Showing papers by "Applied Biosystems published in 2000"

Evaluation of the Applied Biosystems automated Taqman polymerase chain reaction system for the detection of meningococcal DNA.

Abstract: In a period where the proportion of culture confirmed cases in the UK has been steadily declining, diagnosis by PCR has been used to increase the number of confirmed cases and provide additional epidemiological data. This report presents a comparative evaluation of the fluorogenic probe-based 5' exonuclease assay (Taqman) using the Perkin-Elmer Applied Biosystems automated sequence detection system 7700 with previously reported polymerase chain reaction enzyme-linked immunosorbent (PCR ELISA) assays for the detection of meningococcal DNA in CSF, plasma and serum samples. Taqman assays developed were based on the detection of a meningococcal capsular transfer gene (ctrA), the insertion sequence IS1106 and the sialytransferase gene (siaD) for serogroup B and C determination and compared with similar assays in a PCR ELISA format. The Taqman ctrA assay was specific for Neisseria meningitidis, however the IS1106 assay gave false positive reactions with a number of non-meningococcal isolates. Sensitivity of the Taqman ctrA, IS1106 and siaD assays testing samples from culture-confirmed cases were 64, 69 and 50%, respectively, compared with 26, 67 and 43% for the corresponding PCR ELISA assays. Improvements to the DNA extraction procedure has increased the sensitivity to 93 and 91% for the TaqMan ctrA and siaD assays, respectively, compared to culture confirmed cases. Since the introduction of Taqman PCR a 56% increase in laboratory confirmed cases of meningococcal disease has been observed compared to culture only confirmed cases. The developed Taqman assays for the diagnosis of meningococcal disease enables a high throughput, rapid turnaround of samples with considerable reduced risk of contamination.

Rapid 5′ Nuclease (TaqMan) Assay for Detection of Virulent Strains of Yersinia enterocolitica

Abstract: We have developed a rapid procedure for the detection of virulent Yersinia enterocolitica in ground pork by combining a previously described PCR with fluorescent dye technologies. The detection method, known as the fluorogenic 5' nuclease assay (TaqMan), produces results by measuring the fluorescence produced during PCR amplification, requiring no post-PCR processing. The specificity of the chromosomal yst gene-based assay was tested with 28 bacterial isolates that included 7 pathogenic and 7 nonpathogenic serotypes of Y. enterocolitica, other species of Yersinia (Y. aldovae, Y. pseudotuberculosis, Y. mollaretti, Y. intermedia, Y. bercovieri, Y. ruckeri, Y. frederiksenii, and Y. kristensenii), and other enteric bacteria (Escherichia, Salmonella, Citrobacter, and Flavobacterium). The assay was 100% specific in identifying the pathogenic strains of Y. enterocolitica. The sensitivity of the assay was found to be >/=10(2) CFU/ml in pure cultures and >/=10(3) CFU/g in spiked ground pork samples. Results of the assay with food enrichments prespiked with Y. enterocolitica serotypes O:3 and O:9 were comparable to standard culture results. Of the 100 field samples (ground pork) tested, 35 were positive for virulent Y. enterocolitica with both 5' nuclease assay and conventional virulence tests. After overnight enrichment the entire assay, including DNA extraction, amplification, and detection, could be completed within 5 h.

Parental contribution and coefficient of coancestry among maize inbreds: Pedigree, RFLP, and SSR data

Abstract: The genetic relationship between inbreds i and j can be estimated from pedigree or from molecular marker data. The objectives of this study were to: (1) determine whether pedigree, restriction fragment length polymorphism (RFLP), and simple sequence repeat (SSR) data give similar estimates of parental contribution and coefficient of coancestry (f ij ) among a set of maize (Zea mays L.) inbreds, and (2) compare the usefulness of RFLP and SSR markers for estimating genetic relationship. We studied 13 maize inbreds with known pedigrees. The inbreds were genotyped using 124 RFLP and 195 SSR markers. For each type of marker, parental contributions were estimated from marker similarity among an inbred and both of its parents, and were subsequently used to estimate f ij . Estimates of parental contribution differed significantly (α<0.05) between pedigree data and either type of marker, but not between the marker systems. The RFLP estimates of parental contribution failed to sum to 1.0, reflecting a higher frequency of non-parental bands with RFLP than with SSR markers. The f ij estimated from pedigree, RFLP, and SSR data were highly correlated (r=0.87–0.97), although significant differences were found among the three sets of f ij estimates. We concluded that pedigree and marker data often lead to different estimates of parental contribution and f ij , and that SSR markers are superior to RFLP markers for estimating genetic relationship. A relevant question is whether or not the inbreds previously genotyped with an older marker system (e.g., RFLP) need to be re-analyzed with a newer marker system (e.g., SSR) for the purpose of estimating genetic relationship. Such re-analysis seems unnecessary if data for the same type of marker are available for a given inbred and both of its parents.

