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Results for “sanger sequencing”

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At least 631 records · Page 35Linked to original sources
PubMed1988

Bidirectional chain-termination nucleotide sequencing: transposon Tn5seq1 as a mobile source of primer sites.

The sequencing of large DNA fragments by the chain-termination method [Sanger et al., Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5467] has generally required extensive manipulations to bring all parts of the fragment near a specific primer-binding site, or the repeated synthesis of new oligodeoxynucleotide primers. Here we develop a more efficient approach, the use of a transposable element to insert primer binding sites at random in the DNA of interest. We constructed a Tn5 derivative called Tn5seq1 with unique DNA segments near each end so that oligodeoxynucleotides matching them could serve as primers for sequencing in each direction from any Tn5seq1 insertion site. Our experiments demonstrate the use of Tn5seq1 for sequencing in pBR322 plasmids and also in uncloned DNAs of the Escherichia coli chromosome. The unique segments near the left and right ends of Tn5seq1 are promoters from phages T7 and SP6, respectively, to permit the efficient transcription of adjacent DNAs in vivo or in vitro.

Bacteriophage lambda↗
PubMed1986

[Primary structure of the 3'-terminal region of the cloned DNA of the bovine leukemia virus].

The nucleotide sequence of the 3'-terminal region of the cloned bovine leukaemia virus cDNA (1474 bp) was elucidated using both Sanger and Maxam-Gilbert techniques. This DNA region contains U3 and R parts of the BLV LTR and an upstream sequence with four open reading frames (ORF) of unknown function. The comparison of the nucleotide substitutions in these ORF with the two variants of proviral BLV DNA suggests that the only pX1 ORF possesses a coding function. The role of the pX1 protein is discussed.

Animals↗
PubMed2003

Software for automated analysis of DNA fingerprinting gels.

Here we describe software tools for the automated detection of DNA restriction fragments resolved on agarose fingerprinting gels. We present a mathematical model for the location and shape of the restriction fragments as a function of fragment size, with model parameters determined empirically from "marker" lanes containing molecular size standards. Automated identification of restriction fragments involves several steps, including: image preprocessing, to put the data in a form consistent with a linear model; marker lane analysis, for determination of the model parameters; and data lane analysis, a procedure for detecting restriction fragment multiplets while simultaneously determining the amplitude curve that describes restriction fragment amplitude as a function of mobility. In validation experiments conducted on fingerprinted and sequenced Bacterial Artificial Chromosome (BAC) clones, sensitivity and specificity of restriction fragment identification exceeded 96% on restriction fragments ranging in size from 600 base pairs (bp) to 30,000 bp. The integrated suite of software tools, written in MATLAB and collectively called BandLeader, is in use at the BC Cancer Agency Genome Sciences Centre (GSC) and the Washington University Genome Sequencing Center, and has been provided to the Wellcome Trust Sanger Institute and the Whitehead Institute. Employed in a production mode at the GSC, BandLeader has been used to perform automated restriction fragment identification for more than 850,000 BAC clones for mouse, rat, bovine, and poplar fingerprint mapping projects.

Animals↗
PubMed2006

Pathogenesis of enteroaggregative Escherichia coli infection.

Enteroaggregative Escherichia coli (EAEC) is emerging as a significant diarrheal pathogen in multiple population groups. Although most commonly associated with pediatric diarrhea in developing countries, EAEC is also linked to diarrhea in adults including HIV-positive patients and travelers and has been a cause of food-borne outbreaks in the industrialized world. Current data suggest that one set of virulence elements is not associated with all EAEC strains, but that combinations of multiple factors prevail. Pathogenesis is believed to be initiated with adherence to the terminal ileum and colon in an aggregative, stacked-brick-type pattern by means of one of several different hydrophobic aggregative adherence fimbriae. Some strains of EAEC may then elaborate cytotoxins including the plasmid-encoded toxin and the enterotoxins, EAST1 and ShET1. An AraC homolog termed AggR regulates several genes contributing to fimbrial biogenesis in 'typical EAEC strains'. AggR has now also been shown to regulate genes on a chromosomal island. Sequencing of the EAEC type strain 042 completed at the Sanger Center has revealed two other chromosomal islands that are being explored for their pathogenetic potential. This article reviews these virulence elements and presents on-going areas of research in EAEC pathogenesis.

