Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “DNA sequencing”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 577 records · Page 32Linked to original sources
PubMed2005

DNA sequencing by indexer walking.

BACKGROUND: There is a need for DNA sequencing methods that are faster, more accurate, and less expensive than existing techniques. Here we present a new method for DNA analysis by means of indexer walking. METHODS: For DNA sequencing by indexer walking, we ligated double-stranded synthetic oligonucleotides (indexers) to DNA fragments that were produced by type IIS restriction endonucleases, which generate nonidentical 4-nucleotide 5' overhangs. The subsequent amplification (30 thermal cycles) of indexed DNA provided a template for automated DNA sequencing with fluorescent dideoxy terminators. The data gathered in the first sequencing reaction permitted further movement into the unknown nucleotide sequence by digestion of analyzed DNA with selected type IIS restriction endonuclease followed by ligation of the next indexer. A library of presynthesized indexers consisting of 256 oligonucleotides was used for bidirectional analysis of DNA molecules and provided universal primers for sequencing. RESULTS: The proposed protocol was successfully applied to sequencing of cryptic plasmids isolated from pathogenic strains of Escherichia coli. The overall error rate for base-calling was 0.5%, with a mean read length of 550 nucleotides. Approximately 1000 nucleotides of high-quality sequence could be obtained per day from a single clone. CONCLUSIONS: Indexer walking can be used as a low-cost procedure for nucleotide sequence determination of DNA molecules, such as natural plasmids, cDNA clones, and longer DNA fragments. It can also serve as an alternative method for gap filling at the final stage of genome sequencing projects.

DNA Primers↗
PubMed2001

Electrophoretic mobility shift scanning using an automated infrared DNA sequencer.

Electrophoretic mobility shift assay (EMSA) is widely used in the study of sequence-specific DNA-binding proteins, including transcription factors and mismatch binding proteins. We have established a non-radioisotope-based protocol for EMSA that features an automated DNA sequencer with an infrared fluorescent dye (IRDye) detection unit. Our modification of the elec- trophoresis unit, which includes cooling the gel plates with a reduced well-to-read length, has made it possible to detect shifted bands within 1 h. Further, we have developed a rapid ligation-based method for generating IRDye-labeled probes with an approximately 60% cost reduction. This method has the advantages of real-time scanning, stability of labeled probes, and better safety associated with nonradioactive methods of detection. Analysis of a promoter from an industrially important filamentous fungus, Aspergillus oryzae, in a prototype experiment revealed that the method we describe has potential for use in systematic scanning and identification of the functionally important elements to which cellular factors bind in a sequence-specific manner.

DNA↗
PubMed1987

Application of cloned satellite DNA sequences to molecular-cytogenetic analysis of constitutive heterochromatin heteromorphisms in man.

The cloned alpha-satellite DNA sequences were used to evaluate the specificity and possible variability of repetitive DNA in constitutive heterochromatin of human chromosomes. Five probes with high specificity to individual chromosomes (chromosomes 3, 11, 17, 18, and X) were in situ hybridized to metaphase chromosomes of different individuals. The stable position of alpha-satellite DNA sequences in heterochromatic regions of particular chromosomes was found. Therefore, the chromosome-specific alpha-satellite DNA sequences may be used as molecular markers for heterochromatic regions of certain human chromosomes. The homologous chromosomes of many individuals were characterized by cytologically visible heteromorphisms of hybridization intensity with chromosome-specific alpha-satellite DNA sequences. A special analysis of hybridization between homologues with morphological differences provided the evidence for a high resolution power of the in situ hybridization technique for evaluation of chromosome heteromorphisms. The approaches for detection of heteromorphisms in cases without morphological differences between homologues are discussed. The results obtained indicate that constitutive heterochromatin of human chromosomes has a variable amount of alpha-satellite DNA sequences. In situ hybridization of cloned satellite DNA sequences may be used as a new general approach to analysis of chromosome heteromorphisms in man.

Base Sequence↗
PubMed2003

The DNA sequence of a gypsy element from Gossypium hirsutum L. and characterization of gypsy elements in three Gossypium species.

