Sequencing reactions for the applied biosystems 373A Automated DNA Sequencer.
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Automation↗
Biomedical subjects
R K Wilson
Publications and source records attributed to R K Wilson.
High-throughput purification of M13 templates for DNA sequencing.
Two variants of methods for high-throughput preparation of single-stranded M13 DNA are described. Both variants are derived from previously described chemistry and are appropriate for purification of M13 templates in 96-deep well plates. In both variants, phenol extraction is replaced by treatment with sodium iodide to disrupt phage proteins prior to ethanol precipitation of M13 DNA. In one of the variants, nonderivatized paramagnetic particles are employed to collect aggregated M13 phage particles and DNA, thereby replacing the need for centrifugation. The other variant omits the magnetic particles and utilizes a centrifuge that can accommodate the 96-deep well plates. Although the purification scheme that uses magnetic separation results in a decreased yield of M13 DNA, it is amenable to robotic automation strategies and thus will be useful for genomic sequencing projects. Performed manually, either method can easily produce 192 templates in a few hours. Although both variants produce DNA of sufficient quantity for automated fluorescent DNA sequencing, the procedure that utilizes magnetic separation provides template DNA of higher quality.
Autoanalysis↗
Duck liver 'malic' enzyme. Expression in Escherichia coli and characterization of the wild-type enzyme and site-directed mutants.
A cDNA for duck liver 'malic' enzyme (EC 1.1.1.40) was subcloned into pUC-8, and the active enzyme was expressed in Escherichia coli TG-2 cells as a fusion protein including a 15-residue N-terminal leader from beta-galactosidase coded by the lacZ' gene. C99S and R70Q mutants of the enzyme were generated by the M13 mismatch technique. The recombinant enzymes were purified to near homogeneity by a simple two-step procedure and characterized relative to the enzyme isolated from duck liver. The natural duck enzyme has a subunit molecular mass of approx. 65 kDa, and the following kinetic parameters for oxidative decarboxylation of L-malate at pH 7.0: Km NADP+ (4.6 microM); Km L-malate (73 microM); kcat (160 s-1); Ka (2.4 microM) and Ka' (270 microM), dissociation constants of Mn2+ at 'tight' (activating) and 'weak' metal sites; and substrate inhibition (51% of kcat. at 8 mM-L-malate). Properties of the E. coli-derived recombinant wild-type enzyme are indistinguishable from those of the natural duck enzyme. Kinetic parameters of the R70Q mutant are relatively unaltered, indicating that Arg-70 is not required for the reaction. The C99S mutant has unchanged Km for NADP+ and parameters for the 'weak' sites (i.e. inhibition by L-malate, Ka'); however, kcat. decreased 3-fold and Km for L-malate and Ka each increased 4-fold, resulting in a catalytic efficiency [kcat./(Km NADP+ x Km L-malate x Ka)] equal to 3.7% of the natural duck enzyme. These results suggest that the positioning of Cys-99 in the sequence is important for proper binding of L-malate and bivalent metal ions.
Amino Acid Sequence↗
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.
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.
Base Sequence↗
Fluorescence chemistries for automated primer-directed DNA sequencing.
A major limitation in the applicability of automated DNA sequencing instruments has been the difficulty in using user-defined oligonucleotide primers which allow sequencing reactions to start at any specific point in a region of interest. Recently, new chemistries have become available for fluorescent labeling which will begin to facilitate the use of any oligonucleotide primer with automated DNA sequencers. In this report, we describe several methods for automated primer-directed DNA sequencing, and compare and discuss the relative merits and limitations of these methods.
Automation↗
Nucleotide sequence analysis of 95 kb near the 3' end of the murine T-cell receptor alpha/delta chain locus: strategy and methodology.
The nucleotide sequence of a region at the 3' terminus of the murine T-cell receptor alpha/delta chain locus is presented. This region, which encodes the constant region genes for alpha and delta chain polypeptides and all 50 joining gene segments for the alpha chain polypeptide, spans 94,647 bp and includes more than 50 noncoding sequence elements important for T-cell receptor gene rearrangement and expression. DNA sequencing of this region included complete analysis of two cosmid clones and five additional restriction fragments using a random subcloning approach with various manual and automated sequencing strategies. The automated sequencing strategies hold considerable promise for future large-scale DNA sequencing efforts.
Animals↗
Organization, structure, and function of 95 kb of DNA spanning the murine T-cell receptor C alpha/C delta region.