Mutation detection by TaqMan-allele specific amplification: application to molecular diagnosis of glycogen storage disease type Ia and medium-chain acyl-CoA dehydrogenase deficiency.

Abstract: We have devised an allele-specific amplification method with a TaqMan fluorogenic probe (TaqMan-ASA) for the detection of point mutations. Pairwise PCR amplification using two sets of allele-specific primers in the presence of a TaqMan probe was monitored in real time with a fluorescence detector. Difference in amplification efficiency between the two PCR reactions was determined by "threshold" cycles to differentiate mutant and normal alleles without post-PCR processing. The method measured the efficiency of amplification rather than the presence or absence of end-point PCR products, therefore allowing greater flexibility in designing allele-specific primers and an ample technical margin for allelic discrimination. We applied the TaqMan-ASA method to detect a prevalent 727G>T mutation in Japanese patients with glycogen storage disease type Ia and a common 985A>G mutation in Caucasian patients with medium-chain acyl-CoA dehydrogenase deficiency. The method can be automated and may be applicable to the DNA diagnosis of various genetic diseases.

Analysis of HIV type 1 protease and reverse transcriptase in antiretroviral drug-naive Ugandan adults.

Abstract: We analyzed plasma HIV-1 from 27 antiretroviral drug-naive Ugandan adults. Previous subtype analysis of env and gag sequences from these samples identified subtypes A, C, D, and recombinant HIV-1. Sequences of HIV-1 protease and reverse transcriptase (RT) were obtained with a commercial HIV-1 genotyping system. Subtypes based on protease sequences differed from gag subtypes for 5 of 27 samples, demonstrating a high rate of recombination between the gag and pol regions. Protease and RT sequences were analyzed for the presence of amino acid polymorphisms at positions that are sites of previously characterized drug resistance mutations. At those sites, frequent polymorphisms were detected at positions 36 and 69 in protease and positions 179, 211, and 214 in RT. Subtype-specific amino acid motifs were identified in protease. Most of the subtype A sequences had the amino acids DKKM at positions 35, 57, 69, and 89, whereas most subtype D sequences had the amino acids ERHL at those positions. Detection of those polymorphisms may provide a useful approach for rapid identification of subtype A and D isolates in Uganda. This analysis significantly increases the number of Ugandan protease and RT sequences characterized to date and demonstrates successful use of a commercial HIV-1 genotyping system for analysis of diverse non-B HIV-1 subtypes.

Luminescence detection workstation

Abstract: A luminescence detecting apparatus comprising a chamber (60) containing a collimator (110), a Fresnel field lens (120), a filter (130), and a camera lens (140), whereupon a focused image is created by the optics onto a charge-coupled device camera (70).

Water-soluble rhodamine dye peptide conjugates

Abstract: The present invention provides novel, water-soluble, red-emitting fluorescent rhodamine dyes and red-emitting fluorescent energy-transfer dye pairs, as well as labeled conjugates comprising the same and methods for their use. The dyes, energy-transfer dye pairs and labeled conjugates are useful in a variety of aqueous-based applications, particularly in assays involving staining of cells, protein binding, and/or analysis of nucleic acids, such as hybridization assays and nucleic acid sequencing.

Applying environmental product design to biomedical products research.

