Showing papers by "Applied Biosystems published in 1991"

Automated molecular biology laboratory.

Abstract: A liquid-handling instrument (11) has a worksurface (22) with registration for modular stations to support containers of liquid, pipette apparatus with a pipette tip (33) coupled to a sensing circuit, a robotic translation system (31) for moving the pipette tip, and a control system with an iconic user interface for programming and editing. A gauge block (24) registered on the worksurface provides for calibration using the sensing tip, and register cavities on the worksurface provide for modular stations. There is a wash station (25) for the pipette tip on the worksurface. An automated laboratory based on the liquid-handling system has heating and cooling and a sealable incubation station (21) as well as a magnetic separation station (26 and 29). Methods are disclosed using the apparatus to convey droplets of liquid, to aspirate with minimum tip contamination, to mix liquids in containers, and to validate the worksurface. A duck-billed closure (253) is disclosed for minimizing evaporation and cross-contamination during processing, and is a part of a container disclosed for storing and transporting liquids.

HBTU activation for automated Fmoc solid-phase peptide synthesis.

Abstract: Excellent results have been obtained for the Fmoc solid-phase syntheses of peptides using the activating reagent 2-(1H-benzotriazol-1-yl)-1,1,3,3,-tetramethyluronium hexafluorophosphate (HBTU). Activation occurs very rapidly in N,N-dimethylformamide and N-methyl-pyrrolidone, optimal solvents for peptide-resin solvation. It has been observed that complete coupling reactions occur in only 10-30 min. Residues such as Arg, Ile, Leu and Val, which often require double coupling by other activation methods, react with high efficiency by single coupling when HBTU is used. The Fmoc/HBTU chemistry has recently been applied to the peptide synthesizers. The incorporation of trityl side-chain protection for Fmoc-Asn and Fmoc-Gln further enhances coupling efficiencies in difficult sequences.

Method of synthesizing sulfurized oligonucleotide analogs

Abstract: A method for synthesizing sulfurized oligonucleotide analogs, such as phosphorothioate and phosphorodithioate analogs, is provided that employs a thiophosphorus compound, such as a thiophosphoric, dithiophosphoric, thiophosphinic, or dithiophosphinic acid disulfide or polysulfide, as a sulfurizing agent. The method of the invention may be used to sulfurize any phosphorous(III)-containing intermediate. Preferably, the method is practiced on a commercial DNA synthesizer using phosphoramidite and/or phosphorthioamidite intermediates.

Duplex stabilities of phosphorothioate, methylphosphonate, and RNA analogs of two DNA 14-mers

Abstract: The duplex stabilities of various phosphorothioate, methylphosphonate, RNA and 2'-OCH3 RNA analogs of two self-complementary DNA 14-mers are compared. Phosphorothioate and/or methylphosphonate analogs of the two sequences d(TAATTAATTAATTA) [D1] and d(TAGCTAATTAGCTA) [D2] differ in the number, position, or chirality (at the 5' terminal linkage) of the modified phosphates. Phosphorothioate derivatives of D1 are found to be less destabilized when the linkage modified is between adenines rather than between thymines. Surprisingly, no base sequence effect on duplex stabilization is observed for any methylphosphonate derivatives of D1 or D2. Highly modified phosphorothioates or methylphosphonates are less stable than their partially modified counterparts which are less stable than the unmodified parent compounds. The 'normal' (2'-OH) RNA analog of duplex D1 is slightly destabilized, whereas the 2'-OCH3 RNA derivative is significantly stabilized relative to the unmodified DNA. For the D1 sequence, at approximately physiological salt concentration, the order of duplex stability is 2'-OCH3 RNA greater than unmodified DNA greater than 'normal' RNA greater than methylphosphonate DNA greater than phosphorothioate DNA. D2 and the various D2 methylphosphonate analogs investigated all formed hairpin conformations at low salt concentrations.

Phosphorothioate oligonucleotides: chemistry, purification, analysis, scale-up and future directions.

Abstract: There is widespread and growing interest in the use of phosphorothioate-modified oligonucleotides as sequence-specific agents to control transcription, splicing, translation and other regulated processes in vitro and in vivo, as pharmaceuticals. These exciting applications are predicated on the resistance of phosphorothioate oligonucleotides to degradation by nucleases, a property that results simply from incorporation of sulfur into the phosphate backbone. The popularity of phosphorothioate oligonucleotide analogs derives from several of their features: relatively easy automated synthesis, uncomplicated purification and handling, plus high solubility in water. Presented here are current preparative and analytical methods, together with a comprehensive comparative analysis of solid-phase and solution processes for manufacturing phosphorothioate oligonucleotides as bulk pharmaceutical compounds for clinical evaluation. A prospective view of the future is offered with the hope of directing attention to important areas of needed research dealing with phosphorothioate oligonucleotide technology in particular, and manufacturing of nucleic acid pharmaceuticals in general.

