Showing papers by "Applied Biosystems published in 1989"

Identification of a neuropeptide hormone that regulates sex pheromone production in female moths.

Abstract: A pheromone biosynthesis activating neuropeptide (PBAN) hormone that controls sex pheromone production in female moths was identified from the brain-subesophageal ganglion complexes of the adult corn earworm, Heliothis zea. PBAN has 33 amino acid residues and a molecular weight of 3900. Its amino acid sequence has no significant homology with any of the fully characterized peptide hormones. The synthetic peptide, at a dose of between 2 and 4 picomoles, induced production of a normal quantity of sex pheromone in ligated H. zea females. The peptide also induced pheromone production in six other species of moths, thus indicating that this or similar peptides may be responsible for the regulation of pheromone production in moths.

Automated system for polynucleotide synthesis and purification

Abstract: A method and system for polynucleotide synthesis are provided which employ solid phase synthesis on a nonswellable porous polystyrene support by phosphoramidite or hydrogen phosphonate chemistries. The polystyrene support gives rise to fewer tritylated failure sequences caused by chain growth from extraneous support sites, and allows lower amounts of monomer reactants to be used to achieve equal or better coupling efficiencies as those achieveable with CPG. The method and system also employ nucleoside intermediates whose exocyclic amines are protected by base-labile groups which permit simultaneous cleavage and deprotection of the completed polynucleotide chain in the presence of the solid phase support. This latter feature allows practical automation of both the synthesis and purification of polynucleotides.

Detection of specific DNA sequences by fluorescence amplification: a color complementation assay.

Abstract: We have developed a color complementation assay that allows rapid screening of specific genomic DNA sequences. It is based on the simultaneous amplification of two or more DNA segments with fluorescent oligonucleotide primers such that the generation of a color, or combination of colors, can be visualized and used for diagnosis. Color complementation assay obviates the need for gel electrophoresis and has been applied to the detection of a large and small gene deletion, a chromosomal translocation, an infectious agent, and a single-base substitution. DNA amplification with fluorescent oligonucleotide primers has also been used to multiplex and discriminate five different amplified DNA loci simultaneously. Each primer set is conjugated to a different dye, and the fluorescence of each dye respective to its amplified DNA locus is scored on a fluorometer. This method is valuable for DNA diagnostics of genetic, acquired, and infectious diseases, as well as in DNA forensics. It also lends itself to complete automation.

Automated DNA sequencing methods involving polymerase chain reaction.

Abstract: Polymerase chain reaction (PCR) as a method for preparing DNA templates has been used for several DNA sequencing applications. An in situ procedure for directly sequencing PCR products by the dideoxy-termination method has been developed by using fluorophore-labeled sequencing primers. Completed sequence reactions were combined and loaded into a single electrophoretic lane of a fluorescence-based DNA sequence analyzer. DNA targets devoid of a universal primer sequence could be sequenced with labeled universal primers by incorporating a universal primer sequence into the PCR product. With this method, the sequence of a 351-bp region in the bacteriophage lambda genome was fully analyzed in a single lane with automatic base identification accuracy of greater than 99%. An unknown sequence, 1.7 kb long, also was sequenced by this procedure, in combination with a "PCR gene walking" strategy. Comparison of the 1110 bases in overlapping sequence data from both strands yielded only two single-base ambiguities. Automated DNA sequence analysis of the highly polymorphic HLA-DQA-1 (alpha) region in the human genome can be performed with this simple methodology. Use of this PCR-sequencing method to analyze DNA extracted from a one-month-old blood sample from an individual who is heterozygous at this locus allowed unambiguous assignment of genotype.

Labeling by simultaneous ligation and restriction

Abstract: Termini of restricted double-stranded DNA fragments are modified by ligating the fragments with terminal phosphate-free double-stranded oligonucleotides having a complementary terminus in the presence of a restriction enzyme and a ligase, where joining of the complementary ends results in loss of the restriction enzyme recognition sequence.