Bacterial Adhesion↗
PubMed2004

Nematode.net: a tool for navigating sequences from parasitic and free-living nematodes.

Nematode.net (www.nematode.net) is a web- accessible resource for investigating gene sequences from nematode genomes. The database is an outgrowth of the parasitic nematode EST project at Washington University's Genome Sequencing Center (GSC), St Louis. A sister project at the University of Edinburgh and the Sanger Institute is also underway. More than 295,000 ESTs have been generated from >30 nematodes other than Caenorhabditis elegans including key parasites of humans, animals and plants. Nematode.net currently provides NemaGene EST cluster consensus sequence, enhanced online BLAST search tools, functional classifications of cluster sequences and comprehensive information concerning the ongoing generation of nematode genome data. The long-term goal of nematode.net is to provide the scientific community with the highest quality sequence information and tools for studying these diverse species.

Animals↗
PubMed2003

GFPE: gene-finding program evaluation.

Gene-finding program evaluation (GFPE) is a set of Java classes for evaluating gene-finding programs. A command-line interface is also provided. Inputs to the program include the sequence data (in FASTA format), annotations of "actual" sequence features, and annotations of "predicted" sequence features. Annotation files are in the General Feature Format promoted by the Sanger center. GFPE calculates a number of metrics of accuracy of predictions at three levels:the coding level, the exon level, and the protein level.

Computational Biology↗
PubMed1972

Nucleotide sequences of two fragments from the coat-protein cistron of bacteriophage R17 ribonucleic acid.

Bacteriophage R17 RNA was labelled with (32)P and was subjected to partial digestion with ribonuclease T(1). The products were fractionated by ionophoresis on polyacrylamide gel. Two fragments were purified and their nucleotide sequences determined by methods involving complete and further partial digestion with ribonucleases A and T(1). Fragment 20 had a sequence that coded for the amino acids in positions 32-53 of the coat protein of the bacteriophage. Fragment 20X, on further purification in 7m-urea, gave rise to two smaller nucleotides whose sequences coded for the amino acids in positions 56-66 and 67-76 of the coat protein. The sequence of the two fragments was such that they could be written in the form of loops stabilized by base-pairing.

Acrylates↗
PubMed1979

Nucleotide sequence coding for the N-terminal region of the matrix protein influenza virus.

After polyadenylation in vitro of the influenza virus RNA segment which contains the coding information for the matrix protein, a cDNA copy can be made using the primer p(dT)8-dA and reverse transcriptase. The sequence of 166 nucleotides of the cDNA was determined by a modification [Brownlee, G. G. & Cartwright, E. M. (1977) J. Mol. Biol, 114, 93--117] of the plus/minus method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441--481] and adaptation of the "dideoxy" method [Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5463--5467] for sequencing DNA. The cDNA sequences is of the same sense as the mRNA for matrix protein and contains a potential initiating codon, d(ATG), at position 26--28. When matrix protein purified from virus particles was digested with chymotrypsin or trypsin and the amino acid compositions of separated peptides determined, one peptide containing nine amino acids found which had a composition corresponding to that predicted by the cDNA sequence following the first methionine codon, confirming that protein synthesis initiates at this position. The compositions of four other peptides matches those predicted from the nucleotide sequence. There is no processing of the N terminus of the protein before incorporation into the virus particle except for removal of the N-terminal methionine and addition of a "blocking" group on the resulting N-terminal serine residue.

Amino Acid Sequence↗
PubMed1998

The Sanger Centre.

Explore the source record for details and available documents.

Academies and Institutes↗
PubMed2000

A preliminary gene map for the Van der Woude syndrome critical region derived from 900 kb of genomic sequence at 1q32-q41.