Chemiluminescence enhancement of enzyme-activated decomposition of enzymatically cleavable chemiluminescent 1,2-dioxetanes

Abstract: Water soluble naturally-occurring and synthetic enhancer substances, generally macromolecular in nature, for example globular proteins that include hydrophobic regions such as bovine serum albumin, and polymeric quaternary ammonium salts such as poly(vinylbenzyltrimethylammonium chloride), which have the ability to inhibit light-emitting fluorophores resulting from the decomposition of chemiluminescent compounds from releasing energy through non-light emitting pathways, are disclosed as permitting the stabilization, and hence increasing the light intensity, of such light-emitting fluorophores in aqueous media as compared to the intensity of the light emitted by the same quantities of such fluorophores in aqueous media in the absence of such enhancer substances. Any chemiluminescent enzymatically cleavable 1,2-dioxetane, for example 3-(2'-spiroadamantane)-4-methoxy-(3"-phosphoryloxy)phenyl-1,2-dioxetane disodium salt, can be used. Auxiliary fluorophores, for example fluorescein and derivatized fluoresceins, that accept energy from fluorophores produced by decomposition of a chemiluminescent compound and in turn emit detectable energy, can also be present. Such enhancer substance/chemiluminescent compound compositions are useful in detecting the presence or determining the concentration of chemical or biological substances in immunoassays, chemical assays and nucleic acid probe assays, and in chemical/physical probe procedures for studying the microstructures of macromolecules.

High-viscosity polymer matrix and methods

Abstract: A supported, substantially uniform matrix for use in electric field-induced separation of molecular components in a sample. The matrix is prepared by forming a viscoelastic polymer matrix and pumping the matrix into a capillary or preparative-scale tube. The polymer type, concentration, and molecular weight are selected to optimize separation of protein or nucleic acid components. The matrix can be expelled from the tube, after fractionation, for analysis and/or recovery of fractionated components.

Biomolecule sample immobilization

Abstract: A filter system for separating and binding biomolecules contained in a solution includes a first filter means for selectively passing components of the solution that are smaller than the biomolecules, thereby concentrating the biomolecules in the solution. Also included is a binding membrane means having an affinity for the biomolecules for binding the biomolecules to the binding membrane means, with the binding membrane means being located where the biomolecules are concentrated by the first filter means. In the preferred mode, the binding membrane means is chosen to selectively bind the biomolecule of interest. According to a preferred method of the invention, biomolecules from a sample volume are bound to a binding membrane by urging the sample volume against a biomolecule-binding support membrane that is in contact with a cut-off filter membrane such that the sample volume will pass through the biomolecule-binding support membrane before passing through the cut-off filter membrane. The concept is to concentrate the biomolecules of interest by means of the cut-off membrane in the vicinity of the binding membrane, thereby increasing the binding yield on the binding membrane. In the preferred mode, the sample volume is urged against the binding membrane by centrifugation.

Low-viscosity polymer solution for capillary electrophoresis

Abstract: A capillary electrophoresis element is disclosed. The elements includes a capillary elctrophoresis tube containing a low-voscosity polymer solution having a selected mesh size and low-solution viscosity. The mesh size may range from 50-100 Å, for separating single-stranded oligonucleotides, to up to 300 Å or greater, for separating relatively large duplex DNA fragments or proteins. Also disclosed is a method for formulating a low-viscosity electrophoresis separation medium having a selected mesh size.

Separation and determination of divalent metal ions with uv-absorbing chelating agents by capillary electrophoresis

Abstract: Capillary electrophoresis separation of several divalent metal ions, as well as iron(III) and silver(I) was examined with chelating agents. The metal chelates separated in the capillary were measured by on-column UV-absorption detection. When 1, 2-cyclohexanediamine-N, N, N' N'-tetraacetic acid(CyDTA) was used as an chelating agent in a carrier solution (pH 9) the order of the migration time (tm) of metal ions was as follows: Ba2+ Zn2+) and Cd2+

Capillary electrophoresis method with polymer tube coating

Abstract: A method for achieving desired electroosmotic flow characteristics in a capillary tube having charged surface groups. An electrolyte solution containing a compound effective to stably alter the charge of the tube walls is drawn into and through the tube during while the electroosmotic flow rate in the tube is being monitored, until a desired electroosmotic flow rate is achieved. The method can be used to optimize electrophoretic separation of charged protein or nucleic acid species in a capillary tube, and to produce capillary tubes with desired charge density properties.

