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.     

Rapid automated batchwize radiochemical separation techniques

The basic principles and specific techniques of rapid, automated radiochemical separation techniques that use batchwize separation methods are reviewed.     

面向丙烯/丙烷吸附分离的金属有机骨架材料的高通量计算筛选

丙烯、丙烷作为分子尺寸相近的共沸物,其分离一直是化工领域研究热点。金属有机骨架(MOFs)材料因其高度可调的孔道结构,在丙烯/丙烷分离应用上已展现出诱人潜能。本文基于Core MOF 2019数据库,采用巨正则蒙特卡洛基高通量计算筛选技术,获得了分离性优异的MOFs结构,发现其拥有适中的丙烯吸附量和较弱的丙烷吸附能力,且骨架孔径为3.70～4.10?、孔隙率中等(0.35～0.44),并揭示了孔道中心吸附位的选择性与丙烯/丙烷分离系数间关系。本研究阐明了高丙烯/丙烷分离性的骨架材料的结构和性能特征,为设计MOFs实现丙烯/丙烷的高效分离提供理论指导和数据支撑。     

High-throughput automated DNA sequencing facility with fluorescent labels at the European Molecular Biology Laboratory.

One of the aims of the facility is to develop and push the automated on-line DNA sequencing gel technology to its limit in sequence throughput, which may be somewhere around 100 kilobases of sequence per device per day. Key new developments were initiated and applied in operation on the European Molecular Biology Laboratory (EMBL) automated sequencer and its commercial version A.L.F. (Pharmacia). Sequencing speed was increased by a factor of 10-20, up to 1500 bases per hour per clone on ultrathin (about 100 microns) gels, while the resolution and reading length were extended to 1000 bases on gels with 50 cm separation length, using fluorescein-15-*dATP as the internal label. With our sequencing strategy, closing about 40% of the sequence with "walking" primers and F-15-*dATP as internal label, we sequenced both strands of a cosmid insert of 38.5 kb in length, each strand twice, in only 430 sequencing reactions and with average reading of 380 bases per reaction.     

High-throughput separation of DNA and proteins by three-dimensional geometry gel electrophoresis: feasibility studies

We report a novel separation method that is applicable to both DNA and protein samples, based on electrophoresis in a three-dimensional (3-D) geometry. In contrast to conventional electrophoresis, samples are applied in a two-dimensional, planar array to one of the surfaces of a 3-D geometry separation medium. Loading onto a plane results in a very high sample capacity. Sample migration and separation occur along the third spatial dimension, which is perpendicular to the loading plane. The key problem of electrophoresis in a 3-D geometry separation setup is that temperature gradients are caused by Joule's heat, affecting the electrical conductivity and viscosity of the separation medium. A means of achieving straight sample migration under these circumstances is to force heat flow through the separation medium parallel to the axis of sample migration. This can be done by dissipating the heat via the electrode sides of the gel and blocking any other heat transfer. The separation of DNA and proteins by this method has been tested using agarose gel electrophoresis, polyacrylamide gel electrophoresis, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Data were acquired off-line by conventional staining methods as well as on-line by detection of laser-induced fluorescence. We describe how to excise samples from the separation medium for preparative purposes. Possible unique applications of this 3-D geometry electrophoresis separation method are also discussed.     

An automated activation analysis data acquisition system

An automated neutron activation analysis data acquisition system has been assembled from commercially available equipment. The modifications of the components needed to make this into a working system are described in the text. The main components of the data acquisition system are a sample changer, a Ge(Li) detector, a magnetic tape deck and a minicomputer based multichannel analyzer. The sample changer has a 200-sample capacity and can handle both solid and liquid samples. Software for controlling the data acquisition system is flexible, yet simple to use. The system has operated reliably for a year and has sharply reduced the effort needed for data acquisition.     

Peptide separation and isolation with an automated amino acid analyzer

A technique is presented for separation and collection of amino acids and peptides using a microcolumn amino acid analyzer. By use of the program and the column selection valve of the amino acid analyzer, and without any modification of the instrumentation, the stream of the eluate is diverted into the reaction coil and absorbance is recorded at regular intervals. The rest of the eluate is collected in a fraction collector for further characterization of the separated peptides. Splitting ratios can be varied by simple alteration of the program. Small amounts of material (1 to 2 nmol) are needed for monitoring the separation and collection of the eluate.     

A new concept of automated radiochemical analysis based on substoichiometric separation

Tracer methods, such as radioisotope dilution, radiometric analysis, concentration dependent distribution, saturation analysis etc., are compared on a basis of radioactivity-mass balance relationships, and their automation is proposed. The requirements of a chemical separation, which is an integral part of these methods, are discussed. It is shown that automation, in addition to its obvious advantages, enables entirely new procedures to be developed, based on chemical separations which do not give reproducible results when performed normally. Simple commercially available apparatus has been used to verify these concepts by determination of traces of mercury. As little as 0.005 ppm of Hg can be determined, the detection limit being about a tenth of this. In the range 2.4-0.03 ppm, 20 samples/hr can be analysed, for lower amounts the sampling rate is 10 samples/hr.     