We have analyzed the organization, structure, and function of the murine T-cell receptor C alpha/C delta region. This region spans 94.6 kb of DNA and contains the C alpha and C delta genes, as well as the V delta 5, J delta 2, and 50 different J alpha gene segments. Within this sequence we have identified 15 new J alpha gene segments, 40 new 5' RNA splice signals, and 40 new DNA rearrangement signals for the J alpha gene segments. The murine C alpha/C delta sequence contains an exceptionally high level of coding sequence with over 5.7% of the total sequence found in the exons. This is much more than that found in the beta-globin locus and the HPRT locus. Using the sequence data obtained from the C alpha/C delta region, we have designed simple assays to test for J alpha gene segment transcription and to determine the level of polymorphism for simple repeat sequences among different inbred strains of mice using the polymerase chain reaction. Furthermore, comparisons of this 95 kb of sequence with the available sequence from homologous regions of other species have led to the identification of a highly conserved sequence that is present throughout vertebrates and in the mouse binds lymphocyte-specific nuclear proteins. Comparisons of a 10-kb region, which includes the C alpha gene in human and mouse, average 66% sequence similarity. These studies support the contention that large-scale DNA sequencing projects of homologous regions of mouse and human will provide powerful new tools for studying the biology and evolution of loci such as the T-cell receptor and for identifying and posing new questions about the functions of conserved sequences.
Amino Acid Sequence↗
Multiple trans-splicing events are required to produce a mature nad1 transcript in a plant mitochondrion.
The mitochondrial gene encoding NADH dehydrogenase subunit 1 (nad1) in Petunia hybrida is split into five exons, a, b, c, d, and e. With the use of a complete restriction map of the 443-kb Petunia mitochondrial genome, we have cloned these exons and mapped their location. Exon a is located 130 kb away from and in the opposite orientation from exons b and c. Exon d maps 95 kb away and in the opposite orientation from exons b and c. Exons d and e are separated by 190 kb. By performing the polymerase chain reaction on Petunia cDNAs, we have shown that transcripts from these five exons are joined via a series of cis- and trans-splicing events to create a mature nad1 transcript. In addition, we have found 23 C----U RNA edit sites in Petunia nad1. RNA editing changes 19 of the amino acids predicted by the genomic sequence.
Amino Acid Sequence↗
Low pressure DNA shearing: a method for random DNA sequence analysis.
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Animals↗
Sequence and expression of a novel human T-cell receptor beta-chain variable gene segment subfamily.
We describe the characterization of a novel subfamily of human T-cell receptor beta-chain variable TCRBV gene segments, detected through homology to a mouse Tcrb-V13 gene segment probe. The new human TCRBV gene segment subfamily TCRBV21 contains three members, all of which have been characterized with respect to nucleotide sequence, chromosomal location, and expression in human thymocytes and peripheral T lymphocytes. Our results indicate that one member of the TCRBV21 subfamily is not expressed in either tissue type although its coding region, promoter, recognition, and splice sequences all appear to be functional at the nucleotide level, possibly indicating regulation at the level of transcription.
Base Sequence↗
Development of an automated procedure for fluorescent DNA sequencing.
We describe here the development of a procedure for complete automation of the dideoxynucleotide DNA sequencing chemistry using fluorescent dye-labeled oligonucleotide primers. This procedure combines rapid preparation of template DNA using a modification of the polymerase chain reaction, automation of the DNA sequencing reactions using a robotic laboratory workstation, and subsequent analysis of the fluorescent-labeled reaction products on a commercial automated fluorescent sequencer. Using this procedure, we were able to produce sufficient quantities of template DNA directly from bacterial colonies or bacteriophage plaques, perform the DNA sequencing reactions on these templates, and load the reaction products on the fluorescent DNA sequencer in a single work day. This scheme for automation of the fluorescent DNA sequencing method allows the fluorescent sequencer to be run at its full capacity every day and eliminates much of the labor required to obtain a high level of data output. Currently, we are able to perform and analyze 16 fluorescent-labeled reactions every day, with an average output of over 7000 bp per sequencer run.
Automation↗
Influence of smoking on radiographic profusion and pleural changes in asbestos-exposed subjects.
The influence of smoking on pleural and parenchymal radiographic changes, particularly in asbestos-exposed cohorts, has been debated. We studied this issue in 440 asbestos-exposed subjects referred for evaluation between 1981 and 1985. Information was obtained on age, exposure, and smoking. Chest radiographs were independently read by three "B" readers under blind conditions and it was determined that 80.5% of the subjects had a history of smoking. The odds ratio for smoking and parenchymal opacities greater than or equal to 1/0 was 1.98 (95% confidence interval, 1.2 to 3.3); this association was not affected by adjustment for asbestos exposure and/or age. A dose-response pattern emerged between increasing pack-years and parenchymal opacities greater than or equal to 1/0; the effect between asbestos exposure and smoking appeared to be additive. No associations were found between smoking and pleural abnormalities. The findings in this investigation are consistent with prior studies showing an influence of smoking on radiographic parenchymal, but not pleural, changes, at least in asbestos-exposed groups. The anatomical equivalent of these radiographic changes and their impact, if any, on the natural history of asbestos-associated disease remains unclear.
