Showing papers by "Applied Biosystems published in 1998"
TL;DR: The scientific rationale for the proposal and the business plan of the company are described, which aims to sequence the human genome four years faster and at lower cost than the government-backed Human Genome Project.
Abstract: The recent public announcement by TIGR and Perkin-Elmer that they intend to form a new company whose goal is to sequence the human genome four years faster and at lower cost than the government-backed Human Genome Project caught the immediate attention of the community. Venter
et al. now describe the scientific rationale for their proposal and outline the business plan of the company.
359 citations
TL;DR: Data show that MicroSeq provides rapid, unambiguous identification of clinical bacterial isolates, and the improved turnaround time provided by genotypic identification systems may translate into improved clinical outcomes.
Abstract: Rapid and accurate identification of bacterial pathogens is a fundamental goal of clinical microbiology, but one that is difficult or impossible for many slow-growing and fastidious organisms. We used identification systems based on cellular fatty acid profiles (Sherlock; MIDI, Inc., Newark, Del.), carbon source utilization (Microlog; Biolog, Inc., Hayward, Calif.), and 16S rRNA gene sequence (MicroSeq; Perkin-Elmer Applied Biosystems Division, Foster City, Calif.) to evaluate 72 unusual aerobic gram-negative bacilli isolated from clinical specimens at the Mayo Clinic. Compared to lengthy conventional methods, Sherlock, Microlog, and MicroSeq were able to identify 56 of 72 (77.8%), 63 of 72 (87.5%), and 70 of 72 (97.2%) isolates to the genus level (P = 0.002) and 44 to 65 (67.7%), 55 of 65 (84.6%), and 58 of 65 (89.2%) isolates to the species level (P = 0.005), respectively. Four Acinetobacter and three Bordetella isolates which could not be identified to the species level by conventional methods were identified by MicroSeq. In comparison to the full 16S rDNA sequences, the first 527 bp provided identical genus information for all 72 isolates and identical species information for 67 (93.1%) isolates. These data show that MicroSeq provides rapid, unambiguous identification of clinical bacterial isolates. The improved turnaround time provided by genotypic identification systems may translate into improved clinical outcomes.
277 citations
Patent•
16 Apr 1998TL;DR: In this article, a process for binding nucleic acid to solid phase matrices exhibiting sufficient hydrophilicity and electropositivity to tightly bind the nucleic acids from a sample is described.
Abstract: This invention is directed to a process for tightly binding nucleic acid to solid phase and corresponding processes for the utilization thereof. Nucleic acid is bound to solid phase matrices exhibiting sufficient hydrophilicity and electropositivity to tightly bind the nucleic acids from a sample. These processes include nucleic acid (double or single stranded DNA and RNA) capture from high volume and/or low concentration specimens, buffer changes, washes, and volume reductions, and enable the interface of solid phase bound nucleic acid with enzyme, hybridization or amplification strategies. The tightly bound nucleic acid may be used, for example, in repeated analyses to confirm results or test additional genes in both research and commercial applications. Further, a method is described for virus extraction, purification, and solid phase amplification from large volume plasma specimens.
185 citations
TL;DR: Using the recently introduced BigDye terminators, large-template DNA can be directly sequenced with custom primers on automated instruments without additional manipulations of template DNA, thereby bypassing tedious subcloning steps.
Abstract: In microbial genome or large-insert clone sequencing projects that use the predominant random subclone sequencing strategy, progress tends to decrease dramatically at late stages as one confronts gaps. At these points, DNA is under-represented or unstable in subclones (E.Y. Chen et al. 1996; Chissoe et al. 1997). Further sequencing with additional random subclones is then inefficient at best, and one must frequently employ alternative cloning systems or additional methods like long-range PCR to recover missing DNA (C.N. Chen et al. 1996). The variability of performance of these methods and the necessity for custom-tailored work tend to hamper the late stages of sequencing efforts. In contrast, if one can sequence directly from genomic DNA (or large-insert clones such as BACs or PACs) with walking primers, cumbersome work to fill gaps could be completed in a much shorter time.
