Unequal crossover recombination - population screening for PHOX2B gene polyalanine polymorphism using CE

Congenital central hypoventilation syndrome (CCHS) is a rare neurological disorder characterized by abnormal autonomic central nervous system control of breathing during sleep. Mutations in the paired-like homeobox 2B (PHOX2B) gene, including point mutation, frameshift, and polyalanine expansion, are associated with the pathogenesis of CCHS. In this study, PHOX2B mutations were analyzed in seven CCHS patients, their family members, and 1520 healthy individuals from the general population using CE to provide high sensitivity and resolution screening for the PHOX2B polyalanine polymorphism. Seven mutations in the PHOX2B gene, including two frameshift mutations and five polyalanine expansions in the 20-residue polyalanine tract, were identified. The various phenotypes observed in CCHS patients with PHOX2B mutations suggest that the size of the expansion allele is associated with the CCHS risk. In addition, significant differences were found in allele and genotype distributions between the healthy individuals. Alleles (GCN)(20) and (GCN)(15) had the highest population incidence rates of 94.84 and 4.51%, respectively, with the remaining alleles, (GCN)(13) and (GCN)(7), accounting for 0.59 and 0.06%, respectively. Therefore, it has been demonstrated that CE can be used to improve the detection of polyalanine expansions in the PHOX2B gene. The attractive alternative method is a promising tool for the detection of disorders involving trinucleotide repeat tracts.     

On-column detection of multiphoton-excited fluorescence in CE using hyphenated cylindrical-square capillaries

Multiphoton-excited fluorescence (MPEF) is a complementary and useful mode of LIF detection in CE with advantages of ultra-low mass detectability and spectral excitability, but it is currently quite limited by its end-column configuration. In this article, we demonstrate a novel strategy of on-column schemes that can greatly facilitate MPEF detection in CE. FITC-labeled amine species were used as the model samples for the evaluation and comparison of those detection scenarios. By using the square capillary instead of the conventional cylindrical one, the on-column MPEF could be readily achieved, with detection sensitivity of 0.72 microM that was comparable with the end-column mode. However, this strategy unfavorably reduced separation efficiency. The theoretical plate number on averaging all the sample peaks was significantly decreased from 283,000 to 19,000/m. To minimize such an influence, a short square capillary acting as an on-column MPEF detection cell was then mounted to a long cylindrical capillary responsible for the CE separation. Results indicated that both high separation efficiency (240,000/m) and better detectability (0.42 microM) were realized simultaneously by using this binary-capillary configuration. Quantitative analysis was performed under the optimized detector configuration and revealed a linear dynamic range of 2 orders of magnitude, with mass detection limit down to the mid-yottomole level.     

Cell cycle-dependent characterization of single MCF-7 breast cancer cells by 2-D CE

The composition of single MCF-7 breast cancer cells is characterized using 2-D CE. Individual MCF-7 cells were aspirated into a 30 mum inner diameter fused-silica capillary and lysed by contact with an SDS-containing buffer. Proteins and other primary amines were fluorescently labeled on-column using the fluorogenic dye 3-(2-furoyl)quinoline-2-carboxaldehyde. Labeled components were separated first according to molecular weight using capillary sieving electrophoresis (CSE) and then by MEKC. Analytes were detected in a sheath-flow cuvette using LIF. The expression profiles for MCF-7 cellular homogenate and a single MCF-7 cell are compared. As a proof-of-principle investigation, variation in expression was also compared within and between G1 and G2/M cell cycle phases for MCF-7 cells. Following their treatment with the viable nuclear stain Hoechst 33342, MCF-7 cells were sorted by flow cytometry on the basis of their ploidy. Sorted cells were then analyzed by 2-D CE. The degree of variability was >2.5 times larger between cells of different phases than between cells of the same phase. In typical 1 h 2-D CE separations using MCF-7 cells, over 100 components are resolved.     

