Showing papers by "Applied Biosystems published in 1994"

Method for detecting nucleic acid amplification using self-quenching fluorescence probe

Abstract: A method is provided for monitoring the progress of nucleic acid amplifications that rely on a nucleic acid polymerase having 5'→3' exonuclease activity. An important feature of the method is providing an oligonucleotide probe having a reporter molecule and a quencher molecule at either end such that the quencher molecule substantially quenches any fluorescence from the reporter whenever the oligonucleotide probe is in a single stranded state and such that the reporter is substantially unquenched whenever the oligonucleotide probe is in a double stranded state hybridized to a target polynucleotide.

High-density multiplex detection of nucleic acid sequences: oligonucleotide ligation assay and sequence-coded separation.

Abstract: We describe a non-isotopic, semi-automated method for large-scale multiplex analysis of nucleic acid sequences, using the cystic fibrosis transmembrane regulator (CFTR) gene as an example. Products of a multiplex oligonucleotide ligation assay (OLA) are resolved electrophoretically from one another and from unligated probes under denaturing conditions with fluorescence detection. One ligation probe for each OLA target carries a fluorescent tag, while the other probe carries an oligomeric non-nucleotide mobility modifier. Each OLA product has a unique electrophoretic mobility determined by the ligated oligonucleotides and the mobility-modifier oligomer arbitrarily assigned (coded) to its target. The mobility range for practical mobility modifiers is much wider than the accessible range from unmodified ligated oligonucleotides of practical length. Each mobility modifier is built from phosphoramidite monomers in a stepwise manner on its associated oligonucleotide using an automated synthesizer. The resulting mobility modifiers lower the probe-target duplex Tm by less than 3 degrees C and retard probe-target annealing by less than 50%, with negligible effect on OLA yield and specificity. This method is especially useful for allelic discrimination in highly polymorphic genes such as CFTR.

The use of transgenic and naturally occurring mutants to understand and manipulate tomato fruit ripening

Abstract: In the years since we last reviewed the use of mutants to study tomato fruit ripening (Grierson et al. 1987), considerable information has been gained by the cloning, sequencing and identification of many mRNAs implicated in this developmental process. Genes involved in cell wall degradation, colour change and ethylene synthesis have been cloned, and antisense techniques have been developed and used to produce genetically engineered mutant fruit deficient in these aspects of ripening (see Gray et al. 1992). Recently, a previously cloned ripening gene has been used to complement a naturally occurring fruit colour mutant, yellow flesh (Fray & Grierson 1993a), and a ripening impaired mutant, ripening inhibitor, has been used to identify several new ripening-related mRNAs (Picton et al. 1993b). The chromosomal region bearing the ripening inhibitor mutation has been subjected to high-resolution mapping (Churchill, Giovannoni & Tanksley 1993) and chromosome walking experiments are in progress to identify this gene.

Improved quantitative PCR using nested primers.

Abstract: Quantitative PCR can often be improved by conducting the amplification with nested primers. First, fewer nonspecific amplification products, which could otherwise interfere with quantitation, are produced. Often, nonspecific products can be eliminated. In these cases, relatively simple nonspecific detection techniques are suitable for quantitation. In addition, nested primer PCR provides intrinsic PCR product carryover protection and generally improves the robustness and lower limit of detection of PCR. For a nested PCR to provide useful quantitative information, it is important that the initial phase of amplification, performed with the outer pair of primers, takes place entirely in the exponential phase. This is generally achieved easily. The major consideration in designing a nested PCR protocol compatible with quantitation is to assure that the maximum concentration of PCR products produced by the outer primers does not exceed approximately 10% the molarity of the outer primers. A simple formula can be used to determine the maximum number of thermal cycles that provide this assurance. Good correspondence was obtained between initial target concentration and final PCR product yield in a nested-primer HIV-1 PCR.

Enzymatic ligation of 3'-amino-substituted olignucleotides

Abstract: Method and kits are provided for detecting one or more target nucleic acids. A first oligonucleotide having a 3' amino group and a second oligonucleotide having a 5' phosphate group are annealed to a contiguous complementary region of a target nucleic acid. Whenever the 3' terminal nucleotides of the first oligonucleotide and the 5' nucleotides of the second oligonucleotide are complementary to the opposing nucleotides on the target nucleic acid, a nucleic acid ligase ligates the first and second oligonucleotides via the formation of a phosphoramidate linkage. The presence of the target nucleic acid is determined by detection of the ligated first and second oligonucleotides.

Uniparental isodisomy for paternal 7p and maternal 7q in a child with growth retardation.