A strategy was developed to isolate a complete gypsy-element from Gossypium hirsutum L. based on the sequence of a RAPD that was polymorphic in near isoganic lines of cotton that varied in leaf shape. A 5998 nt clone was isolated and its gene order and sequence confirmed it was a gypsy-type retroelement. Sequences homologous to the gag portion of this clone were also found in Gossypium herbaceum L. and Gossypium raimondii L. A portion of the open reading frame of the integrase gene was amplified from three Gossypium species under investigation. PCR products of the expected size were amplified from all three Gossypium species. G. raimondii sequences were statistically more degenerate than sequences from either G. hirsutum or G. herbaceum. Finally, analysis of the open reading frames of selected integrase clones from the three Gossypium species revealed a second gypsy element based on similarity of the deduced amino acid sequence.

Base Sequence↗
PubMed1994

Z curves, an intutive tool for visualizing and analyzing the DNA sequences.

A novel method mapping the DNA or RNA sequence into a folding curve in three dimensional space, the Z curve, has been proposed based on the symmetry of the regular tetrahedrons. There exists a unique Z curve for a given DNA sequence, on the contrary, the DNA sequence can be uniquely determined by the given Z curve. The properties of the Z curves have been studied in great details. The symmetry, the periodicity, the local motif, and the global feather of the distribution of bases of the DNA sequences are reflected by the rich folding structures of the Z curves. The Z curves may be smoothed by the B-spline functions of different orders. Therefore, the Z curves may have any resolution by choosing the suitable spline functions. The higher the order of the B-spline function chosen, the lower the resolution of the Z curve. So, the Z curves are suitable for visualizing and analyzing the DNA sequences with any length. The study of the Z curves develops further a new area to visualizing and analyzing the DNA sequences by a geometrical approach. The method of the Z curves may be strengthened by using the ripe mathematical tools of geometry on the one hand; and by using the powerful technique of the computer graphics on the other hand.

Algorithms↗
PubMed1990

Restriction enzymes have limited access to DNA sequences in Drosophila chromosomes.

Sequence specific DNA binding proteins in eukaryotic cells must efficiently locate their binding sites in chromosomes. Restriction enzymes provide a simple model system with which to investigate the factors which influence this process. We have used P element mediated transformation to introduce a DNA fragment containing a set of characterized restriction sites into the Drosophila germline. Embryonic nuclei prepared from these transgenic animals were treated with restriction enzymes to probe the accessibility of the target restriction sites. The results show that the insert is within an accessible region of the chromosome and that restriction sites within the inserted sequence can be cut. However, the rate of cutting is biphasic. At each restriction site, a fraction of the chromosomes is cut rapidly after which the remainder is refractory. Similar levels of incomplete cutting are obtained when the same P element construct is examined at a different chromosomal location, when different sequence elements are introduced into the P element vector or when the experiment is carried out on nuclei from different embryonic stages. These results are discussed in terms of how sequence specific DNA binding proteins may locate their genomic targets in vivo.

Animals↗
PubMed2006

Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators.

DNA sequencing by synthesis (SBS) on a solid surface during polymerase reaction offers a paradigm to decipher DNA sequences. We report here the construction of such a DNA sequencing system using molecular engineering approaches. In this approach, four nucleotides (A, C, G, T) are modified as reversible terminators by attaching a cleavable fluorophore to the base and capping the 3'-OH group with a small chemically reversible moiety so that they are still recognized by DNA polymerase as substrates. We found that an allyl moiety can be used successfully as a linker to tether a fluorophore to 3'-O-allyl-modified nucleotides, forming chemically cleavable fluorescent nucleotide reversible terminators, 3'-O-allyl-dNTPs-allyl-fluorophore, for application in SBS. The fluorophore and the 3'-O-allyl group on a DNA extension product, which is generated by incorporating 3'-O-allyl-dNTPs-allyl-fluorophore in a polymerase reaction, are removed simultaneously in 30 s by Pd-catalyzed deallylation in aqueous buffer solution. This one-step dual-deallylation reaction thus allows the reinitiation of the polymerase reaction and increases the SBS efficiency. DNA templates consisting of homopolymer regions were accurately sequenced by using this class of fluorescent nucleotide analogues on a DNA chip and a four-color fluorescent scanner.

Base Sequence↗
PubMed1993

Analysis of DNA sequences.