As an example, in a recent project to sequence the 750-kb genome of Ureaplasma urealyticum (J. Glass, in prep.) assemblage of ∼13,000 sequence reads and combinatorial PCR reactions to join contigs left two gaps. No λ pUC, or M13 subclones were recovered that spanned the gaps, nor were PCR products derived with any of several sets of flanking primers. The difficulty of cloning these segments is probably attributable to repeated sequences in and near the two gaps, but the high sensitivity of the recently introduced BigDye terminator (Rosenblum et al. 1997) permitted direct sequencing of the gap regions on genomic U. urealyticum DNA templates. Using the conditions described in this report, two gaps of 259 and 121 bp were sequenced from both strands with walking primers to complete the project of 751,723 bp.
Direct sequencing was further tested for larger templates, and good results were reproducibly obtained with 1.2-Mb Mycoplasma fermentans, 2.3-Mb Streptococcus pneumoniae, and 4.6-Mb Escherichia coli genomic DNA (see example in Fig. Fig.1).1). In addition, several difficult gaps in sequencing projects with BAC clones, ranging in size from 140 to 250 kb, have also been filled in this manner. Essentially the method is applicable whenever 2–3 μg of high-quality large-template DNA is available.
Figure 1
Sequencing of E. coli K12 strain genomic DNA with BigDye terminators. Approximately 3 μg of E. coli DNA was sequenced with an apaG gene primer (5′-GTTCCCACACTCATTCATTA) using the conditions described in the text.
173 citations
TL;DR: The SZ-primed,eaeA-targeted 5′ nuclease detection system demonstrated that it can be integrated readily into standard culturing procedures and that the assay can be useful as a rapid, automatable process for the presumptive identification of E. coli O157:H7 in ground beef and potentially in other food and environmental samples.
Abstract: Presumptive identification of Escherichia coli O157:H7 is possible in an individual, nonmultiplexed PCR if the reaction targets the enterohemorrhagic E coli (EHEC) eaeA gene In this report, we describe the development and evaluation of the sensitivity and specificity of a PCR-based 5' nuclease assay for presumptively detecting E coli O157:H7 DNA The specificity of the eaeA-based 5' nuclease assay system was sufficient to correctly identify all E coli O157:H7 strains evaluated, mirroring the previously described specificity of the PCR primers The SZ-primed, eaeA-targeted 5' nuclease detection assay was capable of rapid, semiautomated, presumptive detection of E coli O157:H7 when >/=10(3) CFU/ml was present in modified tryptic soy broth (mTSB) or modified E coli broth and when >/=10(4) CFU/ml was present in ground beef-mTSB mixtures Incorporating an immunomagnetic separation (IMS) step, followed by a secondary enrichment culturing step and DNA recovery with a QIAamp tissue kit (Qiagen), improved the detection threshold to >/=10(2) CFU/ml Surprisingly, immediately after IMS, the sensitivity of culturing on sorbitol MacConkey agar containing cefeximine and tellurite (CT-SMAC) was such that identifiable colonies were demonstrated only when >/=10(4) CFU/ml was present in the sample Several factors that might be involved in creating these false-negative CT-SMAC culture results are discussed The SZ-primed, eaeA-targeted 5' nuclease detection system demonstrated that it can be integrated readily into standard culturing procedures and that the assay can be useful as a rapid, automatable process for the presumptive identification of E coli O157:H7 in ground beef and potentially in other food and environmental samples
173 citations
TL;DR: A chimeric protein consisting of CXC-chemokine receptor 4 and the green fluorescent protein (GFP) was used for studying receptor localization and trafficking in real time in stably transduced HeLa, U-937, CEM, and NIH/3T3 cells, suggesting that the protective effect of chemokines against HIV infection can be attributed not only to competition for binding but also to depletion of the co-receptor molecules from the cell surface.
170 citations
TL;DR: Real Time RT-PCR appears to offer advantages over previously used quantitative RT-pcR methods by providing absolute quantitation of the target sequence, expanding the dynamic range of quantitation to over six orders of magnitude, eliminating the post- PCR processing, and reducing labor and carryover contamination.