Abstract: The principal themes for the Biomedical Research and the Environment Conference Committee on Environmental Economics in Biomedical Research include the following: healthcare delivery companies and biomedical research organizations, both nonprofit and for-profit, need to improve their environmental performance; suppliers of healthcare products will be called upon to support this need; and improving the environmental profile of healthcare products begins in research and development (R&D). The committee report begins with requirements from regulatory authorities (e.g., U.S. Environmental Protection Agency [EPA], the U.S. Food and Drug Administration), and the healthcare delivery sector). The 1998 American Hospital Association and EPA Memorandum of Understanding to reduce solid waste and mercury from healthcare facilities is emblematic of these requirements. The dominant message from the requirements discussion is to ensure that R&D organizations do not ignore customer, environmental, and regulatory requirements in the early stages of product development. Several representatives from healthcare products manufacturers presented their companies' approaches to meeting these requirements. They reported on efforts to ensure that their R&D processes are sensitive to the environmental consequences from manufacturing, distributing, using, and disposing of healthcare products. These reports describe representatives' awareness of requirements and the unique approaches their R&D organizations have taken to meet these requirements. All representatives reported that their R&D organizations have embraced environmental product design because it avoids the potential of returning products to R&D to improve the environmental profile. Additionally, several reports detailed cost savings, sustainability benefits, and improvements in environmental manufacturing or redesign, and increased customer satisfaction. Many companies in healthcare delivery are working to improve environmental performance. Fundamental to these efforts is the necessity of motivating suppliers to improve the environmental profile of new products used in the healthcare delivery sector.

Analysis and Purification of Synthetic Nucleic Acids Using HPLC

Abstract: HPLC is a powerful and popular method for analyzing and purifying biomolecules. Reversed-phase HPLC allows a high-capacity method for purification, and uses volatile buffer systems that simplify product recovery. Anion-exchange HPLC provides better resolution and a more predictable elution pattern. This unit presents protocols that are optimized for HPLC of oligonucleotides. Because of the resolution limits of both reversed-phase and anion-exchange HPLC, it can be used for oligonucleotides of up to approximately 50 nt in length.

Extended rhodamine compounds useful as fluorescent labels

Abstract: Extended rhodamine compounds exhibiting favorable fluorescence characteristics having structure (I) or (II) are disclosed. In addition, novel intermediates for synthesis of these dyes are disclosed, such intermediates having structure (III). In addition, methods of making and using the dyes as fluorescent labels are disclosed.

Parallel sample introduction electrospray mass spectrometer with electronic indexing through multiple ion entrance orifices

Abstract: An interface apparatus, for coupling a plurality of ion sources to a mass spectrometer has a plurality of ion sources for generating a plurality of ion beams. An inlet device for passing ion beams into the mass spectrometer is provided as is a device or mechanism for selecting one of the ion beams for passage through into the mass spectrometer and for blocking the other ion beams. An outlet provides a connection to a mass spectrometer. A corresponding method is provided.

A Novel High Throughput Chemiluminescent Assay for the Measurement of Cellular Cyclic Adenosine Monophosphate Levels

Abstract: The second messenger 3', 5'-cyclic AMP (cAMP) is a highly regulated molecule that is governed by G protein-coupled receptor activation and other cellular processes. Measurement of cAMP levels in cells is widely used as an indicator of receptor function in drug discovery applications. We have developed a nonradioactive ELISA for the accurate quantitation of cAMP levels produced in cell-based assays. This novel competitive assay utilizes chemiluminescent detection that affords both a sensitivity and a dynamic assay range that have not been previously reported with any other assay methodologies. The assay has been automated in 96- and 384-well formats, providing assay data that are equivalent to, if not better than, data generated by hand. This report demonstrates the application of this novel assay technology to the functional analysis of a specific G protein-coupled receptor, neuropeptide receptor Y1, on SK-N-MC cells. Our data indicate the feasibility of utilizing this assay methodology for monitoring cAMP levels in a wide range of functional cell-based assays for high throughput screening.

A new papillomavirus of possums (Trichosurus vulpecula) associated with typical wart-like papillomas.

Abstract: A previously unknown, cutaneous papillomavirus (Papovaviridae) in a brushtail possum (Trichosurus vulpecula) was demonstrated. This represents one of the first viruses reported in this species. Possum papillomas were identified by typical wart-like appearance and histology. Papillomavirus particles were detected by electron microscopy in tissue homogenates following purification and negative staining. The polymerase chain reaction amplified a conserved portion of the L1 gene which was purified and sequenced. Comparison of the DNA and deduced amino acid sequence from the possum papillomavirus with other papillomavirus sequences, together with phylogenetic analysis, indicated that this was a new papillomavirus.

Polyacrylamide gel electrophoresis (PAGE) of synthetic nucleic acids.

Abstract: Protocols are given for analysis of oligonucleotides by PAGE, using either methylene blue staining or radiolabeling to mark the oligonucleotide. In addition, a separate protocol is provided for purification by PAGE.

