On the separation, detection and quantification of pectin derived oligosaccharides by capillary electrophoresis

Having previously reported that capillary electrophoresis can be used as a tool for the analysis of partially methyl-esterified oligogalacturonides we now describe a method that improves the resolution of individual oligomers, and detail a more rigorous quantification scheme that uses an internal standard and takes into account the relative molecular absorbance of different partially methyl-esterified species. The internal consistency of the method is subsequently demonstrated by performing the quantification of an endo-polygalacturonase pectin digest before and after de-methylation of the resultant oligomers.     

Sequence-specific amplification polymorphisms (SSAPs): a multi-locus approach for analyzing transposon insertions

Multiple copies of transposable elements, inserted at random around the host genome, can be used as molecular markers. Sequence-specific amplification polymorphisms (SSAPs) amplify the region between a PCR primer site near the end of an element and an adjacent restriction site in the flanking genomic DNA. Each amplified insertion is revealed as a band on a sequencing gel or capillary electrophoresis, and a genomic DNA sample produces a characteristic fingerprint of bands. Here, we explain the SSAP marker development method, which can be performed for any species, with recommendations for experimental parameters in several plant species. It takes about 1-2 weeks to complete the whole SSAP marker procedure.     

Molecular sexing of African rhinoceros

We report the development of a fast and reliable PCR-based method for sex identification of African Rhinoceros (Ceratotherium simum and Diceros bicornis) that could easily be incorporated into fluorescent short tandem repeat (STR) profiling. A single primer pair, consisting of a fluorescently labelled forward primer and an unlabelled reverse primer, is used to co-amplify homologous fragments of a zinc finger (ZF) protein intron which exhibits size polymorphism between the X and Y chromosomes. In both species, the amplified ZFX and ZFY amplicons differ in size by 7 bp and can thus be differentiated by capillary electrophoresis. Blood, tissue, horn, and faecal samples were correctly sexed using this method. Cross species testing also demonstrated that this method could be used to sex Indian rhinoceros (Rhinoceros unicornis) samples.     

Dispersion of protein solutions in short, open capillary tubes as a method for rapid molecular weight determination

A method has been developed by which the molecular weight of proteins and other freely diffusing species can be estimated on the basis of chromatographic peak shapes developed by injection of a sample into an open capillary tube in a liquid chromatography system. In chromatographic peaks obtained from such a system, there are contributions from both convection and diffusion. Thus, peak shape is dependent upon the diffusion coefficient of the molecular species, the flow rate, and the length of the capillary tube. In the work reported here it has been found that for samples of different proteins ranging from 2000 to 14,000 molecular weight, each injected at the same mobile phase flow rate, the ratio (R) of h1, the height of the peak primarily due to convection, to h2, the height of the "makeup" peak, primarily due to diffusion from the capillary wall, is a direct measure of protein molecular weight. Linear plots of R vs molecular weight are obtained under certain conditions.     

Determination of fatty acid compositions of Bacillus cereus and related bacteria: a rapid gas chromatographic method using a glass capillary column.

A rapid gas chromatographic method for the determination of fatty acid compositions of Bacillus cereus and related bacteria is presented. By the use of a free fatty acid phase-coated glass capillary column, the complete separation of fatty acids, including the branched ones, was achieved. The method enables a more distinct differentiation of Bacillus species than can be obtained with packed columns.     

Determination of fatty acid compositions of Bacillus cereus and related bacteria: a rapid gas chromatographic method using a glass capillary column.

A rapid gas chromatographic method for the determination of fatty acid compositions of Bacillus cereus and related bacteria is presented. By the use of a free fatty acid phase-coated glass capillary column, the complete separation of fatty acids, including the branched ones, was achieved. The method enables a more distinct differentiation of Bacillus species than can be obtained with packed columns.     

Determination of triazolam in serum by deactivated metal capillary gas chromatography with electron-capture detection

A method for the determination of trace amounts of triazolam in serum by deactivated metal capillary gas chromatography with electron-capture detection was established. The column used exhibits excellent thermostability in high-temperature analysis and easy handling and a long lifetime of the column and well shaped peaks on the chromatograms are obtained. With the metal capillary column, it was found to be easier to maintain suitable analytical conditions for the routine assay of triazolam than with a fused-silica column. With this method, 0.5 ng/ml of triazolam in serum can be determined. The method is useful for pharmacokinetic and therapeutic purposes.     