Abstract: The AML1/ETO fusion transcript can be detected by reverse transcription polymerase chain reaction (RT-PCR) in patients with t(8;21)-associated acute myeloid leukemia (AML) in long-term complete remission (CR). Quantitation of the amount of the fusion transcript during CR may therefore be more predictive of cure or relapse than a simple qualitative assessment. Real Time PCR, a fluorometric-based technique, allows simple and rapid quantitation of a target sequence during the extension phase of PCR amplification, in contrast to end-point quantitative methods. Six patients with t(8;21)(q22;q22) AML, who achieved CR were studied by Real Time RT-PCR at different time intervals following diagnosis and high-dose cytarabine and anthracycline-based induction therapy. Five patients had a diagnostic bone marrow (BM) sample available for molecular analysis. Each patient showed > or = 10(3) copies of the AML1/ETO fusion transcript at diagnosis, and each showed a 2- to 4-log decrease in copy number following successful induction chemotherapy. This is comparable to the log-fold reduction in leukemic blasts that is thought to occur in patients successfully cytoreduced into CR by induction chemotherapy. The sixth patient showed a relatively high copy number immediately following successful remission induction chemotherapy, which continued to increase during early CR and was later followed by relapse. Real Time RT-PCR appears to offer advantages over previously used quantitative RT-PCR methods by providing absolute quantitation of the target sequence, expanding the dynamic range of quantitation to over six orders of magnitude, eliminating the post-PCR processing, and reducing labor and carryover contamination. These features make this an attractive method to prospectively evaluate the prognostic value of AML1/ETO fusion transcript quantitation in a larger patient population with t(8;21)(q22;q22) AML in CR.
166 citations
TL;DR: A low viscosity (ca. 75cP) solution using polydimethylacrylamide (PDMA) was developed for separating DNA sequencing extension products by capillary electrophoresis (CE) and gave a length-of-read (LOR) value of approximately 600 bases in about 2 h using four-color sequencing.
Abstract: A low viscosity (ca. 75cP) solution using polydimethylacrylamide (PDMA) was developed for separating DNA sequencing extension products by capillary electrophoresis (CE). This medium gave a length-of-read (LOR) value of approximately 600 bases in about 2 h using four-color sequencing in 50 microm capillary at 42 degrees C under a field of 160 V/cm. This medium also works in bare capillaries by noncovalently coating the surface to suppress both electroosmotic flow (EOF) and DNA-capillary wall interactions, and eliminates the need for complicated covalent coatings. At least 100 successive sequencing runs were performed in the same capillary by simply pumping fresh medium after every run, without requiring any reconditioning of the capillary surface between runs. The thermal stability of the noncovalent coating can be improved by adding small amounts of high molecular weight PDMA to the separation medium. The advantages of low viscosity separation media and uncoated capillaries are of paramount importance to develop high-throughput instruments for DNA sequencing.
165 citations
TL;DR: A sensitive and specific assay aimed at measuring 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) has been developed by associating a reversed-phase liquid chromatographic separation with an electrospray tandem mass spectrometric detection.
164 citations
Patent•
27 Oct 1998TL;DR: In the absence of a target sequence, PNA molecular beacons facilitate efficient energy transfer between the linked donor and acceptor moieties of the probe, which can be used to detect, identify or quantitate the target sequence in a sample as discussed by the authors.
Abstract: This invention is directed to methods, kits and compositions pertaining to PNA Molecular Beacons. PNA Molecular Beacons comprise self-complementary arm segments and flexible linkages which promote intramolecular or intermolecular interactions. In the absence of a target sequence, PNA Molecular Beacons facilitate efficient energy transfer between the linked donor and acceptor moieties of the probe. Upon hybridization of the probe to a target sequence, there is a measurable change in at least one property of at least one donor or acceptor moiety of the probe which can be used to detect, identify or quantitate the target sequence in a sample.
161 citations
TL;DR: The precision of the 310 capillary electrophoresis system, coupled with software developed for automated genotyping of alleles based on the use of an allelie ladder, allows for accurate genotypesing of STR loci.
Abstract: Automated fluorescence analysis of polymerase chain reaction (PCR)-amplified short tandem repeat (STR) systems by capillary electrophoresis (CE) is becoming an established tool both in forensic casework and in the implementation of both state and national convicted offender DNA databases. A new capillary electrophoresis instrument, the ABI Prism 310 Genetic Analyzer, along with the Performance Optimized Polymer 4 (POP-4) provides an automated and precise method for simultaneously analyzing ten flourescently labeled STR loci from a single PCR amplification kit, which provides a power of discrimination of approximately one in five billion from a single PCR amplification. Data are presented on sizing precision, sizing accuracy, and resolution for the STR loci in the AmpFlSTR Profiler™ kit. Sizing accuracy is highly dependent on the electrophoresis system, and therefore the reporting of alleles based on the nucleotide size obtained from an electrophoresis system is not recommended for forensic work. The precision of the 310 capillary electrophoresis system, coupled with software developed for automated genotyping of alleles based on the use of an allelie ladder, allows for accurate genotyping of STR loci. Sizing precision of ⩽ 0.16 nucleotide standard deviation was obtained with this system, thus allowing for accurate genotyping of length variants that differ in length by a single nucleotide.