Synthesis of 1,2-dioxetanes and intermediates therefor

Abstract: Compounds having formula (I), wherein T is a polycycloalkylidene group (e.g., adamant-2-ylidene); R is a C₁₋₂₀ alkyl, aralkyl or cycloalkyl group; and Y is a fluorescent chromophore (e.g., m-phenylene), produced by reacting a compound having formula (II) with an R-ylating agent (e.g., R₂SO₄) in the presence of an alkali metal alkoxide in a polar aprotic solvent. Also, compounds having formula (III) are produced by reacting a compound having formula (IV) with formula (V), wherein X is an electronegative leaving group (e.g., a halogen anion such as chloride ion) in the presence of a Lewis base (e.g., a trialkyl-amine) dissolved in an aprotic organic solvent (e.g., benzene or toluene). Also, compounds having formula (VI) are produced by reacting a compound of formula (VII) with a tetra-O-acylated-O-hexopyranoside halide, then hydrolysing off the protective acyl groups. The aforementioned compounds and procedures are useful in the synthesis of enzyme-cleavable 1,2-dioxetane ring systems that can serve as members of a binding pair employed, for example, in chemiluminescent immunoassays, DNA probe assays, and direct assays for an enzyme.

Method of nucleic acid extraction

Abstract: An automated apparatus is provided which implements a new method of extracting and purifying nucleic acids from cells without the use of centrifugation. In the method, a lysate is created by treating the cells with proteinase K in the presence of a lysis buffer having a high concentration of a salt. The lysate is mixed with a phenol-based solvent system, thereby creating an emulsion. The emulsion is heated to promote phase separation. Similarly, the rate of phase separation is also enhanced by increasing the surface area of the emulsion. Once the phase separation is complete, the lower organic phase is removed and the upper aqueous phase is repeatedly extracted with the phenol-based solvent a preselected number of times, and is finally extracted using chloroform. The remaining aqueous phase is then dialyzed to further purify and concentrate the nucleic acid solution. Two preferred embodiments of apparatus are presented to accomplish this extraction.

Automated protein hydrolysis system

Abstract: Protein analysis apparatus has porous sample supports enclosed in holes in a sample slide. Slides are delivered to hydrolysis and derivatizer stations by a rotating turret such that moveable heads at the stations form closed reaction chambers with the slides, sealing around the holes in the slides and enclosing the sample supports. Tubings connected to the heads deliver substances for reaction, derivatization and cleaning. Protein samples applied to sample supports at one position on the turret are delivered sequentially to hydrolysis and derivatizer stations, and operations are computer controlled. Derivatives are automatically delivered for chromatographic analysis, and supports are automatically cleaned with solvents and purge gas after derivatization and before return to a sample load position. Closed reaction chambers formed by the moveable heads can be heated and pressurized as required for processing.

The investigation of Fmoc-cysteine derivatives in solid phase peptide synthesis.

Abstract: Fmoc-Cys(t-Bu)-OH, Fmoc-Cys(Acm)-OH, and Fmoc-Cys(Trt)-OH exhibit excellent synthesis characteristics when used in Fmoc solid phase peptide synthesis on the Applied Biosystems Model 431A peptide synthesizer. The actual 5% scavenger mixture will vary according to the particular amino acid residues present. As was previously mentioned, an anisole/ethanedithiol/ethylmethylsulfide mixture (3:1:1) works well as a general scavenger solution for TFA cleavage of Fmoc synthesized peptide resins. It also may be possible to use lower acid (TFA) concentrations. The syntheses and workups of the peptide Somatostatin utilizing these derivatives demonstrate the ease of using these cysteine derivatives with the Fmoc chemistry approach. The use of either the t-Bu or the Acm moiety produces a peptide containing protected thiol groups after cleavage with 95% TFA. The Fmoc-Cys(Trt)-OH derivative is efficiently deprotected using 95% TFA. This investigation should provide further insight into synthesis options and cleavage protocols when working with cysteine-containing peptides.

On‐line negative ion liquid chromatography/mass spectrometry for the analysis of bile acids using low‐ and high‐resolution mass spetrometry

Abstract: Liquid chromatographic/mass spectrometric analysis of a mixture of bile acids has been carried out using a double-focusing mass spectrometer. A solution of polyethylene glycol was added post-column using a second pump and a micro vortex mixer. The mass spectrometer was tuned to give a 10 000 resolution, and mass measurement accuracies of greater than 3 mmu were obtained on-line.