Microfabricated polymer chip for capillary gel electrophoresis

A polymer (PDMS: poly(dimethylsiloxane)) microchip for capillary gel electrophoresis that can separate different sizes of DNA molecules in a small experimental scale is presented. This microchip can be easily produced by a simple PDMS molding method against a microfabricated master without the use of elaborate bonding processes. This PDMS microchip could be used as a single use device unlike conventional microchips made of glass, quartz or silicon. The capillary channel on the chip was partially filled with agarose gel that can enhance separation resolution of different sizes of DNA molecules and can shorten the channel length required for the separation of the sample compared to capillary electrophoresis in free-flow or polymer solution format. We discuss the optimal conditions for the gel preparation that could be used in the microchannel. DNA molecules were successfully driven by an electric field and separated to form bands in the range of 100 bp to 1 kbp in a 2.0% agarose-filled microchannel with 8 mm of effective separation length.     

Simultaneous determination of 19 intracellular nucleotides and nucleotide sugars in Chinese Hamster ovary cells by capillary electrophoresis

Twelve nucleotides and seven nucleotide sugars in Chinese Hamster ovary (CHO) cells were determined by capillary electrophoresis (CE). The CE operating conditions of buffer pH value, ion strength, capillary temperature, polymer additive and cell extraction method were investigated. Optimum separation was achieved with 40 mM sodium tetraborate buffer (pH 9.5) containing 1% (w/v) polyethylene glycol (PEG) at a capillary temperature of 22 degrees C. Acetonitrile and chloroform were used for intracellular extraction. This method can be used to monitor intracellular carbohydrate metabolism.     

Separation of bovine serum albumin and its monoclonal antibody from their immunocomplexes by sodium dodecyl sulfate–capillary gel electrophoresis and its application in capillary electrophoresis-based immunoassay

A non-competitive immunoassay was performed by sodium dodecyl sulfate–capillary gel electrophoresis with UV detection using bovine serum albumin (BSA) and monoclonal anti-BSA. BSA, anti-BSA and their immunocomplexes were well resolved under non-denaturing conditions. A linear calibration curve was obtained and can be used for the quantification of anti-BSA. The limit of detection of anti-BSA was 0.1 μM under the present conditions. Compared with capillary zone electrophoresis, we believed that this method has the potential to be used as a more general format for performing capillary electrophoresis-based immunoassay of medium- and large-sized analytes.     

High Resolution Melting Analysis Is a More Sensitive and Effective Alternative to Gel-Based Platforms in Analysis of SSR - An Example in Citrus

High resolution melting curve analysis (HRM) has been used as an efficient, accurate and cost-effective tool to detect single nucleotide polymorphisms (SNPs) or insertions or deletions (INDELs). However, its efficiency, accuracy and applicability to discriminate microsatellite polymorphism have not been extensively assessed. The traditional protocols used for SSR genotyping include PCR amplification of the DNA fragment and the separation of the fragments on electrophoresis-based platform. However, post-PCR handling processes are laborious and costly. Furthermore, SNPs present in the sequences flanking repeat motif cannot be detected by polyacrylamide-gel-electrophoresis based methods. In the present study, we compared the discriminating power of HRM with the traditional electrophoresis-based methods and provided a panel of primers for HRM genotyping in Citrus. The results showed that sixteen SSR markers produced distinct polymorphic melting curves among the Citrus spp investigated through HRM analysis. Among those, 10 showed more genotypes by HRM analysis than capillary electrophoresis owing to the presence of SNPs in the amplicons. For the SSR markers without SNPs present in the flanking region, HRM also gave distinct melting curves which detected same genotypes as were shown in capillary electrophoresis (CE) analysis. Moreover, HRM analysis allowed the discrimination of most of the 15 citrus genotypes and the resulting genetic distance analysis clustered them into three main branches. In conclusion, it has been approved that HRM is not only an efficient and cost-effective alternative of electrophoresis-based method for SSR markers, but also a method to uncover more polymorphisms contributed by SNPs present in SSRs. It was therefore suggested that the panel of SSR markers could be used in a variety of applications in the citrus biodiversity and breeding programs using HRM analysis. Furthermore, we speculate that the HRM analysis can be employed to analyse SSR markers in a wide range of applications in all other species.     