Recent developments in the statistical analysis of DNA sequences are reviewed. The pace with which sequence data are being generated and analysed has increased with the growth of the human genome project. Two areas of activity are emphasized: attention to error rates in recorded sequences, and heterogeneity in structure of sequences. There is now empirical evidence suggesting error rates in the range 0.1%-1%, and such rates will affect evolutionary studies since these are about the rates at which DNA sequences from different individuals are expected to differ. Heterogeneity for such quantities as base composition, or lengths between successive subsequences of specified types, may be sufficient to account for observed long-range correlations between bases. The need for statistical models and analyses of DNA sequence data will continue, and will offer interesting challenges.

Algorithms↗
PubMed1990

repa, a repetitive and dispersed DNA sequence of the filamentous fungus Podospora anserina.

The sequences of homologous DNA regions of two wild-type strains of the fungus Podospora anserina, revealed in one strain the presence of a 349bp insertion leading to a RFLP. This DNA sequence is repeated in the genome and some of its locations are different in various wild-type strains. This DNA element exhibits structural similarities with the yeast solo delta, sigma or tau elements.

Ascomycota↗
PubMed2003

DNA sequence variation in the mitochondrial control region of subterranean mole rats, Spalax ehrenbergi superspecies, in Israel.

The complete mitochondrial control region was sequenced for 60 individuals representing different populations for each of the four species of the subterranean mole rat Spalax ehrenbergi superspecies in Israel: Spalax galili (2n = 52), S. golani (2n = 54), S. carmeli (2n = 58), and S. judaei (2n = 60). The control region of all species and populations is very similar both in length (979 to 983 bp) and in base composition. As in agreement with previous surveys on mitochondrial control regions on mammals, the mole rat control region can be divided into a central domain and two flanking domains, ETAS (extended termination associated sequences) and CSB (conserved sequence blocks). Along with the common conserved blocks found in these domains (ETAS1, ETAS2, CSB1, CSB2, and CSB3), we have also detected in all individuals an ETAS1-like and a CSB1-like element, both in the ETAS domain. The most conserved region was the central domain, followed by the CSB and ETAS domains, showing important differences in the four species analyzed. Phylogenetic analysis supported the existence of two clades. One clade contained individuals belonging to Spalax galili (2n = 52) and S. golani (2n = 54), separated in two different branches depending on the species. The other clade contained individuals belonging to S. carmeli (2n = 58) and S. judaei (2n = 60) mixed together, suggesting a more recent event of speciation. Within species we have observed a southward trend of increasing variability. These results have been explained as a consequence of the adaptation of the species to ecological factors such as aridity and temperature stresses.

Animals↗
PubMed1989

An oligonucleotide hybridization approach to DNA sequencing.

We have proposed a DNA sequencing method based on hybridization of a DNA fragment to be sequenced with the complete set of fixed-length oligonucleotides (e.g., 4(8) = 65,536 possible 8-mers) immobilized individually as dots of a 2-D matrix [(1989) Dokl. Akad. Nauk SSSR 303, 1508-1511]. It was shown that the list of hybridizing octanucleotides is sufficient for the computer-assisted reconstruction of the structures for 80% of random-sequence fragments up to 200 bases long, based on the analysis of the octanucleotide overlapping. Here a refinement of the method and some experimental data are presented. We have performed hybridizations with oligonucleotides immobilized on a glass plate, and obtained their dissociation curves down to heptanucleotides. Other approaches, e.g., an additional hybridization of short oligonucleotides which continuously extend duplexes formed between the fragment and immobilized oligonucleotides, should considerably increase either the probability of unambiguous reconstruction, or the length of reconstructed sequences, or decrease the size of immobilized oligonucleotides.

Base Sequence↗
PubMed1997

An aromatic stacking interaction between subunits helps mediate DNA sequence specificity: operator site discrimination by phage lambda cI repressor.