TL;DR: In this article, temperature uniformity and time constant measurements of an 18-vessel array using encapsulated liquid crystals suspended in the liquid were obtained by measuring the temperature distribution in the reacting liquid.
Abstract: Polymerase chain reaction (PCR) using micromachined structures promises improved temperature uniformity and cycling time together with decreased reagent and sample volumes. Thermal design of these structures will benefit from measurements of the temperature distribution in the reacting liquid. We report measurements of temperature uniformity and time constant in a microfabricated 18-vessel array using encapsulated liquid crystals suspended in the liquid. Separate sets of crystals are used to image temporal and spatial temperature variations near the processing thresholds of 55/spl deg/C and 95/spl deg/C with a resolution of 0.1/spl deg/C. While the thermometry technique developed here is particularly useful for characterizing microfabricated PCR systems, it can also aid with the thermal design of a broad variety of microfluidic devices.
TL;DR: Comparative sizing of known CEPH samples performed showed the usefulness of the system for genotyping with standard deviations of 0.24 nucleotide, or better, and the presence of denaturants and, more importantly, operation at 60 degrees C was mandatory for high-precision and high-resolution sizing operation.
Abstract: Genotyping, as applied to linkage mapping, human identification, or mapping of genetic traits, mandates electrophoretic separation systems that enable a user to identify alleles with high precision to obtain a correct genotype. For 2-bp microsatellites or short tandem repeats (STRs), standard deviations of ±0.3 nucleotide are required to ensure with 99.7% probability the identity or dissimilarity of tested alleles. A complete system, consisting of commercially available laser-induced fluorescence capillary electrophoresis (ABI PRISM 310) and performance optimized polymer 4 (POP-4), was evaluated for microsatellite separations. POP-4 is a low viscosity polymer for use in uncoated fused microbore silica capillaries. It separates DNA fragments that differ in size by 1 nucleotide up to 250 nucleotides and that differ in size by 2 nucleotides for fragments up to at least 350 nucleotides in length in about 30 min. The presence of denaturants and, more importantly, operation at 60°C was mandatory for high-precision and high-resolution sizing operation. Reproducible separation performance was achieved in excess of 100 injections per capillary with resulting standard deviations in the range of 0.04 to 0.17 nucleotide. Comparative sizing of known CEPH (Centre d’Etudes du Polymorphisme Humaine) samples performed at 22 independent test sites showed the usefulness of the system for genotyping with standard deviations of 0.24 nucleotide, or better.
Patent•
27 Oct 1998TL;DR: In the absence of a target sequence, Linear Beacons facilitate efficient energy transfer between the donor and acceptor moieties linked to opposite ends of the probe as mentioned in this paper, which can be used to detect, identify or quantitate the target sequence in a sample.
Abstract: This invention is directed to methods, kits and compositions pertaining to Linear Beacons. In the absence of a target sequence, Linear Beacons facilitate efficient energy transfer between the donor and acceptor moieties linked to opposite ends of the probe. Upon hybridization of the probe to a target sequence, there is a measurable change in at least one property of at least one donor or acceptor moiety of the probe which can be used to detect, identify or quantitate the target sequence in a sample.
TL;DR: All such studies indicate that the AmpFISTR Blue PCR Amplification Kit will reproducibly yield specific and sensitive results.