Abstract: Uniparental isodisomy resulting from the simultaneous presence of isochromosomes of the p and q arms of a chromosome and absence of a normal homologue is an exceptionally rare event. We have observed a growth-retarded female infant in whom the normal chromosome 7 homologues were replaced by what appeared cytogenetically to be isochromosomes of 7p and 7q. Polymorphic microsatellite loci spanning the length of 7p and 7q were analyzed in the proband and her parents to ascertain the parental origin and extent of heterozygosity of the proband's rearranged chromosomes. These studies demonstrated that the 7p alleles of the proband were derived only from the father, the 7q alleles were derived only from the mother, and there was homozygosity for all chromosome 7 loci analyzed. The mechanisms leading to the formation of the proband's isochromosomes could reflect abnormalities of cell division occurring at meiosis, postfertilization mitosis, or both. We believe that the present case may result from incomplete mitotic interchange in the pericentromeric regions of chromosome 7 homologues, with resolution by sister-chromatid reunion in an early, if not first, zygotic division. Phenotypically, our proband resembled three previously reported cases of maternal isodisomy for chromosome 7, suggesting that lack of paternal genes from 7q may result in a phenotype of short stature and growth retardation.

Capillary zone electrophoresis of oligosaccharides derivatized with various aminonaphthalene sulfonic acids

Abstract: Malto-oligosaccharides were derivatized via their reducing ends with different aminoaphthalene mono-, di- and trisulfonic acids by reductive amination. The derivatives were then separated by capillary zone electrophoresis in uncoated fused silica capillaries, using 50 mM triethylammonium phosphate buffer, pH 2.5, as running electrolyte. The effect of degree of charge on speed of analysis and resolution was studied for different aminonaphthalene mono-, di- and trisulfonic acids. Under the conditions used, a higher degree of charge on the derivatives provided both faster analyses and higher resolution. Investigation of the electrophoretic behavior of derivatized oligosaccharides obtained from bovine pancreatic ribonuclease B gave insight into the possibility of applying such electrophoretic systems to the analysis of more complex carbohydrates. The resolution of positional isomers under the conditions described indicated that the high resolving power of this technique allows separations not strictly based on the effects of charge and mass of the analytes, but on structural characteristics as well. The relationship between electrophoretic mobility and molecular structure was investigated for the different derivatives.

High resolution dna sequencing method using low viscosity medium

Abstract: A DNA sequencing method for use in sequencing a DNA target sequence up to 300 bases, preferably up to 500 bases or greater in length, by electrophoretically separating a mixture of single-stranded DNA sequencing fragments in a capillary tube. The method employs an aqueous denaturing solution comprising between about 4 and about 7 weight percent linear polyacrylamide molecules having an average molecular weight of between about 20 and about 100 kilodaltons. The low-viscosity of the solution allows rapid loading and reloading of such solution into the capillary tube.

Capillary electrophoresis as a technique to analyze sequence-induced anomalously migrating DNA fragments

Abstract: Sequence-induced anomalous migration of double-stranded (ds) DNA in native gel electrophoresis is a well known phenomenon. The retardation of migration is more obvious in polyacrylamide compared with agarose gels, and is greatly affected by the concentration of the gel and the temperature. This anomalous migration results in a difference between calculated and actual sizes of the affected DNA fragments. A low viscosity polymer solution (DNA Fragment Analysis Reagent) under investigation for use in dsDNA analysis by capillary electrophoresis is shown to be useful for the visualization of anomalies in migration of dsDNA fragments. Comparable with traditional slab gel systems, the retardation effect, indicative of bent or curved DNA, is strongly dependent on polymer concentration and separation temperature. These dependencies have implications on the accurate sizing of dsDNA fragments with unknown sequences and secondary structures.

Plasma clearance and immunologic properties of long-acting superoxide dismutase prepared using 35,000 to 120,000 dalton poly-ethylene glycol.

Abstract: Some biological properties of bovine and recombinant human Cu, Zn superoxide dismutase (bSOD and rhSOD)-poly-ethylene glycol (PEG) adducts prepared by coupling 1–9 strands of high molecular weight PEG (35,000–120,000 daltons) are compared to SOD adducts coupled with 7 or 15 strands of low molecular weight PEG (5,000 daltons).

Time-of-flight mass spectrometer with an aperture enabling tradeoff of transmission efficiency and resolution

Abstract: not available for EP0587707Abstract of corresponding document: US5300774A time-of-flight mass spectrometer in which sample ions are generated from a target and are focussed into an ion beam that is incident onto a detector. A barrier that defines an aperture in the path of the ion beam is positioned to block ions having an extra large deviation from an average time-of-flight of the ions, thereby improving resolution. The aperture can be adjusted to adjust a tradeoff between sensitivity and resolution. Alternatively, the position of the aperture or the bias on an einzel lens can be adjusted to control this resolution.

Matrix‐assisted laser desorption/ionization: Dependence of the ion yield on the laser beam incidence angle

Abstract: Some basic properties of matrix-assisted laser desorption/ionization (MALDI) have been investigated by varying the angle between the pulsed UV-laser beam and the target surface, thus determining its influence on the MALDI process. The minimum deposited laser energy needed for protein ion production (the threshold energy) and the protein-yield dependence on the laser energy have been determined for incidence angles between 35° and 60° with respect to the target surface normal. The measurements demonstrate that neither the threshold energy nor the dependence of the protein yield on the laser energy vary with the incidence angle within the range studied. Hence the corresponding parameters that involve the area of irradiation, namely the threshold laser fluence as well as the ion-yield dependence on fluence have a cosine dependence on the incidence angle. Furthermore, the threshold energy at constant incidence angle scales with the mass of the analyte molecule for proteins in the range from 5700 to 66 000 u.