Determination of micro‐RNA in cardiomyoblast cells using CE with LIF detection

Micro‐RNAs (miRNAs) are small, endogenous, singlestranded, and noncoding RNAs. The miRNAs have been found to perform important functions in many cellular processes, such as development, proliferation, differentiation, and apoptosis. Circulating miRNAs have been proposed as emerging biomarkers in diseases such as cancer, diabetes, and cardiovascular disease including acute myocardial infarction (AMI). In this study, we developed CE with LIF (CE‐LIF) using fluorescence‐labeled DNA probe for determination of low abundance miRNA in cell extracts. The target miRNA is miRNA‐499, a biomarker candidate of AMI with low abundance in biological samples. In order to measure the trace level of miRNA, we optimized the hybridization conditions such as hybridization time, temperature, and buffer solution. The highest fluorescence intensity of the hybridized miRNA‐499 was found when hybridization was conducted at 40°C in 50 mM Tris‐acetate (pH 8.0) buffer containing 50 mM NaCl, and 10 mM EDTA for 15 min. The hybridized miRNA‐499 was detected in cultured H9c2 cardiomyoblast cells and the analysis of miRNA‐499 was completed within 1 h using CE‐LIF. These results showed the potential of CE for fast, specific, and sensitive high‐throughput analysis of low‐abundance miRNAs in cell extracts, biofluids, and tissues.     

Genotyping of single nucleotide polymorphism in MDM2 genes by universal fluorescence primer PCR and capillary electrophoresis

Single nucleotide polymorphism (SNP) 309 in the promoter region of the murine double minute 2 (MDM2) gene plays an important role in human tumorigenesis. We established a simple and effective CE method for SNP detection in the MDM2 gene. We designed one universal fluorescence-based nonhuman-sequence primer and one fragment-oriented primer, which were combined in one tube, and proceeded with the polymerase chain reaction (PCR). The amplicons were analyzed by capillary electrophoresis using single-strand conformation polymorphism method. PCR fragments generated from this two-in-one PCR displayed either T/T or G/G homozygosity or T/G heterozygosity. A total of 304 samples were blindly genotyped using this developed method, which included the DNA from 138 healthy volunteers, 43 chronic myeloid leukemia (CML) patients, and 123 colorectal cancer (CRC) patients. The results were confirmed by DNA sequencing and showed good agreement. The SSCP-CE method was feasible for SNP screening of MDM2 in large populations.     

Structural factors determining DNA length limitations in conformation-sensitive mutation detection methods

Numerous mutations and polymorphisms in human genes remain to be identified using reliable methods. Of the available mutation scanning methods those dependent on structural change-induced mobility shifts are highly effective. Their efficiency is, however, DNA length-sensitive and the reasons for that are poorly understood. In this study, we explain why scanning genes for mutations is less effective in longer DNA fragments, and reveal the factors which are behind this effect. We have performed a systematic analysis of the same sequence variants of exon 11 of the BRCA1 gene in DNA fragments of three different lengths using the combined single-strand conformation polymorphism (SSCP) and heteroduplex analysis (DA) by capillary electrophoresis (CE). There are two major structural factors responsible for the reduced mutation detection rate in long amplicons. The first is increased contribution from other secondary structure modules and domains in longer fragments, which mask the structural change induced by the mutation. The second is higher frequency of single-nucleotide polymorphisms (SNPs) including common polymorphisms in longer fragments. This makes it necessary to distinguish the structural effect of the mutation from that of each polymorphic variant, which is often difficult to achieve. Taking these factors into account, an efficient scanning of genes for sequence variants by conformation-sensitive methods may be performed.     

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.     

A multiplex single nucleotide polymorphism genotyping method using ligase‐based mismatch discrimination and CE‐SSCP

Accuracy, simplicity, and cost‐effectiveness are the most important criteria for a genotyping method for SNPs compatible with clinical use. One method developed for SNP genotyping, ligase‐based discrimination, is considered the simplest for clinical diagnosis. However, multiplex assays using this method are limited by the detection method. Although CE has been introduced as an alternative to error prone microarray‐based detection, the design process and multiplex assay procedure are complicated because of the DNA size‐dependent separation principle. In this study, we developed a simple and accurate multiplex genotyping method using reaction condition‐optimized ligation and high‐resolution CE‐based SSCP. With this high‐resolution CE‐SSCP system, we are able to use similar‐sized probes, thereby eliminating the complex probe design step and simplifying the optimization process. We found that this method could accurately discriminate single‐base mismatches in SNPs of the tp53 gene, used as targets for multiplex detection.     

