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At least 613 records · Page 34Linked to original sources
PubMed1990

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↗
PubMed1977

A new method for sequencing DNA.

DNA can be sequenced by a chemical procedure that breaks a terminally labeled DNA molecule partially at each repetition of a base. The lengths of the labeled fragments then identify the positions of that base. We describe reactions that cleave DNA preferentially at guanines, at adenines, at cytosines and thymines equally, and at cytosines alone. When the products of these four reactions are resolved by size, by electrophoresis on a polyacrylamide gel, the DNA sequence can be read from the pattern of radioactive bands. The technique will permit sequencing of at least 100 bases from the point of labeling.

Adenine↗
PubMed2004

VSQual: a visual system to assist DNA sequencing quality control.

A lack of pliant software tools that support small- to medium-scale DNA sequencing efforts is a major hindrance for recording and using laboratory workflow information to monitor the overall quality of data production. Here we describe VSQual, a set of Perl programs intended to provide simple and powerful tools to check several quality features of the sequencing data generated by automated DNA sequencing machines. The core program of VSQual is a flexible Perl-based pipeline, designed to be accessible and useful for both programmers and non-programmers. This pipeline directs the processing steps and can be easily customized for laboratory needs. Basically, the raw DNA sequencing trace files are processed by Phred and Cross_match, then the outputs are parsed, reformatted into Web-based graphical reports, and added to a Web site structure. The result is a set of real time sequencing reports easily accessible and understood by common laboratory people. These reports facilitate the monitoring of DNA sequencing as well as the management of laboratory workflow, significantly reducing operational costs and ensuring high quality and scientifically reliable results.

Database Management Systems↗
PubMed1993

The effect of zinc on the secondary structure of d(GA.TC)n DNA sequences of different length: a model for the formation *H-DNA.

Alternating d(GA.TC)n DNA sequences are known to undergo transition to *H-DNA in the presence of zinc. Here, the effect of zinc on the secondary DNA structure of d(GA.TC)n sequences of different length (n = 5, 8, 10 and 19) was determined. Short d(GA.TC)n sequences form *H-DNA with a higher difficulty than longer ones. At bacterial negative superhelical density (- sigma = 0.05), zinc still induces transition to the *H-DNA conformation at a d(GA.TC)10 sequence but shorter sequences do not form *H-DNA. Transition to *H-DNA at a d(GA.TC)8 sequence is observed under conditions which destabilize the DNA double helix such as high negative supercoiling or low ionic strength. Our results indicate that a first step in the transition to *H-DNA is the formation of a denaturation bubble at the centre of the repeated DNA sequence, suggesting that the primary role of zinc is to induce a local denaturation of the DNA double helix. Subsequently, zinc might also participate in the stabilization of the altered DNA conformation through its direct interaction with the bases. Based on these results a model for the formation of *H-DNA is proposed.

Base Sequence↗
PubMed1986

Control of replication of the broad host range plasmid RSF1010: the incompatibility determinant consists of directly repeated DNA sequences.

A 500 bp DNA fragment located in the vicinity of the origin of replication of plasmid RSF1010 was cloned into the plasmid vector pBR322 and shown to exhibit incompatibility against parental RSF1010. The rightmost region of this fragment contains three perfect 20 bp direct repeats and a fourth half-repeat of 11 bp, as shown by DNA sequencing. Deletion of the four repeats from the cloned fragment resulted in complete loss of incompatibility whereas partial deletion of the repeated sequence resulted in a concurrent decrease in the expression of incompatibility. We conclude that the incompatibility determinant of RSF1010 is defined by the four repeats and also that the incompatibility expressed is not very strong, since the presence of about 1.5 times as many copies of the repeated sequence as are normally in a cell does not cause a total switch off of RSF1010 replication, but only a 40% reduction in the rate of replication.

DNA Replication↗
PubMed1980

Intragenomic DNA sequence homologies in the chicken and other members of the class Aves: DNA re-association under reduced stringency conditions.

We have investigated the intragenomic DNA sequence homologies of twelve species of birds representing five orders, and emphasizing Galliformes. This study differs in two important ways from the classical approaches taken in constructing and evaluating phylogenies based on DNA sequence similarities. Comparisons are made on the basis of sequence homologies within genomes of related birds, rather than between genomes. DNA is reassociated at 50 degrees C in 0.5M phosphate buffer; these conditions allow formation and detection of duplexes containing more mismatch than would normally be permitted using more stringent conditions, affording an opportunity to observe more ancient sequence homologies. Thermal stability profiles of DNA duplexes formed under these conditions are the basis of comparison; three general patterns were observed. This approach emphasizes differences in sequence composition between genomes while the more traditional method of intergenomic tracer DNA hybridization at higher stringency emphasizes sequence similarities. No correlation was found between taxonomic position and intragenomic sequence composition, either within or between lineages. The thermal stability profiles of DNA duplexes formed within avian genomes did not reflect the biological similarities inferred from morphology, karyotype, and studies of interspecific hybridization. While all of the differences observed could have occurred over geological time, it was surprising that the genomes of the domestic chicken and the Red Jungle Fowl (Gallus gallus) differ in their sequence compositions. It appears that amolification/reduction events and/or positional changes occur rather often during evolution of a lineage.

