Showing papers by "Applied Biosystems published in 1992"

DNA sequencing with dye-labeled terminators and T7 DNA polymerase: effect of dyes and dNTPs on incorporation of dye-terminators and probability analysis of termination fragments.

Abstract: The incorporation of fluorescently labeled dideoxynucleotides by T7 DNA polymerase is optimized by the use of Mn2+, fluorescein analogs and four 2'-deoxyribonucleoside 5'-O-(1-thiotriphosphates) (dNTP alpha S's). The one-tube extension protocol was tested on single-stranded templates, as well as PCR fragments which were made single-stranded by digestion with T7 gene 6 exonuclease. Dye primer sequencing using four dNTP alpha S's was shown to give uniform termination patterns which were comparable to four dNTPs. Efficiency of the polymerase also appeared to improve with the dNTP alpha S's. A mathematical model was developed to predict the pattern of termination based on enzyme activity and ratios of ddNTP/dNTPs. This method can be used to optimize sequencing reactions and to estimate enzyme discrimination constants of chain terminators.

Automated DNA sequencing and analysis of 106 kilobases from human chromosome 19q13.3

Abstract: A total of 116,118 basepairs (bp) derived from three cosmids spanning the ERCC1 locus of human chromosome 19q13.3 have been sequenced with automated fluorescence-based sequencers and analysed by polymerase chain reaction amplification and computer methods. The assembled sequence forms two contigs totalling 105,831 bp, which contain a human fosB proto-oncogene, a gene encoding a protein phosphatase, two genes of unknown function and the previously-characterized ERCC1 DNA repair gene. This light band region has a high average density of 1.4 Alu repeats per kilobase. Human chromosome light bands could therefore contain up to 75,000 genes and 1.5 million Alu repeats.

Rapid and reliable fluorescent cycle sequencing of double-stranded templates.

Abstract: Automated DNA sequencing is an extremely valuable technique which requires very high quality DNA templates to be carried out successfully. While it has been possible to readily produce large numbers of such templates from M13 or other single-stranded vectors for several years, the sequencing of double-stranded DNA templates using the ABI 373 DNA Sequencer has had a considerably lower success rate. We describe how the combination of a new fluorescent, dideoxy sequencing method, called cycle-sequencing, coupled with modifications to template isolation procedures based on Qiagen columns, makes fluorescent sequencing of double-stranded templates a reliable procedure. From a single five milliliter culture enough DNA can be isolated (up to 20 micrograms) to do 4-8 sequencing reactions, each of which yields 400-500 bases of high quality sequence data. These procedures make the routine use of double-stranded DNA templates a viable strategy in automated DNA sequencing projects.

Synthesis of carbon-linked glycopeptides as stable glycopeptide models

Abstract: A C-glycosyl analog of β-Gal-O-Ser has been synthesized and incorporated by automated solid-phase peptide synthesis into a hydrolytically stable, α-helical C-glycopeptide

Separation and Determination of Alkaline-Earth Metal Ions as UV- Absorbing Chelates with EDTA by Capillary Electrophoresis. Determination of Calcium and Magnesium in Water and Serum Samples

Abstract: Capillary electrophoresis of alkaline-earth metal ions was examined with a UV-absorbing chelating agent. The metal1 chelates separated in a capillary were measured by on-column UV-absorptive detection. When ethylenediamine-tetraacetic acid (EDTA) was used as an chelating agent in a carrier solution (pH 9.2), the order of the migration time (t(m)) of metal ions was as follows: Ba(2+)

A new approach to template purification for sequencing applications using paramagnetic particles.

Abstract: A new method using traditional hybridization methodology, coupled with the new magnetic particle technology, has been developed for DNA purification, specifically for sequencing applications. The method is similar to the reverse hybridization blot system; however, a specific oligonucleotide probe was attached to the paramagnetic particle instead of a sheet membrane. The target DNA containing the complementary sequence of the probe hybridizes to the probe that is attached to the bead and is then magnetically removed from solution, washed and collected. This system eliminates the need of organic extractions and precipitation/concentration steps. The entire hybridization-purification system can be done in a 1.5-ml microcentrifuge tube making the method ideal for automation. M13 phage clones were purified with this method, both by manual means and by using the CATALYST 800 Molecular Biology LabStation fitted with a prototype magnetic station, and then sequenced. DNA sequencing results obtained with this system were reproducible and gave excellent length of read with low background.

The use of fluorescence detection and internal lane standards to size PCR products automatically.

Abstract: We have developed chemical procedures, optical and electrophoretic instrumentation and computer software automate the analysis of polymerase chain reaction (PCR) products. DNA molecules labeled with up to four different fluorescent dyes are analyzed within a single electrophoresis gel lane. A size calibration curve is created for each electrophoresis lane from the electrophoretogram of uniquely labeled DNA fragments belonging to an internal lane standard that co-electrophoreses with the PCR products. The unknown molecular lengths of PCR products are automatically calculated from the calibration curve. Data from control experiments with DNA segments of known molecular length demonstrate the accuracy and precision of such sizing. This system has been applied to the analysis of PCR products for research in the areas of human identification, genetic mapping and genetic disease.

Chemical synthesis of RNA using fast oligonucleotide deprotection chemistry.

Abstract: The exocyclic amine protecting groups in oligonucleotide synthesis which require 8-16 hours at 55 degrees C for deprotection in ammonia have been replaced with more labile base protecting groups (dimethylformamidine for adenine and guanine and isobutyryl for cytosine). Using these fast oligonucleotide deprotecting groups which require 2-3 hours at 55 degrees C for complete deprotection, a new set of cyanoethyl phosphoramidite ribonucleoside monomers and supports has been developed. Ribozymes and substrate RNAs which were synthesized with these phosphoramidites were assayed and were found to have full catalytic (biological) activity.