Identification and relative quantification of adenosine to inosine editing in serotonin 2c receptor mRNA by CE

A new method has been developed allowing the identification and relative quantification of different forms of mRNA after RNA editing. This method was applied to the serotonin 2c receptor mRNA that potentially exhibits 32 different forms after adenosine to inosine editing at five different sites located in a row of 13 nucleotides. CE was used to characterize fluorescently labeled ssDNA molecules on the basis of their conformational polymorphism. The relative amount of these 32 mRNA forms has been estimated by measuring the fluorescence intensity of each individual DNA strand. Accuracy of quantification was established by diluting one form into another or into a mixture of cDNA, showing linear and precise proportion of each form (0.06 相似文献   

Analysis of dideoxyadenosine triphosphate by CE with fluorescence detection. I. Derivatization through the phosphate group

A CZE method was developed, which separates 2',3'-dideoxyadenosine-5'-triphosphate (ddATP) from other metabolites and endogenous nucleotides at high concentrations (20-200 microg/mL) to allow UV detection. To enhance sensitivity, fluorescence detection which requires prior derivatization of compounds was examined. Precapillary derivatization of ddATP in the presence of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDAC) with dansyl ethylenediamine (dansyl EDA) was faster and stable compared to that of 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylenediamine (BODIPY FL EDA). Reaction conditions, reagent concentrations and detection parameters were optimized and highest derivatization efficiency was achieved in 0.1 M 1-methylimidazole buffer (pH 8.0) with 140 mM EDAC in 1-methylimidazole buffer and 30 mM dansyl EDA in DMF for 90 min at 60 degrees C. Dansyl EDA derivatives of ddATP, 2'-deoxyadenosine-5'-triphosphate (dATP) and ATP were comigrating with the CZE method; therefore, a MEKC method was developed and optimized for repeatable separations. Upon dansylation, sensitivity of ddATP with fluorescence detection (LOQ = 12 ng/mL) was 160 times higher than UV detection (LOQ = 1.9 microg/mL).     

Use of multiplex PCR and CE for gene dosage quantification and its biomedical applications for SMN, PMP22, and alpha-globin genes

Many genetic diseases are caused by the presence of point mutations, small insertions, and deletions in respective genes, and the number of diseases known to be caused by deletions and duplications involving large DNA genomes is increasing. These changes lead to underexpression or overexpression of the gene, according to changes in gene dosage. The methods for the detection of point mutations, small insertions, and deletions are well established, but the detection of larger genomic deletions or duplications is more difficult. Due to the lack of efficient and technically feasible protocols for gene dosage quantification, we describe a diagnostic protocol employing a combination of available methods. The efficient and accurate gene dosage quantification platform is combined with multiplex PCR and CE, and applied to detect dosages of several genes, including SMN, PMP22, and alpha-globin genes. The reliability of this novel methodology shows that it is a relatively speedy and low-cost procedure and a significant tool for genetic diagnosis. Its sensitivity and specificity for identifying deletion and duplication genotypes approach 100%. Moreover, once we establish this powerful system, we will further apply this technique to the rapid detection of trisomy syndromes and microdeletion syndromes, including trisomy 13, Down syndrome, DiGeorge syndrome, and others.     