Animals↗
PubMed2007

Synthesis of DNA-sequence-selective hairpin polyamide platinum complexes.

Two DNA-sequence-selective hairpin polyamide platinum(II) complexes, containing pyrrole and imidazole heterocyclic rings, have been synthesised by different methods. A six-ring complex, selective for (A/T)GGG(A/T) DNA sequences, was made by using solid-phase synthesis, whilst an eight-ring complex, selective for (A/T)CCTG(A/T) DNA sequences, was made by utilising standard wet chemistry. Solid-phase synthesis resulted in a significantly higher yield, required less purification and is more efficient than the wet synthesis; as such, it is the preferred method for further work. The metal complexes were characterised by (1)H and (195)Pt NMR spectroscopy and ESI mass spectrometry. The two compounds provide a foundation for the synthesis of more complex molecules containing multiple hairpins and/or platinum groups.

DNA↗
PubMed1992

A more stringent choice of primers can improve the performance of fluorescent automated DNA sequencers.

Some primers frequently used in the double-stranded dideoxy DNA sequencing technique with radioactive markers are not suited for fluorescent detection. In fact oligonucleotides have a different annealing efficiency related to their base sequence, and this is reflected in nonequivalent results, particularly in fluorescent automated DNA sequencing. We present a method for the evaluation of primer performance in automated DNA sequencers and show its application to the search for a better set of primers for pBluescript vector.

Base Sequence↗
PubMed1995

Specific stimulation of recA-independent plasmid recombination by a DNA sequence at a distance.

Recombination between directly repeated DNA sequences can occur via both recA-dependent and recA-independent mechanisms. They are differentially affected by the length of the repeat and the distance separating the repeats, respectively. Interestingly, a 623 base-pairs long DNA sequence of the plasmid pBR322 was found to stimulate specifically recA-independent recombination between tandem repeats. Analysis of this stimulating sequence has revealed the following features. (1). It is cis-acting. (2). No specific region of it appears to be essential for the effect. Moreover, a neutral sequence of comparable size was able to substitute for the sequence in influencing recombination. (3). The sequence affects recombination between tandem repeats within the tetA but not the bla gene of pBR322. (4). The sequence exerts its effect in a position-dependent manner. (5). It changes not only the frequency but also the products of recombination. Our results provide an example that recA-independent recombination can be influenced by DNA sequences at a distance.

Antiporters↗
PubMed2006

Taxonomic reliability of DNA sequences in public sequence databases: a fungal perspective.

BACKGROUND: DNA sequences are increasingly seen as one of the primary information sources for species identification in many organism groups. Such approaches, popularly known as barcoding, are underpinned by the assumption that the reference databases used for comparison are sufficiently complete and feature correctly and informatively annotated entries. METHODOLOGY/PRINCIPAL FINDINGS: The present study uses a large set of fungal DNA sequences from the inclusive International Nucleotide Sequence Database to show that the taxon sampling of fungi is far from complete, that about 20% of the entries may be incorrectly identified to species level, and that the majority of entries lack descriptive and up-to-date annotations. CONCLUSIONS: The problems with taxonomic reliability and insufficient annotations in public DNA repositories form a tangible obstacle to sequence-based species identification, and it is manifest that the greatest challenges to biological barcoding will be of taxonomical, rather than technical, nature.

DNA, Fungal↗
PubMed1985

Human papillomavirus type 16 DNA sequence.

The complete nucleotide sequence of HPV16 DNA (7904 bp) cloned from an invasive cervical carcinoma was determined. Homology comparisons allowed us to align the major open reading frames with the other published papilloma virus DNA sequences. The general organization of the open reading frames is similar to that of the other four papillomavirus (BPV1, HPV1a, HPV6b, CRPV) already sequenced. The sequence reveals an interruption of the reading frame coding for a suspected E1 protein.

Base Sequence↗
PubMed2006

Novel numerical and graphical representation of DNA sequences and proteins.

We have introduced novel numerical and graphical representations of DNA, which offer a simple and unique characterization of DNA sequences. The numerical representation of a DNA sequence is given as a sequence of real numbers derived from a unique graphical representation of the standard genetic code. There is no loss of information on the primary structure of a DNA sequence associated with this numerical representation. The novel representations are illustrated with the coding sequences of the first exon of beta-globin gene of half a dozen species in addition to human. The method can be extended to proteins as is exemplified by humanin, a 24-aa peptide that has recently been identified as a specific inhibitor of neuronal cell death induced by familial Alzheimer's disease mutant genes.