Application specific capillary electrophoresis

Abstract: A capillary electrophoresis system incorporates an application specific cassette (11) having run buffers and capillary column (27) selected for the specific separation procedure, with buffer reservoirs (19, 21) sealed for storage and shipment. A serving apparatus (41) is provided to accept the cassette (11) and perform the specific electrophoresis procedure until buffer is depleted or the performance of the separation column (27) deteriorates. The serving apparatus (41) comprises liquid transfer apparatus (55), removable covers (35) for the reservoirs (19, 21, 23, 25) of the cassette, electrodes (87, 123, 133) for establishing voltage potential, and a control system (69).

Genetic polymorphisms of the CST2 locus coding for cystatin SA.

Abstract: A new genetic polymorphism of cystatin SA has been identified in human submandibular-sublingual saliva by means of basic gel electrophoresis and immunoblotting with anti-cystatin S. Two proteins, SA1 and SA2, are given by two alleles of CST2, viz., CST2*1 and CST*2. Inheritance is controlled by two codominant alleles at an autosomal locus. This hypothesis is supported by studies of 16 families 32 children. Gene frequencies for CST2*1 and CST2*2 are 0.935 and 0.065, respectively (n = 341). Eighteen amino acids determined among 20 N-terminal residues of cystatin SA2 are identical with the sequence encoded by CST2. Three forms of cystatin S (mono-phosphorylated cystatin S, di-phosphorylated cystatin S, and non-phosphorylated cystatin S) are present in the 341 saliva samples tested.

Acceptor-substrate recognition by N-acetyl-glucosaminyltransferase-V: Role of the mannose residue in βDGlcNAc(1→2)αDMan(1→6)βDGlcOR

Abstract: βGlcNAc(1→2)αMan(1→6)βGlc-O(CH 2 ) 7 CH 3 ( 4 ) is an acceptor specific for N-acetylglucosaminyltransferase-V (GlcNAcT-V), a branching enzyme controlling the biosynthesis of cell-surface Asn-linked oligosaccharides. Three analogs of 4 , where the central mannose residue has been O-methylated at O-3, at O-6, and where the 6-OH group was replaced by fluorine, were chemically synthesized, characterized by NMR-spectroscopy and kinetically evaluated as substrates for GlcNAcT-V. Along with results obtained using previously described derivatives of 4 , the conclusion is drawn that none of the OH groups on the Man residue are critical for recognition by the enzyme. These results should simplify the design of inhibitors for this important tumor-associated enzyme.

A Facile Method for the Release, Labeling and ce Analysis of Glycoprotein Oligosaccharides

Abstract: Publisher Summary This chapter describes a facile method for the release, labeling, and capillary electrophoresis (CE) analysis of glycoprotein oligosaccharides. It describes a finger printing method in which N-linked oligosaccharides of glycoproteins are rapidly and effectively released and labeled, facilitating their analytical separation by high-performance liquid chromatography (HPLC) or CE. In this method, oligosaccharides are enzymatically released using peptide- N -glycosidase F, followed by chemical derivatization with the chromophore l-phenyl-3-methyl-5-pyrazolone. PNGase F releases accessible Asn-linked oligosaccharides by cleaving β-aspartylglucosylamine bonds, resulting in the release of intact oligosaccharides. During the process, the asparagine residue to which the carbohydrate was attached is converted to aspartic acid. The protocol developed requires no purification of the released oligosaccharides prior to labeling, and the oligosaccharides once labeled are ready for analysis by CE and/or HPLC immediately following a simple liquid–liquid extraction used for their clean-up. Increasing the PNGase F concentration up to 10-fold did not increase the extent of deglycosylation past the end point that was rapidly achieved with much less enzyme.

Separation of two isoforms (Ser7/Thr7) of natural sarafotoxin-a by capillary electrophoresis : mass spectrometry and synthesis

Abstract: Each isoform was identified within the mixture in the natural sarafotoxin-a: Ser7 and Thr7-SRTX-a has been identified by chemical sequencing and recently by the sequencing of c-DNAs encoding for the sarafotoxins family. Mass spectrometry (MS) of the natural SRTX-a displayed the presence of two isoforms as noted by chemical and c-DNA sequencing. In order to evaluate the role of the 7 th residue in the bioactivity of SRTX-a we carried out the separation of Ser7 and Thr7-SRTX-a from the natural SRTX-a isolated from the venom successicely by gel filtration, ion-exchange and reversed-phase HPLC. The capillary electrophoresis in micellar conditions (MECC) enabled us the separation of the Ser7/Thr7-SRTX-a in an analytical range of peptide only. Due to the low range of the CE process in the quantitative recovery of two isoforms, each isoform was then synthesized by solid phase for the bioassays and further spectroscopic studies. The MW and migration time of each synthetic isoform were found to fit with t...