzyme, the analyte was converted into a product with a high electrophoretic mobility, the migration time for which was a function of the position of the enzyme reactor. This approach was applied to the separation and determination of acetaldehyde and pyruvate. Finally, the proposed method was validated with the determination of ethanol, acetaldehyde, and pyruvate in beer and wine samples. 相似文献
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Pressure-assisted CE (PACE) was applied to determine the previously inaccessible complete set of pK values for folic acid and eight related multiprotic compounds. PACE allowed the determination of all acidity macroconstants at low (相似文献
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