Van der Woude syndrome (VWS) is a common form of syndromic cleft lip and palate and accounts for approximately 2% of all cleft lip and palate cases. Distinguishing characteristics include cleft lip with or without cleft palate, isolated cleft palate, bilateral lip pits, hypodontia, normal intelligence, and an autosomal-dominant mode of transmission with a high degree of penetrance. Previously, the VWS locus was mapped to a 1.6-cM region in 1q32-q41 between D1S491 and D1S205, and a 4.4-Mb contig of YAC clones of this region was constructed. In the current investigation, gene-based and anonymous STSs were developed from the existing physical map and were then used to construct a contig of sequence-ready bacterial clones across the entire VWS critical region. All STSs and BAC clones were shared with the Sanger Centre, which developed a contig of PAC clones over the same region. A subset of 11 clones from both contigs was selected for high-throughput sequence analysis across the approximately 1.1-Mb region; all but two of these clones have been sequenced completely. Over 900 kb of genomic sequence, including the 350-kb VWS critical region, were analyzed and revealed novel polymorphisms, including an 8-kb deletion/insertion, and revealed 4 known genes, 11 novel genes, 9 putative genes, and 3 psuedogenes. The positional candidates LAMB3, G0S2, HIRF6, and HSD11 were excluded as the VWS gene by mutation analysis. A preliminary gene map for the VWS critical region is as follows: [see text] 41-TEL. The data provided here will help lead to the identification of the VWS gene, and this study provides a model for how laboratories that have a regional interest in the human genome can contribute to the sequencing efforts of the entire human genome.

Animals↗
PubMed2003

Analysis of pancreatic polypeptide cDNA from the house musk shrew, Suncus murinus, suggests a phylogenetically closer relationship with humans than for other small laboratory animal species.

Pancreatic polypeptide was isolated and sequenced from endocrine cells of the pancreas from an insectivore, the house musk shrew, Suncus murinus. The primary sequence was APLEPAYPGD(10)NATPEQMAQY(20)AAELRKYINM(30)VTRPRYamide. This is the first polypeptide hormone to be characterised from this species and is typical of the primary sequences of pancreatic polypeptide of other animals, being a C-terminal-amidated peptide with 36 residues. Comparison with several vertebrate sequences shows that it has more in common with the human form than do the forms from common laboratory animals such as rabbits, rats, mice and guinea-pigs.

Amino Acid Sequence↗
PubMed1973

Use of DNA polymerase I primed by a synthetic oligonucleotide to determine a nucleotide sequence in phage fl DNA.

A sequence of 50 residues in f1 DNA has been determined by the extension of a chemically synthesized octadeoxyribonucleotide by Escherichia coli DNA polymerase I, with radioactive nucleoside triphosphates and f1 DNA template. The polymerized product was synthesized either in the presence of manganese and a mixture of ribo- and deoxyribotriphosphates or in a magnesium-containing reaction with one or more of the four triphosphates absent. The sequence determination depended largely on fractionation of the polymerized products by two-dimensional "homochromatography." This approach and the techniques for the subsequent sequence analysis should be of general use for determining other sequences of DNA. Several features of this sequence suggest that it is located in an intercistronic region of f1 DNA.

Base Sequence↗
PubMed1991

Resonance ionization spectroscopy for multiplex sequencing of tin-labeled DNA.

A method is described for synthesis of a tin reagent, triethylstannylpropanoic acid (TESPA), and its attachment to oligonucleotide primers. Except for the expected mobility retardation, the presence of [116Sn]-TESPA did not affect the sequencing ladder on electrophoresis gels. By using [120Sn]-TESPA and [35S]-dTTP simultaneously in the Sanger procedure, DNA bands on an electrophoresis gel were first located by autoradiography and then by resonance ionization spectroscopy to demonstrate the coincidence of the signals. Previous results using stable isotopes as labels on model compounds are now confirmed by their use in actual DNA sequencing products.

Autoradiography↗
PubMed1998

A sequence highly similar to PNG is located on chromosome 22q12 in intron 15 of the LIMK-2 gene.