Cell Death↗
PubMed1995

Human gamma X satellite DNA: an X chromosome specific centromeric DNA sequence.

The cosmid clone, CX16-2D12, was previously localized to the centromeric region of the human X chromosome and shown to lack human X-specific alpha satellite DNA. A 1.2 kb EcoRI fragment was subcloned from the CX16-2D12 cosmid and was named 2D12/E2. DNA sequencing revealed that this 1,205 bp fragment consisted of approximately five tandemly repeated DNA monomers of 220 bp. DNA sequence homology between the monomers of 2D12/E2 ranged from 72.8% to 78.6%. Interestingly, DNA sequence analysis of the 2D12/E2 clone displayed a change in monomer unit orientation between nucleotide positions 585-586 from a "tail-to-head" arrangement to a "head-to-tail" configuration. This may reflect the existence of at least one inversion within this repetitive DNA array in the centromeric region of the human X chromosome. The DNA consensus sequence derived from a compilation of these 220 bp monomers had approximately 62% DNA sequence similarity to the previously determined gamma 8 satellite DNA consensus sequence. Comparison of the 2D12/E2 and gamma 8 consensus sequences revealed a 20 bp DNA sequence that was well conserved in both DNA consensus sequences. Slot-blot analysis revealed that this repetitive DNA sequence comprises approximately 0.015% of the human genome, similar to that found with gamma 8 satellite DNA. These observations suggest that this satellite DNA clone is derived from a subfamily of gamma satellite DNA and is thus designated gamma X satellite DNA. When genomic DNA from six unrelated males and two unrelated females was cut with SstI or HpaI and separated by pulsed-field gel electrophoresis, no restriction fragment length polymorphisms were observed for either gamma X (2D12/E2) or gamma 8 (50E4) probes. Fluorescence in situ hybridization localized the 2D12/E2 clone to the lateral sides of the primary constriction specifically on the human X chromosome.

Base Sequence↗
PubMed1988

The interaction of proteins encoded by Drosophila homeotic and segmentation genes with specific DNA sequences.

The ANT-C gene cluster is part of a network of genes that govern pattern formation in the development of Drosophila. The ANT-C genes encode proteins that contain a conserved 60 amino acid sequence, the homeodomain. Here we show that the homeodomains encoded by two of the ANT-C loci confer sequence-specific DNA-binding activity. The DNA sequence specificities of the Dfd and ftz homeodomains appear to overlap completely in vitro, indicating that differences in regulatory specificity among ANT-C and BX-C proteins (assuming that differences exist) must be a consequence of the nonconserved protein sequences found outside of the homeodomains. Deletions that remove sequences from either end of the ftz homeodomain abolish DNA-binding activity, consistent with the commonly held assumption that the homeodomain is a structural domain. The relevance of in vitro DNA-binding experiments to the regulatory function of ftz is supported by our finding that a temperature-sensitive ftz mutation that causes a pairwise fusion of embryonic segments also reduces the affinity of the ftz homeodomain for DNA. Restriction fragments containing ftz homeodomain binding sites were identified within a 90 kb stretch of DNA extending the Antp P1 and P2 promoters. Binding sites appear to be clustered near the P1 promoter but also occur near P2 and in the region between the two. The task remains of determining which of these sequences mediate regulation of Antp by ftz or by other genes that encode closely related homeodomains.

Animals↗
PubMed1980

The white pock (mu) mutants of rabbit poxvirus. III. Terminal DNA sequence duplication and transposition in rabbit poxvirus.

The restriction fragment patterns of the DNA of three white pock (mu) nonhost range mutants RPmu mutants) of rabbit poxvirus (RPV) show the presence of net insertions of DNA. Two of the mutants (RPmu21 and RPmu7) contain two molar quantities of 13 X 10(6) and 15 X 10(6) daltons, respectively, of the left-most viral DNA sequences. The extra copy of these sequences was inserted into the right-most region of the viral genome which in both cases had been modified by deletion of 10 X 10(6) daltons of the original extreme right-most DNA sequences. Hence two mutants with "left-hand" mirror image termini of 13 X 10(6) and 15 X 10(6) daltons of DNA were generated with an overall net increase in mass of 3 X 10(6) and 5 X 10(6) daltons of DNA to yield genome molecular weights of 121 X 10(6) and 123 X 10(6) for RPmu21 and 7, respectively. A third mutant (RPmu81g) contained 28 X 10(6) daltons of the right-most parental DNA sequences inserted into the left end of the genome, which had deleted 6 X 10(6) daltons of the extreme left-hand sequences. This variant contains "right-hand" mirror image termini of 28 X 10(6) daltons of DNA and a net increase in molecular weight of 22 X 10(6) daltons of DNA to yield a genome molecular weight of 140 X 10(6). The cross-linked end of the transposed sequences is retained in all cases. Our data suggest that the terminal DNA sequences at either end of the virus are not essential for a productive infection in certain host cells and that the pathogenicity of orthopoxviruses may be related in part to a series of varied and interchangeable DNA sequences located at both extremes of a high conserved genome core.