Abstract: Studies were performed as recommended by the Technical Working Group on DNA Analysis Methods (TWGDAM) committee to validate the AmpFISTR Blue PCR Amplification Kit for forensic casework applications. The kit coamplifies the tetranucleotide short tandem repeat (STR) loci D3S1358, vWA, and FGA. The dye-labeled amplification products were electrophoresed and detected directly using the ABI PRISM™ 377 DNA Sequencer or the 310 Genetic Analyzer. CEPH family studies demonstrated Mendelian inheritance of these loci and probability of identity values from population studies were 1/4,830 (African-American), 1/5,479 (U.S. Caucasian), and 1/3,443 (U.S. West Coast Hispanic). In all studies examining different body tissues and fluids, the expected genotypes were observed. Studies to determine and test the PCR reagent components and thermal cycling parameters demonstrated specificity, sensitivity, and balance over a wide range of conditions. Reliable results were obtained from DNA quantities as low as 0.25 ng. A variety of environmental studies were performed, as forensic samples are often exposed to different environmental conditions and substances which may degrade DNA or inhibit the amplification process. Highly degraded samples demonstrated that FGA was the first locus to become undetectable, followed by vWA, and then D3S1358; this is the expected pattern according to locus size. In studies of PCR inhibition, the pattern in which the loci became undetectable was different; FGA was the first locus to become undetectable, followed by D3S1358, and then vWA. Single versus multiple locus amplifications revealed no benefit to single locus analysis, even in cases of degradation or inhibition. The occurrence of preferential amplification was very rare, particularly in noncompromised, unmixed samples. Artifact peaks were not observed in any instance. Mixture studies confirmed the ability to detect mixed DNA samples and included the characterization of stutter and peak height ratios; the limit of detection was 1:10 for 1 ng total genomic DNA and 1:30 for 5 ng. DNA extracted from nonprobative case evidence was successfully amplified and genotyped. All such studies indicate that the AmpFISTR Blue PCR Amplification Kit will reproducibly yield specific and sensitive results.
TL;DR: This publication is the first report on the preparation of the TOM complex from plant mitochondria, and sequence analysis of cDNAs encoding the 7-kDa protein revealed significant sequence homology of this protein to TOM7 from yeast.
TL;DR: There was no distinguishable difference in performance of probes synthesized using the dye-labeled support compared with traditional post-synthetic attachment of rhodamine in PCR for real time detection of a specific PCR product.
Abstract: A fast cleaving non-nucleosidic tetramethylrhodamine dye-labeled support has been developed for automated synthesis of double dye-labeled oligodeoxyribonucleotides in high yield. A mixture (1:1:2) of t-butylamine:methanol:water is used for cleavage and deprotection of dye-labeled oligodeoxyribonucleotides without any degradation or modification of dyes and nucleobases. The cleavage rate of oligodeoxyribonucleotides is significantly increased by using a diglycolate ester linkage instead of the commonly used succinate linkage. These double dye-labeled probes are used in PCR for real time detection of a specific PCR product. Using a 54-exonuclease assay, detected on the ABI PRISM 7700 Sequence Detection System, there was no distinguishable difference in performance of probes synthesized using the dyelabeled support compared with traditional postsynthetic attachment of rhodamine.
TL;DR: The FIA-ionspray-MS-MS method is a fast, quantitative, sensitive, and highly specific alternative method to drug-screening by immunoassays, high-performance liquid chromatography, and GC-MS.
Abstract: A fast analytical approach for the simultaneous quantitative screening for illicit drugs in serum and urine without tedious chromatographic separation steps was developed by combining solid-phase extraction (SPE) followed by flow-injection analysis (FIA) with ionspray-ionization and tandem mass spectrometry (MS-MS) detection using a PE Sciex API 300 triple-quadrupole MS. MS-MS analysis was performed by sequentially isolating the precursor ions of the analytes and their deuterated standards with subsequent fragmentation and monitoring of one fragment ion for each substance. A multiple-reaction monitoring experiment was set up for morphine (MO), codeine (COD), amphetamine (AMP), benzoylecgonine (BZE), and their deuterated analogues. For method evaluation, serum samples spiked with 2-1000 ng of each drug and deuterated standards were extracted by mixed-mode SPE, redissolved in CH3CN-NH4OAc-buffer, and directly injected by flow injection into the ionspray source. The specificity of this new method was demonstrated by testing compounds with similar chemical structure for interferences from the analytes of interest (e.g., dihydromorphone, morphine glucuronide, and 6-monoacetylmorphine with MO; dihydrocodeine and hydrocodone with COD; cocaine [COC] and ecgonine methylester with BZE; methamphetamine with AMP). The possibility of interferences of such compounds with the FIA-ionspray-MS-MS screening method is discussed. Spiked serum samples and serum and urine samples from drug addicts and victims of drug abuse were analyzed with FIA-MS-MS and, after derivatization, with gas chromatography-mass spectrometry (GC-MS). Comparable quantitative results were obtained with both methods; no interferences with metabolites or other compounds were found. The FIA-ionspray-MS-MS method is a fast, quantitative, sensitive, and highly specific alternative method to drug-screening by immunoassays, high-performance liquid chromatography, and GC-MS. It can be used for the simultaneous detection of different drugs and metabolites such as opiates, COC, AMP derivatives, and many other drugs.