In silico analysis of nonsynonymous single nucleotide polymorphisms of the human adiponectin receptor 2 (ADIPOR2) gene

Polymorphisms of the ADIPOR2 gene are frequently linked to a higher risk of developing diseases including obesity, type 2 diabetes and cardiovascular diseases. Though mutations of the ADIPOR2 gene are detrimental, there is a lack of comprehensive in silico analyses of the functional and structural impacts at the protein level. Considering the involvement of ADIPOR2 in glucose uptake and fatty acid oxidation, an in silico functional analysis was conducted to explore the possible association between genetic mutations and phenotypic variations. A genomic analysis of 82 nonsynonymous SNPs in ADIPOR2 was initiated using SIFT followed by the SNAP2, nsSNPAnalyzer, PolyPhen-2, SNPs&GO, FATHMM and PROVEAN servers. A total of 10 mutations (R126W, L160Q, L195P, F201S, L235R, L235P, L256R, Y328H, E334K and Q349H) were predicted to have deleterious effects on the ADIPOR2 protein and were therefore selected for further analysis. Theoretical models of the variants were generated by comparative modeling via MODELLER 9.16. A protein structural analysis of these amino acid variants was performed using SNPeffect, I-Mutant, ConSurf, Swiss-PDB Viewer and NetSurfP to explore their solvent accessibility, molecular dynamics and energy minimization calculations. In addition, FTSite was used to predict the ligand binding sites, while NetGlycate, NetPhos2.0, UbPerd and SUMOplot were used to predict post-translational modification sites. All of the variants showed increased free energy, though F201S exhibited the highest energy increase. The root mean square deviation values of the modeled mutants strongly indicated likely pathogenicity. Remarkably, three binding sites were detected on ADIPOR2, and two mutations at positions 328 and 201 were found in the first and second binding pockets, respectively. Interestingly, no mutations were found at the post-translational modification sites. These genetic variants can provide a better understanding of the wide range of disease susceptibility associated with ADIPOR2 and aid the development of new molecular diagnostic markers for these diseases. The findings may also facilitate the development of novel therapeutic elements for associated diseases.     

Single nucleotide polymorphism analysis in the human phosphatase PTPrj gene using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry

Data derived from analysis of single nucleotide polymorphisms (SNPs) are being applied in many diverse fields, from medical studies of disease mechanisms and individual drug response, to population genetics for tracking migration and mixing of ancestral groups and also in forensic science for the identification of human remains and identification of individuals from bodily samples. All these applications have in common the need to generate data for multiple loci from large numbers of samples. Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) is a promising platform for the generation of such data and we present a simple, flexible and robust technique for SNP determination. We demonstrate these features by typing two SNPs (Q276P and R326Q) in the human phosphatase gene PTPrj, which has been implicated in the aetiology of colon, lung, breast and thyroid cancers. A nucleotide depletion primer extension assay using no commercial kits or dideoxyNTPs was used to genotype a panel of DNAs derived from thyroid cancer patients and normal volunteers. The results obtained were in perfect agreement with those generated via restriction fragment length polymorphism analysis. No significant association was noted between possession of either allelic variant and a disease state, but the technique was validated as simple, flexible and appropriate for application in this context. Furthermore, it was highly cost-effective and required minimal optimisation, rendering it ideal for this type of pilot study.     

A 2‐D‐HPLC‐CE platform coupled to ESI‐TOF‐MS to characterize the phenolic fraction in olive oil

A 2‐D‐HPLC/CE method was developed to separate and characterize more in depth the phenolic fraction of olive oil samples. The method involves the use of semi‐preparative HPLC (C18 column 250×10 mm, 5 μm) as a first dimension of separation to isolate phenolic fractions from commercial extra‐virgin olive oils and CE coupled to TOF‐MS (CE‐TOF‐MS) as a second dimension, to analyze the composition of the isolated fractions. Using this method, a large number of compounds were tentatively identified, some of them by first time, based on the information concerning high mass accuracy and the isotopic pattern provided by TOF‐MS analyzer together with the chemical knowledge and the behavior of the compounds in HPLC and CE. From these results it can be concluded that 2‐D‐HPLC‐CE‐MS provides enough resolving power to separate hundreds of compounds from highly complex samples, such as olive oil. Furthermore, in this paper, the isolated phenolic fractions have been used for two specific applications: quantification of some components of extra‐virgin olive oil samples in terms of pure fractions, and in vitro studies of its anti‐carcinogenic capacity.     