Sequence specific DNA binding by regulatory proteins provides the basis for regulation of initiation of transcription. A great deal of progress has been made toward understanding sequence specific recognition by individual protein subunits. An additional level of control that needs to be understood is that due to coupling between the subunits of oligomeric regulatory proteins. An example is the bacteriophage lambda cI repressor, a dimeric protein that regulates the lysogenic to lytic genetic switch of the phage. Two levels of specificity are critical to this regulation. First, like all transcriptional regulators, dimers distinguish operator from nonspecific DNA. Direct readout of the DNA sequence by the recognition helix is considered the well understood mechanism for this. However, differential affinity for O(R)1, O(R)2 and O(R)3 is equally critical to the switch because it mediates opposing regulation of divergent promoters. Site specificity at this second level is less well understood. Conformational adaptation by both the repressor and the different operators appears to be important. To evaluate how subunit-subunit interactions are involved in this process, we investigated the effects on both dimer stability and operator binding of amino acid substitutions at the contacts between the symmetrically related helices-5 in the dimer interface. Substitutions for Tyr88 alter dimer stability and greatly perturb differential operator affinity, but generally do not affect operator versus non-operator specificity. The pattern of these effects suggests that the geometry of the face-to-face aromatic stacking interaction between symmetrically related Tyr88 in each subunit, a group in the dimer interface but far removed from the DNA binding interface, plays a critical role in operator discrimination. Conformational changes in the tertiary structure of the subunits appears to be involved. By contrast, the significant effect of I84S substitution is to greatly decrease affinity for all three operators. Presumably, the altered packing of the dimer interface causes a quarternary structural change that moves the two helix-turn-helix motifs out of register with successive DNA major grooves.

Bacteriophage lambda↗
PubMed1998

Membrane-mediated sample loading for automated DNA sequencing.

A new and improved sample loading method for DNA sequencing gels using oligo/polynucleotide binding membranes is described. The labeled DNA sequencing fragments were spotted onto a membrane sample loader, which was then placed in contact with the precast polyacrylamide separation gel. In this way, the time-consuming sample well loading by the tedious pipetting procedure was avoided. The spotted DNA fragments remain immobilized on the membrane until the separation process is initiated by the application of the electric field (an active DNA release mechanism). This novel technique enables sample loading outside of the separation platform, thereby allowing full utilization of various automated sample preparation and liquid handling (robotics) systems, resulting in "real" automated DNA sequencing. The loaded membranes can be stored for more than 24 hours for later use.

Automation↗
PubMed2001

Capillary gel electrophoresis with sinusoidal voltammetric detection: a strategy to allow four-"color" DNA sequencing.

A novel detection strategy for DNA sequencing applications that utilizes a frequency-based electrochemical method is reported. Sinusoidal voltammetry is used to selectively identify four unique redox molecules that are covalently attached to the 5'-end of a 20-base sequencing primer. The tags used in this work are ferrocene derivatives with different substituents attached to the ferrocene ring, where the electron-donating or -withdrawing character of the substituent alters the half-wave potential of the modified ferrocene. Therefore, each tag has a unique SV frequency spectrum that can be easily identified in the frequency domain. In this work, the discrimination of one tag versus all others is accomplished through a "phase-nulling" technique. The signal for each tag is selectively eliminated while the other three responses remain virtually unchanged. This analysis scheme allows for the selective identification of each tagged oligonucleotide eluting in sieving polymer capillary gel electrophoresis with a separation efficiency of 2 x 10(6) theoretical plates per meter. This separation efficiency is sufficient to perform "low-resolution" DNA sequencing; the conditions used in this work have not yet been optimized for high-resolution sequencing applications.

DNA↗
PubMed1983

A comparative study on the genes for three porins of the Escherichia coli outer membrane. DNA sequence of the osmoregulated ompC gene.

The DNA sequence of the ompC gene which encodes one of the outer membrane porins has been determined. The gene appears to encode a secretory precursor of OmpC protein consisting of a total of 367 amino acid residues with a signal peptide of 21 amino acid residues at its NH2-terminal end. The 5' end noncoding region including the promoter of the ompC gene is extremely [A-T]-rich, and the codon usage in the ompC gene is unusual as are those in genes for other abundant outer membrane proteins. The promoter sequence of the ompC gene was compared with that of the ompF gene, both of which are controlled by the osmoregulatory operon, ompB. The deduced amino acid sequence of the OmpC protein showed extensive homology with that of the other porins (OmpF and PhoE proteins). The homology in the primary amino acid sequences, as well as the coding DNA sequences among the porins, indicates that the structural genes for the three porins evolved from a common ancestral gene. Comparison of the amino acid sequences among the OmpC, OmpF, and PhoE porins will be discussed with regard to structure and function.

Amino Acid Sequence↗