Automatic Sampling and Analysis of Organics and Biomolecules by Capillary Action-Supported Contactless Atmospheric Pressure Ionization Mass Spectrometry

Contactless atmospheric pressure ionization (C-API) method has been recently developed for mass spectrometric analysis. A tapered capillary is used as both the sampling tube and spray emitter in C-API. No electric contact is required on the capillary tip during C-API mass spectrometric analysis. The simple design of the ionization method enables the automation of the C-API sampling system. In this study, we propose an automatic C-API sampling system consisting of a capillary (∼1 cm), an aluminium sample holder, and a movable XY stage for the mass spectrometric analysis of organics and biomolecules. The aluminium sample holder is controlled by the movable XY stage. The outlet of the C-API capillary is placed in front of the orifice of a mass spectrometer, whereas the sample well on the sample holder is moved underneath the capillary inlet. The sample droplet on the well can be readily infused into the C-API capillary through capillary action. When the sample solution reaches the capillary outlet, the sample spray is readily formed in the proximity of the mass spectrometer applied with a high electric field. The gas phase ions generated from the spray can be readily monitored by the mass spectrometer. We demonstrate that six samples can be analyzed in sequence within 3.5 min using this automatic C-API MS setup. Furthermore, the well containing the rinsing solvent is alternately arranged between the sample wells. Therefore, the C-API capillary could be readily flushed between runs. No carryover problems are observed during the analyses. The sample volume required for the C-API MS analysis is minimal, with less than 1 nL of the sample solution being sufficient for analysis. The feasibility of using this setup for quantitative analysis is also demonstrated.     

Ups and downs of protein crystallization: studies of protein crystals by high-performance capillary electrophoresis

High-performance capillary electrophoresis is a high-technology micro-separation method. Short run time, full automation and minute amounts of sample make it a very attractive technique. In this report we describe studies of protein crystals by capillary electrophoresis. We show how high-performance capillary electrophoresis can be used effectively for rapid evaluation and examination of the protein solution used for crystallization, the protein crystals (solubilized) and surrounding mother liquor. With coated capillaries, the runs were reproducible and disturbing effects, such as electroendosmosis and interaction of the proteins with the capillary wall, were suppressed efficiently. We recommend this new technique as a powerful and routine companion to protein crystallography.     

Ultra-rapid procedure to test for gamma-hydroxybutyric acid in blood and urine by gas chromatography-mass spectrometry

Gamma-hydroxybutyric acid (GHB) is a substance naturally present within mammal species. Properties of a neurotransmitter or neuromodulator are generally suggested for this substance. GHB is therapeutically used as an anaesthetic, but can be used for criminal offences (date-rape drug). It appears that the window of detection of GHB is very short in both blood and urine, and therefore its presence is very difficult to prove after a rape case. Twenty microl of blood or urine were pipetted into a glass tube, followed by 20 microl GHB-d(6) and 45 microl acetonitrile. After vortexing and efficient centrifugation, the supernatant was collected and evaporated to dryness. The residue was derivatized with BSTFA+1% TMCS for 20 min at 70 degrees C. After injection on a 30-m HP5 MS capillary column, GHB (m/z 233, 204 and 147) and GHB-d(6) (m/z 239) were identified by mass spectrometry. The procedure was linear from 1 to 200 mg/l for both blood and urine. Precisions were in the range 4 to 11%. The method appears simple, specific and rapid as an accurate result can be obtained within 1 h.     

Identification of point mutations in mixtures by capillary electrophoresis hybridization.

We have developed a rapid method for unambiguous identification and mutant fraction determination of individual mutants in mixtures of DNA sequence variants each differing by one or a few nucleotides. This method has applications to such diverse areas as interpretation of mutational spectra, screening of populations for polymorphisms and identification of species in environmental mixtures. In our approach, a mixture of unknown sequences labeled with a fluorescent dye is combined with a set of predetermined sequences (standards) representing the variants to be assayed. Labeling the standards with another dye allows the two sets of variants to be measured independently. Using constant denaturing capillary electrophoresis, the sequence variants are separated as individual peaks on the basis of differential melting equilibria. The unknown sequence variants are initially identified based on co-migration with particular standards. This preliminary identification is verified by hybridization of the unknown variants with the co-migrating standards within the capillary. We demonstrate the use of capillary electrophoresis hybridization to dissect complex mutational spectra of human cells in culture.     