Biopolymer synthesis apparatus and method

Abstract: A cartridge containing a dry polymer composition carrying trapped biopolymer subunits for use in synthesis of biopolymer chains provides a discrete unit for stepwise supply of subunits, such as protected amino acids, in an automated apparatus for biopolymer synthesis. The composition is swellable in organic solvent to release and supply the subunits to a growing biopolymer chain immobilized on a polymer support. An automated synthesizer uses the cartridges for sequential subunit supply and monitors conductivity of deprotection reagents to control cycling and timing of steps.

PHOX2A and PHOX2B genes are highly co-expressed in human neuroblastoma.

Abstract: The detection of PHOX2B mutations in a small proportion of patients affected with either familial or sporadic neuroblastoma (NB), has arisen interest on the possible pathogenic role of this gene in the disease determination. In this light, we have carried out a quantitative expression analysis of PHOX2B and its paralogue PHOX2A on a panel of NB cell lines and NB tumour samples to identify a possible differential expression between NB cells and their normal counterpart (adrenal medulla cells). Our results revealed that both PHOX2A and PHOX2B are over-expressed in tumour samples and NB cell lines. Particularly, the expression levels of the two genes in NB cell lines show a highly significant correlation, suggesting their possible synergistic role or a coordinated expression regulation. Furthermore, PHOX2 gene over-expression in NB tumours and cell lines suggests these genes may be widely involved in NB development through either a direct mechanism of up-regulation or a failure in maintaining proper transcript levels after embryonic development.

Horizontal polyacrylamide gel electrophoresis

Abstract: An electrophoresis apparatus has a gel film cast between two plates and buffer reservoirs at each end of the film with electrodes connectable to an external power supply for providing electromotive force for driving electrophoresis. The reservoirs are configured to wet the ends of the gel film and submerge the electrodes with the apparatus positioned either horizontally or vertically, so gel films can be cast horizontally with sample wells formed in the end of the gel between the plates. Samples may be added to the wells and run into the gel with the apparatus positioned vertically, and the analytical separation may be performed with the apparatus again positioned horizontally, such as in an automatic scanning apparatus.

Fluorescent dye phosphoramidite labelling of oligonucleotides.

Abstract: A series of fluorescein phosphoramidites (FAM) have been synthesized for use on automated DNA synthesizers. After coupling of the FAM reagents to the 5' hydroxyl of the oligonucleotide on the DNA synthesizer, the excess reagent is removed by washing the solid support. The dye, and its linkage to the oligonucleotide, are stable during the conditions of DNA synthesis and cleavage/deprotection conditions. Purification is attained with the OPC (Oligonucleotide Purification Cartridge), a polystyrene based affinity matrix, which selectively retains hydrophobic oligonucleotide conjugates. Analysis by MicroGel capillary electrophoresis effectively separates fluorescent dye labelled oligonucleotides from unlabelled products.

Crystal and molecular structure of the alternating dodecamer d(GCGTACGTACGC) in the A-DNA form: comparison with the isomorphous non-alternating dodecamer d(CCGTACGTACGG)

Abstract: The crystal structure of the alternating dodecamer d(GCGTACGTACGC) (5'-GC) has been determined to a resolution of 2.55A using oscillation film data. The crystals belong to space group P6(1) 22, a = b = 46.2A, c = 71.5A with one strand in the asymmetric unit, and are isomorphous with a previously described non-alternating dodecamer, d(CCGTACGTACGG) (5'-CC). Refinement by X-PLOR/NUCLSQ gave a final R factor of 14.2% for 1089 observations. The molecule adopts the A-DNA form. The interchange of the terminal base pairs in the two dodecamers results in differences in the intermolecular contacts and may account for the differences in the bending. This dodecamer shows an axial deflection of 30 degrees, in the direction of the major groove compared to 20 degrees in 5'-CC and may be a consequence of additional contacts generated in 5'-GC by the interchange of end base pairs. The high helical axis deflection appreciably influences the local helical parameters. The molecule exhibits relatively high inclination angles, and has a narrow major groove. The helical parameters when described relative to the dyad-related hexamer halves of the molecule give more reasonable values. The crystal packing, local helical parameters, torsion angles, and hydration are described and also compared with the non-alternating 5'-CC dodecamer.

Automated system for providing a sequence of chemicals to a reaction process

Abstract: An automated system for providing a preselected sequence of chemicals to a reaction. This apparatus includes a track on which a set of cartridges are placed in a preselected order corresponding to an order in which they are to be used in a reaction process. These cartridges are moved past a point at which these chemicals are extracted for use in the process. The chemicals are preferably in liquid form and are contained in containing having a top seal through which a needle can penetrate to extract chemicals for use in the process. These containers preferably contain the aliquot portion needed for the process, thereby providing a mechanism for providing accurate amounts of each chemical to the process.

Capillary detector cell having imaging elements positioned to optimize sensitivity

Abstract: A detector cell, having a capillary entrance elbow through which a beam of exposing light can be directed into the bore of a central leg of the detector cell capillary. Ray traces, to determine the effect of the entrance elbow on the beam of exposing light are used to optimize performance of this detector cell. Back ray tracing is implemented to illustrate an optimized embodiment. Forward ray tracing is also illustrated and can be used to determine system performance for a range or choices of a single parameter or for concurrent ranges of choices of a subset of the parameters of the beam of exposing light.