Base Sequence↗
PubMed1978

The single-copy DNA sequence polymorphism of the sea urchin Strongylocentrotus purpuratus.

The single-copy DNA sequence difference between individual sea urchins of the species Strongylocentrotus purpuratus has been estimated by comparing the thermal stability of reassociated DNA duplexes from two individuals with that for DNA from an individual. Thermal stability was measured by hydroxyapatite thermal chromatography, S1 nuclease resistance after heating in a solvent which neutralizes the effect of DNA base composition, and spectrophotometric melting. One pair of individuals appear to differ from each other in about 4% of the nucleotide pairs of their single-copy DNA sequence. The differences in DNA sequence among individuals in local populations are not distinguishably smaller than those among populations as far apart as 2000 kilometers along the Pacific coast of North America.

Animals↗
PubMed2006

Microbial source tracking by DNA sequence analysis of the Escherichia coli malate dehydrogenase gene.

Criteria for sub-typing of microbial organisms by DNA sequencing proposed by Olive and Bean were applied to several genes in Escherichia coli to identify targets for the development of microbial source tracking assays. Based on the aforementioned criteria, the icd (isocitrate dehydrogenase), and putP (proline permease) genes were excluded as potential targets due to their high rates of horizontal gene transfer; the rrs (16S rRNA) gene was excluded as a target due to the presence of multiple gene copies, with different sequences in a single genome. Based on the above criteria, the mdh (malate dehydrogenase) gene was selected as a target for development of a microbial source tracking assay. The mdh assay was optimized to analyze a 150 bp fragment corresponding to residues G191 to R240 (helices H10 and H11) of the Mdh catalytic domain. 295 fecal isolates (52 horse, 50 deer, 72 dog, 52 seagull and 69 human isolates) were sequenced and analyzed. Target DNA sequences for isolates from horse, dog plus deer, and seagull formed identifiable groupings. Sequences from human isolates, aside from a low level (ca. 15%) human specific sequence, did not group; nevertheless, other hosts could be distinguished from human. Positive and negative predictive values for two- and three-way host comparisons ranged from 60% to 90% depending on the focus host. False positive rates were below 10%. Multiple E. coli isolates from individual fecal samples exhibited high levels of sequence homogeneity, i.e. typically only one to two mdh sequences were observed per up to five E. coli isolates from a single fecal sample. Among all isolates sequenced from fecal samples from each host, sequence homogeneity decreased in the following order: horse>dog>deer>human and gull. For in-library isolates, blind analysis of fecal isolates (n=12) from four hosts known to contain host specific target sequences was 100% accurate and 100% reproducible for both DNA sequence and host identification. For blind analysis of non-library isolates, 18/19 isolates (94.7%) matched one or more library sequences for the corresponding host. Ten of eleven geographical outlier fecal isolates from Florida had mdh sequences that were identical to in-library sequences for the corresponding host from California. The mdh assay was successfully applied to environmental isolates from an underground telephone vault in California, with 4 of 5 isolates matching sequences in the mdh library. 146 sequences of the 645bp mdh fragment from five host sources were translated into protein sequence and aligned. Seven unique Mdh protein sequences, which contained eight polymorphic sites, were identified. Six of the polymorphic sites were in the NAD+ binding domain and two were in the catalytic domain. All of the polymorphic sites were located in surface exposed regions of the protein. None of the non-silent mutations of the Mdh protein were in the 150bp mdh target. The advantages and disadvantages of the assay compared to established source tracking methods are discussed.

Animals↗
PubMed1994

The genome reconstruction manager: a software environment for supporting high-throughput DNA sequencing.

A new software system designed for use in high-throughput DNA sequencing laboratories is described. The Genome Reconstruction Manager (GRM) was developed from requirements derived from ongoing large-scale DNA sequencing projects. Object-oriented principles were followed in designing the system, and tools supporting object-oriented system development were employed for its implementation. GRM provides several advances in software support for high-throughput DNA sequencing: support for random, directed, and mixed sequencing strategies; a novel system for fragment assembly; a commercial object data-base management system for data storage; a client/server architecture for using network computational servers; and an underlying data model that can evolve to support fully automatic sequence reconstruction. GRM is currently being deployed for production use in high-throughput DNA sequencing projects.

Algorithms↗