TL;DR: Although the shifts in migration times for the p53 mutations relative to the corresponding wild‐type strands could be successfully detected by either SG or CE analysis, the individual electrophoresis run times were about tenfold faster and more automated with capillary electrophoreis.
Abstract: We have analyzed five p53 single point mutations by single strand conformation polymorphism using capillary electrophoresis (CE-SSCP) and have compared these measurements to measurements obtained by slab gel electrophoresis (SG-SSCP). PCR primers were used for amplification of specific exons for mutation detection. 5' Primers were labeled with FAM (5-carboxyfluorescein) and 3' primers were labeled with JOE (2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein). CE-SSCP was performed using the Perkin Elmer ABI PRISM 310 Genetic Analyzer with GeneScan Software and the Beckman P/ACE 5510 CE equipped for laser-induced fluorescence detection. Although the shifts in migration times for the p53 mutations relative to the corresponding wild-type strands could be successfully detected by either SG or CE analysis, the individual electrophoresis run times were about tenfold faster and more automated with capillary electrophoresis. The CE-SSCP measurements were performed at temperatures ranging from 10 to 60 degrees C on a prototype instrument. For mutations measured at ambient temperature (25 degrees C), characteristic shifts in direction and magnitude were observed in the migration times of both strands of all mutations relative to the wild type. This demonstrated the ability of CE at ambient temperature to resolve these mutations. However, the magnitude and direction of shifts in migration time varied with temperature in a discrete pattern for each mutation and resulted in a temperature-specific profile for each mutation. This demonstrated that extended temperature control will be an important advantage in resolving single point mutations by CE-SSCP. In addition, by using CE, discrete intra-strand isoforms could be easily observed at different temperatures. The combination of mutation-specific temperature profiling and analysis of isoforms by CE-SSCP should be of help to the diagnostic community in the detection of genetic mutations.
TL;DR: The basis for the unusual properties of the catalytic subunit (C) of ram sperm cAMP-dependent protein kinase was investigated and it was found that the different amino terminus of sperm C may be related to a unique requirement for localization of the “free” C subunit within the sperm flagellum.
TL;DR: Three recently published works represent the first descriptions of a potential new family of 5-trans-membrane cell surface glycoproteins that may indeed be the homologue of mouse prominin.
TL;DR: The results indicate that this spectrofluorometric method and FRET probes can be used to estimate the efficacy of hybridization between a probe and its target site within highly ordered structures.
Abstract: We previously developed a method for monitoring the integrity of oligonucleotides in vitro and in vivo by quantitating fluorescence resonance energy transfer (FRET) between two different fluorochromes attached to a single oligonucleotide. As an extension of this analysis, we examined changes in the extent of FRET in the presence or absence of target nucleic acids with a specific sequence and a higher-ordered structure. In this system FRET was maximal when probes were free in solution and a decrease in FRET was evidence of successful hybridization. We used a single-stranded oligodeoxyribonucleotide labeled at its 5'-end and its 3'-end with 6-carboxyfluorescein and 6-carboxytetramethylrhodamine, respectively. Incubation of the probe with a single-stranded complementary oligonucleotide reduced the FRET. Moreover, a small change in FRET was also observed when the probe was incubated with an oligonucleotide in which the target site had been embedded in a stable hairpin structure. The decrease in the extent of FRET depended on the length of the stem region of the hairpin structure and also on the higher-ordered structure of the probe. These results indicate that this spectrofluorometric method and FRET probes can be used to estimate the efficacy of hybridization between a probe and its target site within highly ordered structures. This conclusion based on changes in FRET was confirmed by gel-shift assays.
Patent•
30 Oct 1998TL;DR: In this paper, a dispensing assembly in the apparatus has a plurality of heads which are spaced for depositing reagents at selected positions in different array areas in a substrate, allowing parallel deposition in several array areas at reagent regions which are closely spaced relative to the spacing between deposition heads in the assembly.