An automated micromethod for the separation of monosaccharides by partition chromatography

Summary The conditions for a separation of monosaccharid.es and their automatic determination on the microscale have been studied. The separation is based upon partition chromatography on an anion-exchange resin in its sulphate form with ethanol -water as the eluent. The eluate is analysed colorimetrically with orcinol as the colour reagent. Microgram quantities in complicated mixtures of monosaccharides can be determined in less than three hours.

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Zusammenfassung Die Arbeitsbedingungen für die Trennung und automatische Bestimmung von Monosacchariden im Mikromaßstab wurden untersucht. Die Trennung wird verteilungschromatographisch an einem Anionenaustauscherharz in Sulfatform mit Äthanol-Wasser als Eluent ausgeführt. Das Eluat wird kolorimetrisch mit Orcin als Farbreagens durchgeführt. In Monosaccharidgemischen lassen sich Mikrogrammengen in weniger als 3 Stunden bestimmen.

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Résumé On a étudié les conditions de séparation des monosaccharides et leur dosage automatique à l'échelle micro. La séparation repose sur la Chromatographie de partage sur une résine échangeuse anionique sous sa forme sulfate, avec le mélange éthanol-eau comme éluant. On analyse par colorimétrie l'éluat avec l'orcinol comme réactif coloré. On peut doser des quantités de l'ordre du microgramme dans des mélanges complexes de monosaccharides en moins de 3 heures.

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The financial support of the Swedish Technical Research Council is gratefully acknowledged. Thanks are also due to Dr.H. W. Holy, Teehnicon Instruments Co., Chertsey, Great Britain, for a gift of specially prepared ion-exchange resin.     

Automated separation of 99Tc using plastic scintillation resin PSresin and openview automated modular separation system (OPENVIEW-AMSS)

Automated methods for the analysis of radionuclides potentially increase laboratory productivity by reducing operator intervention and increasing the number of samples that can be treated in a given time. To this end, here we report a new openview automated modular separation system which can be used in combination with PSresin, in this case, for the analysis of ⁹⁹Tc. Quality parameters of this method using the automated system were comparable to those obtained manually and quantification of water samples spiked with low levels of ⁹⁹Tc resulted in deviations lower than 10% for all the samples analysed.

     

Development of a relatively cheap and simple automated separation system for a routine separation procedure based on extraction chromatography

A robust analytical method has been developed in our laboratory for the separation of radionuclides by means of extraction chromatography using an automated separation system. The proposed method is both cheap and simple and provides the advantageous, rapid and accurate separation of the element of interest. The automated separation system enables a shorter separation time by maintaining a constant flow rate of solution and by avoiding clogging or bubbling in the chromatographic column. The present separation method was tested with two types of samples (water and urine) using UTEVA-, TRU- and Sr-specific resins for the separation of U, Th, Am, Pu and Sr. The total separation time for one radionuclide ranged from 60 to 100 min with the separation yield ranging from 68 to 98% depending on the elements separated. We used ICP-QMS, multi-low-level counter and alpha spectroscopy to measure the corresponding elements.     

A fully automated catecholamines analyzer based on cartridge extraction and HPLC separation

Summary A fully automated analyzer is described for the HPLC analysis of catecholamines. Firstly urinary and plasma catecholamines are reacted with diphenylboronic acid giving a complex without requiring a pH-meter step. This complex is strongly retained on a C 18 cartridge and elution with acetic acid solutions shows recoveries higher than 90 %. The catecholamines are eluted also by the mobile phase employed for the HPLC’ analysis. An intelligent autosampler makes the complex and fills a loop with all the solvents necessary for the sample cleanup. It then inverts the flow and pumps the sample and the solvents throught the cartridge. In the elution step the disposable extraction cartridge is positioned on stream with the HPLC analytical column by an automatic sampler. The separation is performed by ion-pair reversed-phase high-performance liquid chromatography, with sodium dodecyl sulphate as pairing ion, a short analytical column and electrochemical detection. The HPLC analysis time is 3 min and the total sample turnover time is 8 min. The recoveries and the precision of the analyzer are given together with correlation with manual HPLC.     

High-throughput microwave-assisted organic synthesis: moving from automated sequential to parallel library-generation formats in silicon carbide microtiter plates

A 48 deep-well microtiter plate system for sealed vessel parallel microwave synthesis is described. The plate consists of a standard 6 x 8 matrix of 48 wells with a maximum working volume of 300 microL and is made out of strongly microwave-absorbing sintered silicon carbide. In combination with an alumina sealing plate equipped with adequate conical bore holes for sample withdrawal, the setup can be used for microwave processing at temperatures up to approximately 200 degrees C and 20 bar of pressure. The microtiter plate setup displays excellent temperature and reaction homogeneity and was used for the generation of a 30-member library of 2-aminopyrimidines.     