Method of C-terminal peptide sequencing

Abstract: A method of C-terminal peptide sequencing. The peptide is reacted with a mixed anhydride of isothiocyanic acid and a carboxylic, carbonic, or sulfonic acid, under basic conditions, to produce a C-terminal peptidyl thiohydantoin. The C-terminal amino acid can be identified by cleaving the thiohydantoin from the peptide and identifying the free amino acid thiohydantoin.

Improved chemistry for oligodeoxyribonucleotide synthesis substantially improves restriction enzyme cleavage of a synthetic 35mer

Abstract: Two DNA duplexes of identical sequence and 35 nt in length were synthesized by an original and a highly improved version of phosphoramidite chemistry. By base composition analysis, DNA synthesized by improved chemistry (termed DMTS-imp) contained no detectable modified bases while DNA synthesized by the original chemistry (termed DMTS-std) had a large number of modifications. Under optimal reaction conditions, HhaI and RsaI cleaved the DMTS-std duplex to 76-77% completion and the DMTS-imp duplex to 96-99% completion. Restriction analysis and piperidine treatment yielded estimates of approximately 3.0% modified nucleotides in DMTS-std and approximately 1.0% in DMTS-imp. Overall, the improvements in chemistry increased the restriction efficiency of synthetic DNA up to 10-fold.

Ultraviolet Photolysis and Ortho-Phthalaldehyde-Mercaptoethanol Derivatization of Pharmaceuticals for Enhanced Fluorescence Detection in HPLC

Abstract: Several classes of nitrogenous pharmaceutical were examined for fluorescence after ultraviolet (UV) radiation induced photolysis followed by reaction with o-phthalaldehyde-2-mercaptoethanol (OPA-MERC), and after UV photolysis alone. Photolyses were examined in water, mixtures of methanol/water (1:1), and acetonitrile/water (1:1). Acetone was assessed as a photosensitizer to enhance photolysis and fluorescence response. Flow injection analysis and high-performance liquid chromatographic techniques were used for several pharmaceuticals. The analytes were subjected to UV photolysis and reaction with OPA-MERC reagent for generation of fluorophores that responded to fluorescence detection. During photolysis, solvent type as well as the presence of photosensitizers seem to play a significant role in the formation of primary amines and fluorophores. Photochemical transformation products of some of the pharmaceutical chemicals are proposed. Analytical figures of merit were determined for some analytes. T...

Synthesis of Anti-Sense Phosphorothioate Analogues for Pharmacokinetic and Pre-Clinical Studies

Abstract: Nudease-resistant oligonucleotides (11 to 28-mers) containing stereorandom phosphorothioate linkages have been recently reported to exhibit potent anti-HIV III effects and sequence-specific inhibition of protein synthesis. Relatively large amounts (100 mg 1g) of these analogues. which are needed for further biological testing and initial pharmacokinetic and pre-clinical studies, were readily obtained by automated hydrogen phosphonate chemistry followed by reversed-phase HPLC and further processing. This chemistry features 1 -adamanetanecarbonyl chloride as the activator, capping with isopropyl phosphite, and more complete sulfurization in only one-step following chain assembly. An automated, quantitative. picomole method for analysis of the analogues in blood samples has been developed.

A dual beam full spectrum multichannel spectrophotometer

Abstract: The present invention relates to an improved dual beam multichannel spectrophotomer employing a simple and novel optical system in combination with photodiode arrays and a unique logrithmic data converter to convert light signals to absorbance. In particular, the optical system utilizes optical elements in a novel arrangement to direct a pair of equivalent sample and reference beams in an essentially parallel formation respectively through a sample and reference cell and to focus and direct the emergent sample and reference beams to a single flat horizonatally ruled grating which disperses each of the sample and reference beams respectively onto a pair of vertically disposed photodiode arrays whereby the light signals are converted into absorbance units (AU) by an unique logarithmic data converter. The spectrophotometer is highly accurate, has very low drift, less than 2 x 10⁻⁴ AU/°C, and very low noise, less than ±2 x 10⁻⁵AU. The dual beam multichannel spectrophotomer is particularly suitable for use in high pressure liquid chromatography to record the absorbance spectrum of the samples as they are being eluted from the chromatographic column.