Abstract: Apparatus for producing a plurality of arrays of reagent regions is disclosed. A dispensing assembly in the apparatus has a plurality of heads which are spaced for depositing reagents at selected positions in different array areas in a substrate. As the heads in the assembly are advanced along array area, the regions in a row in that array are successively filled, allowing parallel deposition in several array areas at reagent regions which are closely spaced relative to the spacing between deposition heads in the assembly. Also disclosed is a method for producing a plurality of arrays using the apparatus.
TL;DR: The first report on the subunit composition of a respiratory enzyme complex from a fern or a horsetail is reported, and an evolutionary model is suggested that assumes a correlation between the presence of an active MPP within the cytochromec reductase complex and the occurrence of chloroplasts.
TL;DR: Finnish HNPCC kindreds are characterized by the predominant involvement of MLH1 and a high rate of shared mutations, offering unique possibilities for mutation screening for both research and diagnostic purposes.
Abstract: Worldwide, the DNA mismatch repair genes MSH2 and MLH1 account for a major share and almost equal proportions of hereditary nonpolyposis colorectal cancer (HNPCC). Furthermore, the predisposing mutation usually varies from kindred to kindred. In this study, we screened 29 verified or putative HNPCC kindreds from Finland for mutations in these two genes and found 8 different mutations, 7 in MLH1 and 1 in MSH2, occurring in 13 families. Four of these mutations were novel. Altogether, we have to date studied 81 kindreds for mutations and 12 different mutations in 52 families have been identified, 10 in MLH1 and 2 in MSH2. These data show that Finnish HNPCC kindreds are characterized by the predominant involvement of MLH1 (49/52, 94% of the families) and a high rate of shared mutations (5/12, 42%) offering unique possibilities for mutation screening for both research and diagnostic purposes. Hum Mutat 11:482–483, 1998. © 1998 Wiley-Liss, Inc.
Patent•
12 Nov 1998TL;DR: In this paper, a serpentine electrophoresis channel for a microchip format is described, where the angles and cross-sections of the two channel portions are such that δtf, the time differential of analyte migration at inner and outer tracks in the first curved portion is equal to δts, and δt, the analyte migrations at outer and inner track in the second curved portion, respectively.
Abstract: A serpentine electrophoresis channel, e.g., for a microchip format, is disclosed. The channel includes pairs of linear segments, e.g., parallel or right-angle segments, each joined by an angled channel region having a first curved channel portion subtending an angle αf>α, where α is the angle between segments in a pair, and a second curved channel portion subtending an angle αs=αf−α. The angles and cross-sections of the two channel portions are such that δtf, the time differential of analyte migration at inner and outer tracks in the first curved portion is equal to δts, the time differential of analyte migration at outer and inner tracks in the second curved portion, respectively.
TL;DR: The full-length dna-pkcs gene was introduced into SX9 and restoration of V(D)J recombination by wild typedna- pkcs cDNA was observed, confirming that SX9 is a noveldna-Pkcs-deficient cell line.
Patent•
20 Mar 1998TL;DR: In this article, an apparatus is provided which includes a sample holder for holding a reaction chamber which includes an optical interface, a fiber optic cable for delivering an excitation beam to a sample housed within the reaction chamber and for receiving light emitted by the sample.
Abstract: An apparatus is provided which includes a sample holder for holding a reaction chamber which includes an optical interface, a fiber optic cable for delivering an excitation beam to a sample housed within the reaction chamber and for receiving light emitted by the sample, and a lens co-axially disposed with the fiber optic cable and positioned outside the reaction chamber for focusing the excitation beam through the optical interface and within a volume of the sample and for collecting and transmitting to the fiber optic cable light emitted within the volume of the sample.
TL;DR: In this article, a real-time fluorescence detection of polymerase chain reaction (PCR) products in various fabricated microstructures using surface chemical analysis has been demonstrated, and the fabrication process method for glass micro-structures for PCR has been optimized.
Abstract: Miniaturization has the potential to impart cost, performance, size, throughput, and ease-of-use benefits to many fields of analysis. We have demonstrated the ability to perform real-time fluorescence detection of polymerase chain reaction (PCR) products in various fabricated microstructures. Utilizing surface chemical analysis, we have optimized the fabrication process method for glass microstructures for PCR. Chemical “cleanliness” supersedes particle issues for these bioanalytical microdevices, a distinct deviation from integrated circuit development from which the microfabrication processes were derived.