On-capillary gap junction for fluorescence detection in capillary electrophoresis

Abstract: A junction reactor (50) that aligns a pair of capillaries (513, 514) substantially collinearly, end-to-end that allows a small gap (512) to be produced between these two ends. An applied voltage difference between the other ends of these two capillaries producces in the gap (512) electric fields lines that extend across the gap (512). Empirical evidence shows that the gap (512) introduces only a small reduction in resolution of an electrophoretic or electrochromatographic separation. The gap (512) enables sample liquid to be coupled between capillaries (513, 514) of different internal diameters and enables on-capillary reactions such as attaching a fluorescent tag to a sample components.

Primary structure and origin of schistosomin, an anti-gonadotropic neuropeptide of the pond snail Lymnaea stagnalis

Abstract: In the pond snail Lymnaea stagnalis infected with the schistosome parasite Trichobilharzia ocellata, a peptide called schistosomin is released from the central nervous system, which counteracts the bioactivity of a number of gonadotropic hormones. This leads to inhibition of the reproductive activities of the infected snail. In order to determine the structure of schistosomin, the neuropeptide was purified from the central nervous system using gel-permeation chromatography and reverse-phase h.p.l.c. The complete primary structure of the peptide was determined by N-terminal sequencing and peptide mapping. Schistosomin is a single-chain molecule of 79 amino acids with a molecular mass of 8738 Da. The peptide contains eight cysteine residues which may give rise to four intramolecular disulphide bridges that fold the peptide into a stable globular structure. A database search did not reveal any known peptides that show significant sequence similarity to schistosomin. By means of immunocytochemistry, the peptide was shown to be localized in the growth-controlling neurosecretory light green cells, which are located in the cerebral ganglia of the central nervous system of Lymnaea. In addition to schistosomin, these neurons are known to produce various insulin-related peptides.

Structure determination of [d(ATATATAUAT)]2 via two-dimensional NOE spectroscopy and molecular dynamics calculations.

Abstract: Proton homonuclear two-dimensional (2D) NOE spectra were obtained for the decamer [d(ATATATAUAT)]2 as a function of mixing time, and proton resonance assignments were made. Quantitative assessment of the 2D NOE cross-peak intensities was used in conjunction with the program MARDIGRAS, which entails a complete relaxation matrix analysis of the 2D NOE peak intensities, to obtain a set of upper and lower bound interproton distance constraints. The analysis with MARDIGRAS was carried out using three initial models: A-DNA, B-DNA and Z-DNA. The distance constraints determined were essentially the same regardless of initial structure. These experimental structural constraints were used with restrained molecular dynamics calculations to determine the solution structure of the decamer. The molecular dynamics program AMBER was run using A-DNA or B-DNA as starting model. The root-mean-square (rms) difference between these two starting models is 0.504 nm. The two starting models were subjected to 22.5 ps of restrained molecular dynamics calculations. The coordinates of the last 10.5 ps of the molecular dynamics runs were averaged to give two final structures, MDA and MDB. The rms difference between these two structures is 0.09 nm, implying convergence of the two molecular dynamics runs. The 2D NOE spectral intensities calculated for the derived structures are in good agreement with experimental spectra, based on sixth-root residual index analysis of intensities. A detailed examination of the structural features suggests that while the decamer is in the B-family of DNA structures, many torsion angle and helical parameters alternate from purine to pyrimidine. with kinks occuring at the U-A steps.

Process and compounds for RNA synthesis

Abstract: A method and compositions are provided for synthesizing polynucleotides wherein the exocyclic amino groups of 5'-O-protected-2'O-alkylsilyl-adenosine phosphoramidite and 5'-O-protected-2'-O-alkylsilylguanosine phosphoramidite monomers are protected with dialkylformamidine. In a preferred embodiment, the ribonucleoside phosphoramidite monomers are activated with ethylthiotetrazole.