5′ Nuclease Assays for the Loci CCR5-+/Δ32, CCR2-V64I, and SDF1-G801A Related to Pathogenesis of AIDS

Abstract: Background: Variations within the human genome play important roles in human disease. To study variations related to susceptibility to AIDS, we have developed 5′ nuclease assays that eliminate post-PCR molecular biology steps. Methods: TaqMan assays based on the 5′ nuclease activity of Taq polymerase and fluorescent resonance energy transfer were developed to score alleles at the biallelic loci CCR5 - + /Δ 32 , CCR2-V64I and SDF1-G801A . For each assay, 72 samples were analyzed. Data collection and analysis were performed on the Prism 7700 Sequence Detection System. For comparison with gel electrophoresis methods, each locus was also scored on a subset of 24 samples, using restriction enzymes or single-strand conformational polymorphism (SSCP). Results: Clear allelic discrimination was obtained on each of the 72 samples for all three TaqMan assays. The TaqMan scores for the subset of 24 samples were concordant with the restriction enzyme and SSCP scores. Conclusions: Because of its simplicity, speed, and potential for automation and miniaturization, TaqMan is an excellent candidate for investigation of genetic variation in clinical, research, and forensic settings.

Base Composition Analysis of Nucleosides Using HPLC

Abstract: In this protocol, nuclease digestion of an oligonucleotide is followed by dephosphorylation and HPLC analysis of the monomers on a reversed-phase C18 column. This method can be used to detect and quantitate a wide variety of nucleobase modifications in oligonucleotides. Integrated areas of the nucleoside chromatogram give precise quantitation of nucleoside composition when the relative extinction coefficient cofactors are applied to the sum of the areas of the four bases. The protocol is also useful for analysis of oligonucleotides containing conjugated moieties and carbohydrate modifications.

Overview of purification and analysis of synthetic nucleic acids.

Abstract: Synthetic nucleic acids are produced routinely for a wide variety of applications, including biological and chemical research, and diagnostic or therapeutic applications. To ensure an adequate level of quality and purity, rapid and convenient analytical methods are necessary. This unit discusses basic principles to guide in the selection of appropriate purification and analysis protocols.

A rapid and simple method for quantitation of urinary hydroxylysyl glycosides, indicators of collagen turnover, using liquid chromatography/tandem mass spectrometry.

Abstract: Some glycosides of hydroxylysine, viz., alpha-1, 2-glucosylgalactosyl-O-hydroxylysine and beta-1-galactosyl-O-hydroxylysine, appear to be good indicators of collagen turnover. A simple liquid chromatography/tandem mass spectrometry (LC/MS/MS) method is proposed for measuring these analytes in urine, with no sample preparation except for a dilution step. Quantitation is performed using external calibration with no internal standard. A preliminary survey indicates good intra- and inter-day reproducibility (better than 5 and 8%, respectively). With the present method, the estimated limits of detection (S/N > 3) in urine are 0.8 and 0.5 microM/L for beta-1-galactosyl-O-hydroxylysine and alpha-1,2-glucosylgalactosyl-O-hydroxylysine, respectively. The method is proposed as a robust tool for a large-scale research investigation on collagen turnover.

Eleven densely clustered genes, six of them novel, in 176 kb of mouse t-complex DNA.

Abstract: Targeted sequencing of the mouse t-complex has started with a 176-kb, gene-rich BAC localized with six PCR-based markers in inversion 2/3 of the highly duplicated region. The sequence contains 11 genes recovered primarily as cDNAs from early embryonic collections, including Igfals (previously placed on chromosome 17), Nubp2 (a fully characterized gene), Jsap1 (a JNK-binding protein), Rsp29 (the mouse homologue of the rat gene), Ndk3 (a nucleoside diphosphate kinase), and six additional putative genes of unknown function. With 50% GC content, 75% of the DNA transcribed, and one gene/16.0 kb (on average), the region may qualify as one of the most gene-dense segments in the mouse genome and provides candidates for dosage-sensitive phenotypes and mouse embryonic lethals mapped to the vicinity.

Receptor function assay for G-protein coupled receptors and orphan receptors by reporter enzyme mutant complementation

Abstract: Methods for detecting G-protein coupled receptor (GPCR) activity; methods of assaying GPCR activity; and methods of screening for GPCR ligands, G-protein-coupled receptor kinase (GRK) activity, and compounds that interact with components of the GPCR regulatory process are described.