Capacitively coupled contactless conductivity detection as an alternative detection mode in CE for the analysis of kanamycin sulphate and its related substances

A method was developed to determine simultaneously kanamycin, its related substances and sulphate in kanamycin sulphate using capacitively coupled contactless conductivity detection. Kanamycin is an aminoglycoside antibiotic that lacks a strong UV-absorbing chromophore. Due to its physicochemical properties, CE in combination with capacitively coupled contactless conductivity detection was chosen. The separation method uses a BGE composed of 40 mM 2-(N-morpholino)ethanesulphonic acid monohydrate and 40 mM L-histidine, pH 6.35. A 0.6 mM N-cetyltrimethyl ammonium bromide (CTAB) solution was added as electroosmotic flow modifier in a concentration below the critical micellar concentration (CMC). Ammonium acetate 50 mg/L was used as internal standard. In total, 30 kV was applied in reverse polarity on a fused-silica capillary (65/41 cm; 75 μm id). The optimized separation was obtained in less than 6 min with good linearity (R(2)=0.9999) for kanamycin. It shows a good precision expressed as RSD on the relative peak areas equal to 0.3 and 1.1% for intra-day and inter-day precision, respectively. The LOD and LOQ are 0.7 and 2.3 mg/L, respectively. Similarly, for sulphate, a good linearity (R(2)=0.9996) and precision (RSD 0.4 and 0.6% for intra-day and inter-day, respectively) were obtained.     

Control of the EOF in CE using polyelectrolytes of different charge densities

The control of the EOF direction and magnitude remains one of the more challenging issues for the optimization of separations in CE. In this work, we investigated the possibility to use variously charged polyanions for a fine-tuning of the EOF using polyelectrolyte multilayers. For that purpose, polyanions of poly(acrylamide-co-2-acrylamido-2-methyl-1-propanesulfonate) (PAMAMPS) with different chemical charge rates varying between 3 and 100% were used. These copolymers are statistic hydrophilic copolymers of acrylamide (AM) and 2-acrylamido-2-methyl-1-propanesulfonate (AMPS). The study of the influence of the chemical charge rate (AMPS molar proportion in the copolymer) on the electroosmotic mobility (mu(eo)) of a capillary modified by a polyelectrolyte bilayer (polycation/PAMAMPS) revealed that the fine-tuning of the EOF was possible, at least for cathodic or slightly anodic EOF (micro(eo) from -5 x 10(-5) to +35 x 10(-5) cm(2)V(-1)s(-1)). Electroosmotic mobility values were compared with the free-draining electrophoretic mobilities of the PAMAMPS constituting the last layer of the capillary coating. The stability of the EOF is discussed in detail on the basis of successive determinations of electroosmotic mobility and migration times. The application to the separation of a model peptide mixture demonstrated the interest (and the simplicity) of this approach for optimizing resolution and analysis time. Experimental resolutions were compared to the theoretical ones that we would obtain on a fused-silica capillary having the same EOF as the coated capillary.     

Separation of amino alcohols using divalent dipeptides as counter ions in aqueous CE

Divalent dipeptides have been introduced as counter ions in aqueous CZE. The dipeptides form ion pairs with amino alcohols in the BGE and facilitate the separation of amino alcohols. High concentrations of dipeptide caused reversed effective mobility for the analytes. The net charge of the dipeptide can be controlled using a buffer or a strong base, and regulates the interaction between the dipeptide and the amino alcohol. A stronger interaction and higher selectivity of amino alcohols was observed when the dipeptides were used as divalent counter ions, than in monovalent or uncharged form. Association constants for ion pairs between divalent dipeptides and amino alcohols can be used to enhance selectivity for amino alcohols in CZE. No chiral separation of amino alcohols was observed when using the dipeptides as ion‐pairing chiral selectors in aqueous BGE, but addition of methanol to the BGE promoted enantioselectivity.     