2,3-disubstituted-1,3,2-oxazaphosphacycloalkanes as nucleic acid linking agents

Abstract: The compounds of the invention include novel linking agents comprising 2-substituted-3-protected-1,3,2-oxazaphosphacycloalkanes and their phosphoramidite precursors. The compounds of the invention further include conjugates of the above linking agents with oligonucleotides and polymer supports. The compounds of the present invention are useful for linking organic moieties, such as fluorescent or chromogenic dyes, to polymer supports and oligonucleotides, particularly single- and double-stranded DNA and RNA fragments.

Imaging as a tool for improving length and accuracy of sequence analysis in automated fluorescence-based DNA sequencing.

Abstract: A new method of signal analysis for automated fluorescence-based DNA sequencing is presented. Signal resolution is a limiting factor in obtaining accurate sequence information beyond 400-450 nucleotides per gel lane. We have developed a computer program for the imaging of DNA bands in sequencing gels. The image analysis shows that distortions in the shapes of the bands decrease resolution of peaks observed served in the standard data plots. Reconstruction of the undistorted band shape prior to signal analysis substantially improves the resolution of peaks and may improve the accuracy and length of the contiguous sequence read. Image analysis identified other factors limiting the accuracy and length of automated DNA sequence analysis and provided a tool for evaluating various remedies. Our techniques should also be applicable in other systems, for example, in gel electrophoresis of proteins and DNA restriction fragments, and in scranning densitometry.

Neuropeptide schistosomin inhibits hormonally-induced ovulation in the freshwater snail Lymnaea stagnalis.

Abstract: This study examines the interaction between the caudodorsal cell hormone (CDCH) and schistosomin, a peptide secreted by the central nervous system of the snail (Lymnaea stagnalis) infected with the avain schistosome Trichobilharzia ocellata. Non-infected snails were injected with synthetic as well as native CDCH in the absence or presence of purified schistosomin. The response to 2 pmol of synthetic CDCH was blocked for 90% by coinjection with 3.5 pmol of schistosomin. The ovulation-inducing activity of extracts of cerebral commissures (the storage area of native CDCH) was also blocked by schistosomin. The degree of inhibition (65%), however, was less than that observed with synthetic CDCH. These results show that schistosomin inhibits ovulation and egg laying in Lymnaea. This explains the decrease or absence of egg laying in schistosome-infected freshwater snails.

A modified reaction cartridge for direct protein sequencing on polymeric membranes.

Abstract: A newly designed reaction vessel implements a vertical cross-flow type reactor with the Applied Biosystems multi-mode reaction cartridge design. This cartridge is designed for sequencing samples on polyvinylidine difluoride-type membranes. The benefits of this design include a reduced reaction chamber volume that results in lower rates of chemical consumption and less risk of sample loss or contamination during sequencing. Visualization of the membrane in the reaction chamber during sequencing facilitates optimization of drying, washing, extraction and transfer times. The cycle modifications described in this report are designed to optimize post-coupling extraction, cleavage and post-cleavage extraction steps during "flow across" conditions for polymeric membranes. Also, efficient washing and drying of membranes allows for a fast cycle time of 30 minutes when using Pulsed Liquid chemistry. Examples of Blott cartridge utility for sequencing polyvinylidine difluoride-bound proteins in the low picomole range are shown by analyzing samples prepared by a two-dimensional purification scheme using the 230A HPEC and sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Method for chromatographic separation of synthetic phosphorothioate oligonucleotides

Abstract: A method is provided for the preparation and/or analysis or synthetic phosphorothioate and -dithioate oligonucleotides. In particular, the method permits separation of fully sulfurized phosphorothioate or -dithioate oligonucleotides from incompletely sulfurized defect species on strong-anion exchange HPLC columns using concentration gradients of novel "soft base" anionic eluents, such as bromide, thiocyanate, and the like.

Genetic typing using automated electrophoresis and fluorescence detection.

Abstract: Multi-color fluorescence detection systems offer unique advantages when compared to single label detection methods for DNA typing, genetic disease testing, population fingerprinting, and DNA mapping. Internal controls are easily used and identified by different color dye labels. Multiple independent samples or multiple analyses of the same sample are run in each lane of a gel. Precision of size assignment and quantification are improved. Here, we will review a variety of methods used to analyze DNA and present the advantages of the multi-color fluorescence dye approach. An automated and quantitative DNA typing assay for human identification is shown. This method is an improvement over previous manual techniques and uses multi-color fluorescence labeling, electrophoresis and real-time detection methodology.