Aged↗
Optimization of asymmetric polymerase chain reaction for rapid fluorescent DNA sequencing.
A high-throughput method for the preparation of single-stranded template DNA, which is suitable for sequence analysis using fluorescent labeling chemistry, is described here. In this procedure, the asymmetric polymerase chain reaction is employed to amplify recombinant plasmid or bacteriophage DNA directly from colonies or plaques. The use of amplification primers located at least 200 base pairs 5' to the site of sequencing primer annealing removes the need for extensive purification of the asymmetric polymerase chain reaction product. Instead, the single-stranded product DNA is purified by a simple isopropanol precipitation step and then directly sequenced using fluorescent dye-labeled oligonucleotides. This method significantly reduces the time and labor required for template preparation and improves fluorescent DNA sequencing strategies by providing a much more uniform yield of single-stranded DNA.
Animals↗
Sequencing reactions in microtiter plates.
We describe a simple and inexpensive method of performing sequencing reactions for 24 single-strand M13 DNA clones in microtiter plates. To simplify elevated temperature incubations during sequencing reactions, two heating blocks were designed to accommodate microtiter plates and fit within common laboratory heating modules. With only slight modification of standard fluorescent and radioisotopic sequencing methods, the sequencing reactions for 24 clones can be done in as little as 40 minutes.
Bacteriophages↗
Bronchoalveolar lavage analysis, gallium-67 lung scanning and soluble interleukin-2 receptor levels in asbestos exposure.
This study examined different markers of lung immunologic and inflammatory responses to previous asbestos exposure. We performed bronchoalveolar lavage (BAL) and gallium-67 (67Ga) lung scans and measured serum and BAL soluble interleukin-2 receptor (IL-2R) and angiotensin-converting enzyme (SACE) levels in 32 subjects with a history of significant asbestos exposure, 14 without (EXP) and 18 with (ASB) radiographic evidence of asbestosis. BAL analysis revealed increases in neutrophils in both ASB and EXP when compared to controls (P less than 0.01), which persisted after adjustment for smoking category. Although significant abnormalities of macrophage and total lymphocyte profiles were not found in the study population, lymphocyte subpopulation analysis revealed elevation of BAL T4/T8 ratios in the entire study group (ASB + EXP) when compared to controls (P less than 0.05), independent of smoking category. 67Ga lung scan activity was increased in 56% of ASB and in 36% of EXP: no correlations between positive scans and different radiological and functional parameters could be found. There was no significant elevation of mean SACE, serum, or BAL IL-2R levels in any of the study categories. These data suggest that asbestos exposure may be associated with parenchymal inflammation, even in the absence of clinical criteria for asbestosis. Abnormalities of gallium uptake and of BAL analysis reflect the clinically inapparent inflammation. The increased BAL T4/T8 ratios observed suggest that abnormal local pulmonary immunoregulation may play a role in the pathogenesis of asbestos-related lung diseases.
Adult↗
Physical maps of the mouse and human immunoglobulin-like loci.
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Animals↗
Presence of the hypermodified nucleotide N6-(delta 2-isopentenyl)-2-methylthioadenosine prevents codon misreading by Escherichia coli phenylalanyl-transfer RNA.
The overall structure of transfer RNA is optimized for its various functions by a series of unique post-transcriptional nucleotide modifications. Since many of these modifications are conserved from prokaryotes through higher eukaryotes, it has been proposed that most modified nucleotides serve to optimize the ability of the tRNA to accurately interact with other components of the protein synthesizing machinery. When a cloned synthetic Escherichia coli tRNAPhe gene was transfected into a bacterial host that carried a defective phenylalanine tRNA-synthetase gene, tRNAPhe was overexpressed by 11-fold. As a result of this overexpression, an undermodified tRNAPhe species was produced that lacked only N6-(delta 2-isopentenyl)-2-methylthioadenosine (ms2i6A), a hypermodified nucleotide found immediately 3' to the anticodon of all major E. coli tRNAs that read UNN codons. To investigate the role of ms2i6A in E. coli tRNA, we compared the aminoacylation kinetics and in vitro codon-reading properties of the ms2i6A-lacking and normal fully modified tRNAPhe species. The results of these experiments indicate that while ms2i6A is not required for normal aminoacylation of tRNAPhe, its presence stabilizes codon-anticodon interaction and thereby prevents misreading of the genetic code.
Adenosine↗
Asbestos-associated disease: a review.
Asbestos and its potential for adversely affecting health remain a source of concern to several sectors of society. Since it rarely occurs in the absence of occupational exposure to asbestos, and because it is potentially preventable, asbestosis was recently defined as a reportable occupational disease in Texas. An overview of the cardinal characteristics of the asbestos minerals and their associated health effects is presented. The role of the primary physician in diagnosis and counseling of individuals with asbestos-associated diseases is addressed.
Asbestosis↗