Identification of inorganic ions in post‐blast explosive residues using portable CE instrumentation and capacitively coupled contactless conductivity detection

Novel CE methods have been developed on portable instrumentation adapted to accommodate a capacitively coupled contactless conductivity detector for the separation and sensitive detection of inorganic anions and cations in post‐blast explosive residues from homemade inorganic explosive devices. The methods presented combine sensitivity and speed of analysis for the wide range of inorganic ions used in this study. Separate methods were employed for the separation of anions and cations. The anion separation method utilised a low conductivity 70 mM Tris/70 mM CHES aqueous electrolyte (pH 8.6) with a 90 cm capillary coated with hexadimethrine bromide to reverse the EOF. Fifteen anions could be baseline separated in 7 min with detection limits in the range 27–240 μg/L. A selection of ten anions deemed most important in this application could be separated in 45 s on a shorter capillary (30.6 cm) using the same electrolyte. The cation separation method was performed on a 73 cm length of fused‐silica capillary using an electrolyte system composed of 10 mM histidine and 50 mM acetic acid, at pH 4.2. The addition of the complexants, 1 mM hydroxyisobutyric acid and 0.7 mM 18‐crown‐6 ether, enhanced selectivity and allowed the separation of eleven inorganic cations in under 7 min with detection limits in the range 31–240 μg/L. The developed methods were successfully field tested on post‐blast residues obtained from the controlled detonation of homemade explosive devices. Results were verified using ion chromatographic analyses of the same samples.     

Highly multiplex and sensitive SNP genotyping method using a three‐color fluorescence‐labeled ligase detection reaction coupled with conformation‐sensitive CE

For the development of clinically useful genotyping methods for SNPs, accuracy, simplicity, sensitivity, and cost‐effectiveness are the most important criteria. Among the methods currently being developed for SNP genotyping technology, the ligation‐dependent method is considered the simplest for clinical diagnosis. However, sensitivity is not guaranteed by the ligation reaction alone, and analysis of multiple targets is limited by the detection method. Although CE is an attractive alternative to error‐prone hybridization‐based detection, the multiplex assay process is complicated because of the size‐based DNA separation principle. In this study, we employed the ligase detection reaction coupled with high‐resolution CE‐SSCP to develop an accurate, sensitive, and simple multiplex genotyping method. Ligase detection reaction could amplify ligated products through recurrence of denaturation and ligation reaction, and SSCP could separate these products according to each different structure conformation without size variation. Thus, simple and sensitive SNP analysis can be performed using this method involving the use of similar‐sized probes, without complex probe design steps. We found that this method could not only accurately discriminate base mismatches but also quantitatively detect 37 SNPs of the tp53 gene, which are used as targets in multiplex analysis, using three‐color fluorescence‐labeled probes.     

CE with on‐line detection by ICP‐MS for studying the competitive binding of zinc against cadmium for glutathione

Development of a feasible method for studying the competitive interaction between a pair of antagonists is essential for understanding the antagonism of trace metals in biological systems. Herein, we report the application of CE on‐line coupled with ICP mass spectroscopy (CE‐ICP‐MS) to investigate the competitive binding of Zn²⁺ against Cd²⁺ for glutathione (GSH), which is related to the detoxification of Cd²⁺ in biological system, and introduce a method to evaluate the kinetics and thermodynamics for the competitive binding of Zn²⁺ against Cd²⁺ for GSH. The CE‐ICP‐MS hybrid technique allows easy and sensitive probing of the competitive binding of Zn²⁺ against Cd²⁺ for GSH and quantitative determination of the important thermodynamic and kinetic parameters of the competitive binding of Zn²⁺ against Cd²⁺ for GSH. Owing to the high sensitivity and element selectivity with multi‐elements detection capacity of ICP‐MS, we detailed the evaluation of the kinetics and thermodynamics describing the competition of Zn²⁺ against Cd²⁺ for GSH from the systematic data obtained by CE‐ICP‐MS. The competitive binding of Zn²⁺ against Cd²⁺ for GSH was demonstrated exothermic and thermodynamically favorable (ΔG=?7.2 kJ/mol) and driven entirely by a large favorable enthalpy decrease (ΔH=?15.1 kJ/mol) but with an unfavorable entropy decrease (ΔS=?25.6 J/mol/K). The kinetic data were fit to a second‐order equation with the reaction rate constant (k) of (2.18±0.10)×10² L/(mol·s) under the simulated physiological condition.