Specific Enzymatic Cleavage at Cystine/Cysteine Residues. The Use of ASP-N Endoproteinase

Abstract: Proteases are currently used in protein structure elucidation for peptide generation and direct sequencing. Endoproteases, such as trypsin and Glu-C protease cleave at well defined sites to yield fragments that are subsequently used in such characterizations.

NMR Relaxation Footprinting: The [Cr(NH3)6]3+ Cation as a Probe for Drug Binding Sites on Oligonucleotides

Abstract: [Cr(NH3)6] (N03)3 was used to probe structural features of the oligonucleotide, d(ATGCdCAT)2 (numbering of strand: 5′ A1T2G3C4G5C6A7T8 3′) and to footprint the binding site of actinomycin D(ActD) in the unique 2:1 ActD/d(ATGCGCAT)2 complex by proton longitudinal relaxation (T1) studies. Longitudinal relaxation rates (1/T1) of the 1H NMR signals were measured before and after the addition of the [Cr(NH3)6]3+ solution to determine the paramagnetic longitudinal relaxation rate (1/T1p = 1/T1(Cr) — l/T1(noCr)). The chromium complex seems to prefer the center of the duplex, since signals for protons on nucleotides in the center of the duplex have the largest 1/T1ps. Larger 1/T′1p values are observed for signals of major groove base protons on G3, C4, G5 and C6 ancfalso for signals of deoxyribose H1′ and H3′ protons which are close to the phosphate backbone (H3′ closer than H1′). We believe that electrostatic forces and hydrogen bonding are the main interactions between the chromium hexaammine cation and d(ATGCGCAT)2. To interpret our data we used distances from computer-generated models for five major binding modes. Four of these involved hydrogen bonding of the ammonia ligands to various sites on the oligonucleotide (phosphate oxygens, base oxygens and base nitrogens). One mode involved the approach of the [Cr(NH3)6]3+ cation into the minor groove of the duplex with no hydrogen bonding. Neither a single binding mode nor an equal weighting of all binding modes appeared to explain the results. Two modes appeared to have the greatest influence. One involved major groove interstrand binding at G3 and G5. The other involved interaction of the cation with a single phosphate group, with all phosphate groups exhibiting this binding mode. Other modes most likely do occur, but from modeling studies these modes appear to be less important.

Solid-phase sequencing method for single- and double-stranded nucleic acids

Abstract: The invention includes a method and apparatus for the sequence analysis of single- and double-stranded nucleic acids on solid phase supports whereby nucleic acids for the purpose of sequencing are terminally elongated with modified nucleic acid units, which themselves serve as units for anchoring to a water insoluble and solid polymer support In the preferred mode, modified nucleotides which are to be attached to a nucleic acid chain by chemical or enzymatically catalyzed reactions are in particular 5-bromo-2'-deoxyuridylate or in general other nucleotides halogenated or otherwise appropriately substituted at the base or sugar moieties, or streptavidin Nucleic acids which can be immobilized in this way are either single-stranded DNA- or RNA-chains or such chains as parts of the double-stranded DNA or RNA Water insoluble and solid supports are polymers such as cellulose, sepharose or sephadex as well as inorganic supports, in particular supports which have a backbone structure formed mainly by silicon and oxygen atoms The water-insoluble solid supports have effector groups, in particular, they may contain immobilized antibodies specific to 5-bromo-2'-deoxyuridylate or other appropriate units capable of selective interaction with other halogenated nucleotides or nucleotides appropriately substituted at the base or sugar moieties The binding of the nucleic acid strands which are to be sequenced to the polymer support is effected by interactions of the modified nucleotide units with the effector groups immobilized to the support, in particular by receptor ligand interactions An apparatus is disclosed for carrying out the method

Device for mixing solutions

Abstract: A device for mixing liquids contains a mixing chamber (1) into which a mixing head propelled around a rotationally axis is designed. Between the wall of the mixing chamber and the mixing head (2) a cleft mixing area is created. Through rotation of the mixing head (2) a shear flux of the injected liquids (11, 12, 13, 14) is created which causes mixing. With this active mixing method minute liquid amounts with differing volumes and viscosities can be mixed with high rapidity. The device is especially suitable for the automated analysis of enzyme kinetics.