An angiographic technique for three-dimensional determination of arterial supply patterns in cadaver soft tissue.

A method for arterial tree mapping that can be used in cadaver soft tissue is presented. In situ angiograms and photographs are supplemented with profile angiograms of relatively narrow bands of tissue from the removed specimen. The described method was better suited for mapping the course and supply patterns of a soft-tissue arterial network than either in situ angiograms or dissection. While practical problems were encountered with most of the solutions used for providing radiopacity or structural support to the vessels, pure barium sulfate was found to be suitable because it filled the vascular tree to the capillary level without leakage during excision of the specimen.     

传粉生物学中几种花蜜采集和糖浓度测定方法的比较

花蜜的研究是花生物学中的一个重要内容,探寻实用的方法将方便野外操作。我们分别还用毛细管、注射器、滤纸条和离心法采集了5种花的花蜜,以比较各种方法的优劣,并用3种旋光测糖仪测量了慈姑Sagittaria trifolia L,的雌雄花的花蜜糖含量。目的是为寻找一种适合小型花的花蜜采集测量方法。结果表明,几种方法的适用性受花的大小、形状、蜜的分泌量及蜜腺位置的影响非常大,不同的花要采用不同的方法。对于一般的野外工作建议用毛细管采集后使用便携式旋光测糖仪测其糖含量。特别小的花和蜜量微小的花可以采用离心法收集。     

Primers<Emphasis Type="Italic">ITS1, ITS2</Emphasis> and<Emphasis Type="Italic">ITS4</Emphasis> detect the intraspecies variability in the internal transcribed spacers and 5.8S rRNA gene region in clinical isolates of fungi

Restriction fragment length polymorphism analysis of the 5.8S rRNA gene and the internal transcribed spacers (ITS1 and ITS2) was used for examination of 66 isolates belonging to 19 species. Intraspecies variability was found in the examined region of 11 species (Candida albicans, C. catenulata, C. colliculosa, C. glabrata, C. kefyr, C. melinii, C. parapsilosis, C. guillermondii, C. solanii, C. tropicalis, Saccharomyces cerevisiae). Region of ITS-5.8S rDNA was amplified using the primers ITS1 and ITS4. The amplicons were digested by HaeIII, HinfI and CfoI. The recognized intraspecies variability was confirmed in the second step, in which the shorter fragments of this region were amplified using primers ITS1 and ITS2 and analyzed by capillary electrophoresis. Considerable intraspecific variability renders this method unsuitable for species identification, whereas it can be useful for epidemiological tracing of isolates.     

昆虫表皮中碳氢化合物在昆虫分类中的应用

碳氢化合物是昆虫表皮蜡层中的主要成分,利用气相色谱技术或气谱质谱联用技术对其进行分析,并以此为依据对昆虫进行分类鉴定,是近十几年来昆虫分类学发展的一个方面,主要用于近缘种及种下类群的研究。该文概述了这类化合物的研究方法及 应用情况     

Whole-body systemic transcapillary filtration rates, coefficients, and isogravimetric capillary pressures in Bufo marinus and Rana catesbeiana

Whole-body and organ-level transcapillary filtration rates and coefficients are virtually unexamined in ectothermal vertebrates. These filtration rates appear to be greater than in mammals when plasma volume shifts and lymphatic function are analyzed. Gravimetric techniques monitoring whole-body mass changes were used to estimate net systemic filtration in Bufo marinus and Rana catesbeiana while perfusing with low-protein Ringer's and manipulating venous pressure. Capillary pressures were estimated from arterial and venous pressures after measuring the venous to arterial resistance ratio of 0.23. The capillary filtration coefficient (CFC) for the two species was 25.2+/-1.47 mL min-1 kg-1 kPa-1. Isogravimetric capillary pressure (Pci), the pressure at which net fluid is neither filtered nor reabsorbed, was 1.12+/-0.054 kPa and was confirmed by an independent method. None of these variables showed a significant interspecific difference. The anuran CFC and Pci are significantly higher than those found using the same method on rats (7.6+/-2.04 mL min-1 kg-1 kPa-1 and 0.3+/-0.37 kPa, respectively) and those commonly reported in mammals. Despite the high CFC, the high Pci predicts that little net filtration will occur at resting in vivo capillary pressures.