In this report we describe a sequence (PNG22) highly similar to the Phospholipase C beta3 Neighboring Gene (PNG). We also report that PNG22 is located in the q12 region on chromosome 22 between markers D22S1144 and D22S280. This finding explains that PNG probes cross hybridize to sequences on chromosome 22. Fine mapping using our sequence data and the complete sequence of a PAC clone (DJ515N1), located in this region, determined that PNG22 is located in intron 15 of the LIMK-2 gene. PNG22 is 93% homologous to PNG, however it do not have the introns described for the PNG gene, instead matching the cDNA sequence. This leads us to suggest that PNG22 probably represents a PNG pseudogene. In this report we also list the exon intron borders and the genomic structure of LIMK-2 and place it on the Sanger Center chromosome 22 Physical map. It also explains the finding that PNG probes cross hybridize to sequences on chromosome 22.

Base Sequence↗
PubMed2001

Dorsal root ganglion neurons show increased expression of the calcium channel alpha2delta-1 subunit following partial sciatic nerve injury.

Neuropathic pain is associated with changes in the electrophysiological and neurochemical properties of injured primary afferent neurons. A mRNA differential display study in rat L(4/5) dorsal root ganglia (DRGs) revealed upregulation of the calcium channel alpha2delta-1 subunit 2 weeks after partial sciatic nerve ligation (Seltzer model of neuropathic pain). The upregulated transcript appeared to represent previously unidentified sequence from the 3'-untranslated region of rat alpha2delta-1 mRNA. In situ hybridization using L(5) DRGs from sham operated rats showed that 73, 40 and 19% of small (<700 microm(2)), medium (700-1100 microm(2)) and large (>1100 microm(2)) neuronal profiles, respectively, expressed alpha2delta-1 mRNA. Two weeks following nerve injury there was a significant ipsilateral increase, both in the percentage of DRG neurons expressing alpha2delta-1 mRNA and in the intensity of the hybridization signal. Comparison of this ipsilateral expression with that in sham animals, revealed that for small, medium and large neurons, respectively, the proportion of neurons labelled increased by 1.2-, 1.8- and 2.7-fold, while the hybridization signal in alpha2delta-1-labelled neurons increased by 2.8-, 2.5- and 3.7-fold. The most intensely labelled neuronal profiles in ipsilateral, sham and contralateral DRGs, were generally those with small cross-sectional areas. The alpha2delta-1 auxiliary subunit is known to modulate calcium channel function in heterologous expression systems via its association with the pore-forming alpha1 calcium channel subunit. Therefore the increased levels of this subunit in the populations of primary afferents described may, via modulation of calcium-dependent processes such as neurotransmitter release and neuronal excitability, influence the processing of sensory information.

Animals↗
PubMed1988

Trypanothione reductase of Trypanosoma congolense: gene isolation, primary sequence determination, and comparison to glutathione reductase.

The gene encoding trypanothione reductase, the redox disulfide-containing flavoenzyme that is unique to the parasitic trypanosomatids (Shames et al., 1986), has been isolated from the cattle pathogen Trypanosoma congolense. Library screening was carried out with inosine-containing oligonucleotide probes encoding sequences determined from two active site peptides isolated from the purified Crithidia fasciculata enzyme. The nucleotide sequence of the gene was determined according to the dideoxy chain termination method of Sanger. The structural gene is 1476 nucleotides long and encodes 492 amino acids. We have identified the active site peptide containing the redox-active disulfide, a peptide corresponding to the histidine-467 region of human erythrocyte glutathione reductase, as well as the flavin binding domain that is highly conserved in all disulfide-containing flavoprotein reductase enzymes. Alignment of five tryptic peptides (80 residues) isolated from the C. fasciculata trypanothione reductase with the primary sequence of the T. congolense enzyme showed 88% homology with 76% identity. Additionally, a sequence comparison of the glutathione reductase from Escherichia coli or human erythrocytes to T. congolense trypanothione reductase reveals greater than 50% homology. A search for the amino acid residues in the primary sequence of trypanothione reductase functionally active in binding/catalysis in human erythrocyte glutathione reductase shows that only the two arginine residues (Arg-37 and Arg-347), shown by X-ray crystallographic data to hydrogen bond to the GS1 glutathione glycyl carboxylate, are absent.

Amino Acid Sequence↗