High-throughput sodium dodecyl sulfate polyacrylamide gel electrophoresis by linking conventional gel plates with automated liquid handler via Gel Adaptor

The automation of SDS-PAGE required substantial investment. To lower this barrier, we devised a cost-effective, simple, and general method where samples are loaded on SDS-PAGE gels held by a newly devised "Gel Adaptor" on a general automated liquid handler. In this method, the liquid handler loads samples on the SDS-PAGE gels held at an angle of 80 degrees on the Gel Adaptor so that the samples can be vertically loaded on the narrow paths of the wells of the slanted gels. This method allows the automated liquid handler to load 144 samples on SDS-PAGE gels in approximately 10 min, greatly reducing the time and cost for high-throughput SDS-PAGE analyses.     

Application of totally automated on-line sample clean up for prostanoid extraction and HPLC separation

Summary The aim of this study has been the evaluation of an automated system for on-line sample preparation using solid phase extraction and HPLC purification for the measurement of prostanoids in urine. We have established the optimum precolumn and column conditions for this analysis. The manual extraction —HPLC procedure furnishes lower recoveries and higher coefficients of variation than those obtained by the automated on-line procedure. The automated system has been applied to prostanoid analysis of human urine samples from subjects exposed to lead.     

High-throughput absolute quantification of proteins using an improved two-dimensional reversed-phase separation and quantification concatemer (QconCAT) approach

Stable isotope dilution–selective reaction monitoring–mass spectrometry (SID-SRM-MS) has been widely used for the absolute quantitative analysis of proteins. However, when performing the large-scale absolute quantification of proteins from a more complex tissue sample, such as mouse liver, in addition to a high-throughput approach for the preparation and calibration of large amounts of stable-isotope-labelled internal standards, a more powerful separation method prior to SRM analysis is also urgently needed. To address these challenges, a high-throughput absolute quantification strategy based on an improved two-dimensional reversed-phase (2D RP) separation and quantification concatemer (QconCAT) approach is presented in this study. This strategy can be used to perform the simultaneous quantification of hundreds of proteins from mouse liver within one week of total MS measurement time. By using calibrated synthesised peptides from the protein glutathione S-transferase (GST), large amounts of GST-tagged QconCAT internal standards corresponding to hundreds of proteins can be accurately and rapidly quantified. Additionally, using an improved 2D RP separation method, a mixture containing a digested sample and QconCAT standards can be efficiently separated and absolutely quantified. When a maximum gradient of 72 min is employed in the first LC dimension, resulting in 72 fractions, identification and absolute quantification experiments for all fractions can be completed within one week of total MS measurement time. The quantification approach developed here can further extend the dynamic range and increase the analytical sensitivity of SRM analysis of complex tissue samples, thereby helping to increase the coverage of absolute quantification in a whole proteome.

Considerations for the automated collection of thermodynamic data in gas chromatography

Thermodynamic modeling of retention times in gas chromatography depends on the accurate estimation of thermodynamic parameters. Previous research has used manual injections of samples with coinjection of a dead time marker to obtain accurate measurements of the retention factor of analytes. Ideally this process would be automated. Herein an approach is presented by which thermodynamic parameters can be estimated both autonomously and accurately. This method also allows for a consistent estimation of thermodynamic parameters regardless of factors such as data system delays and the nature of the void time marker employed. Ignoring these factors can lead to significant errors in the prediction of retention times when using thermodynamic models.     

High-throughput study of phenytoin solid dispersions: formulation using an automated solvent casting method, dissolution testing, and scaling-up

A high-throughput experimentation method for studying the dissolution of phenytoin, a poorly water soluble drug, was developed and validated. Solid dispersions with 12 excipients (7 polymers and 5 surfactants) were prepared and tested. Each excipient was screened with three drug loadings: 10, 20, and 40% (w/w). Each solid dispersion was prepared in triplicate, for a total of 108 samples. The drug dissolution was studied in simulated gastric fluid without pepsin plus 1% sodium laurylsulfate. This study led to the identification of three improved formulations, exhibiting an extent of dissolution higher than 90% after both 30 and 60 min. The HTE results could be reproduced at a larger scale using a conventional solvent evaporating method, proving the reliability of the HTE protocol.     

Computer analysis of automated Edman degradation and amino acid analysis data

Computer programs are described that allow facile analysis of data from a protein sequencer and amino acid analyzer. The sequencer program provides automated sequence interpretation while requiring minimal user interaction. The program serves as a powerful aid in deciphering mixture sequences and allows routine monitoring of sequencer performance. The computer program for amino acid analysis data provides the following calculations: mole percent, protein concentration and residues per mole with comparison between theoretical and calculated values. A plot of molecular weight versus deviation from integer values is calculated providing a measure of peptide or protein purity.