Structural determinants for the efficient and specific interaction of thioredoxin with 2-oxoacid dehydrogenase complexes

Abstract: Specificity and efficiency of thiored oxin action upon the 2-oxoacid dehydrogenase complexes are studied by using a number of thiored oxins and complexes. Bacterial and mammalian pyruvate and 2-oxoglutarate dehydrogenase systems display similar row of preference to thioredoxins that may result from thioredoxin binding to the homologous or common dihydrolipoamide dehydrogenase components of the complexes. The most sensitive tothioredoxin is the complex whose component exhibits the highest sequence similarity to eukaryotic thioredoxin reductase. Hence, thioredoxin binding to the complexes may be related to that in the thioredoxin reductase, a dihydrolipoamide dehydrogenase homolog. The highest potency of mitochondrial thioredoxin to affect the mitochondiral complexes is revealed. A 96–100% conservation of the mitochondrial thioredoxin structure is shown within the four known sequences and the N-terminus of the pigheart protein determined. Eleven thioredoxins tested biochemically are analyzed by multiple sequence alignment and homology modeling. Their effects correlate with the residues at the contact between the α 3/310 and α 1 helices, the length of the α 1 helix and charges in the α2–β3 and β4–β5 linkers. Polarization of the thioredoxin molecule and its active site surroundings are characterized. Thioredoxins with a highly polarized surface around the essential disulfide bridge (mitochondiral, pea f, and Arabidopsis thaliana h3) show low cross-reactivity as compared to the species with a decreased polarization of this area (e.g., from Escherichia coli). The strongest polarization of the whole molecule results in the highest magnitude of the electrostatic dipole vector of mitochondrial thioredoxin. Thiored oxins with the dipole orientation similar to that of the latter have the affinities for the 2-oxoacid dehydrogenase complexes, proportional to the dipole magnitudes. Thioredoxin with an opposite dipole orientation shows no effect. Activating and inhibitory thioredoxin disulfides are distinguished by the charges of the residues 13/14 (α1 helix(, 51 (α2–β3 linker), and 83/85 (β4–β5 linker), changing the dipole direction. The results show that the thioredoxin-target interplay may be controlled by the long-range interactions between the electrostatic dipole vectors of the proteins and the degree of their interface polarization.

Cartridge methods for oligonucleotide purification.

Abstract: Protocols are given for purification of oligonucleotides by dimethoxytrityl-sensitive and affinity desalting methods. The protocols are applicable for many of the convenient disposable products available for rapid oligonucleotide purification, clean-up by selective adsorption, and elution on solid-phase media. Many of these products are prepackaged, single-use cartridges or columns filled with affinity or size-exclusion media.

Automated, solid-phase coupling of rhodamine dye acids to 5' amino oligonucleotides.

Abstract: A convenient method has been developed for directly labeling the 5' amino group of oligonucleotides on solid-support with rhodamine dyes.

Water-soluble rhodamine dye and its conjugate

Abstract: PROBLEM TO BE SOLVED: To provide a dye useful in various aqueous base applications, especially in dyeing cells, and in assay including the analyses of nucleic acids such as a bonding of proteins and/or hybridization assay and nucleic acid sequence determination, and energy transfer dye pairs and labeled conjugates. SOLUTION: This new water soluble red color-emitting fluorescent rhodamine dye. the red color-emitting energy transfer dye pairs, the labeled conjugates comprising the same and a method for using them are provided. COPYRIGHT: (C)2006,JPO&NCIPI

Maldi ion source with a pulse of gas, apparatus and method for determining molecular weight of labile molecules

Abstract: A mass spectrometer instrument for determining the molecular weight of labile molecules of biological importance, in particular heavy molecules, such as proteins, peptides or DNA oligomers, is disclosed. The instrument includes a MALDI ion source that is enclosed in a chamber with an inlet for admitting a gas and an ion sampling aperture for limiting gas flow from the chamber. The elevated pressure of the source in the range from 0.1 to 10 torr causes low energy collisions between the gas and the ions that can cause rapid collisional cooling of the excited ions, thereby improving the stability of the produced ions. The formation of clusters of ions (e.g., protein ions) with matrix material is broken without fragmenting the ions by increasing the downstream gas temperature to between 150 and 250°C. Operating the source at laser energy at least two times higher than the threshold value of ion formation and using a high repetition rate laser significantly improve sensitivity of analysis and speed of data acquisition.