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At least 649 records · Page 36Linked to original sources
PubMed1989

Specific-primer-directed DNA sequencing using automated fluorescence detection.

Automated fluorescence-based DNA sequence analysis offers the possibility to undertake very large scale sequencing projects. Directed strategies, such as the specific-primer-directed sequencing approach ('gene walking'), should prove useful in such projects. Described herein is a study involving the use of this approach in conjunction with automated fluorescence detection on a commercial instrument (ABI 370A DNA sequencer). This includes procedures for the rapid chemical synthesis and purification of labeled primers, the design of primer sequences that are compatible with the commercial analysis software, and automated DNA sequence analysis using such primers. A set of four fluorophore-labeled primers can be reliably synthesized in a twenty-four hour period, and greater than 300 nucleotides of analyzed new sequence obtained using this set in an additional twenty-four hours. Scale-up of these procedures to take advantage of the full capabilities of the sequencer is, at present, too slow and costly to be suitable for routine sequencing, and therefore the use of specific-primers is best suited to the closure of gaps in extended sequence produced using random cloning and sequencing strategies.

Autoanalysis↗
PubMed1983

Perfect palindromic lac operator DNA sequence exists as a stable cruciform structure in supercoiled DNA in vitro but not in vivo.

A perfect palindromic 66-base pair (bp) DNA sequence derived from the lac operator and cloned into plasmid pMB9 [Betz, J. L. & Sadler, J. R. (1981) Gene 13, 1-12] can exist in a 66-bp linear form or as two 33-bp cruciform arms. The fraction of the sequence in the cruciform depends on the superhelical density of the plasmid DNA. Relaxed DNA contains no cruciforms. The palindrome in the cruciform structure is cut by EcoRI endonuclease at the base of the cruciform arms, releasing 33-bp fragments; when in the linear form only 66-bp fragments are produced. The cruciform structure is fixed by trimethylpsoralen crosslinks in the cruciform arms. This together with the EcoRI cutting provides an assay for the cruciform structures in the DNA of living cells. Using this assay we show that the cruciform structure rarely if ever exists in vivo, but after DNA isolation greater than 90% of the sequence is in cruciforms. Results suggest that the plasmid DNA as organized in vivo either lacks sufficient torsional tension to form this cruciform or the palindrome is restrained in the linear form by other bound molecules.

Base Sequence↗
PubMed1989

1-Tuple DNA sequencing: computer analysis.

A new method of DNA reading was proposed at the end of 1988 by Lysov et al. According to the authors' claims it has certain advantages as compared to the Maxam-Gilbert and Sanger methods, which are revealed by automation and rapidity of DNA sequencing. Nevertheless its employment is hampered by a number of biological and mathematical problems. The present study proposes an algorithm that allows to overcome the computational difficulties occurring in the course of the method during reconstruction of the DNA sequence by its l-tuple composition. It is shown also that the biochemical problems connected with the loss of information about the l-tuple DNA composition during hybridization are not crucial and can be overcome by finding the maximal flow of minimal cost in the special graph.

Algorithms↗
PubMed1991

The study of stacking energy for natural DNA sequences.

The stacking energies between bases of DNA for both A and B conformation have been calculated by Aida & Nagata (1986, Int. J. Quantum Chem. 29, 1253-1261). For naturally occurring DNA sequences, by assuming that the average stacking energy of A conformation is completely identical with that of B, the lowest average stacking energy for double strand DNA has been calculated and found to be equal to -7.38 kcal M-1. This conclusion has been confirmed by the data of stacking energy of 112 promoter sequences of Escherichia coli and some other natural DNA sequences. Our result shows that the natural DNA sequences are bi-stable. One stable-state is the A conformation, the other is B. It is shown that the result is only applicable to the transcription processes that regulate the gene expression. Through the interaction with RNA polymerase in the processes of transcription, the conformation of DNA might undergo a transition of B----A----B.

Animals↗
PubMed1992

Evolutionary relationships of the salmonid fish genus Salvelinus inferred from DNA sequences of the first internal transcribed spacer (ITS 1) of ribosomal DNA.

DNA sequences of the ribosomal DNA (rDNA) first internal transcribed spacer region (ITS 1) of six species of the salmonid fish genus Salvelinus (alpinus, malma, confluentus, leucomaenis, fontinalis, and namaycush) and the closely related species Hucho perryi were determined. Phylogenetic analysis of the aligned sequences by both phenetic and cladistic methods with H. perryi as an outgroup generated one best topology which pairs S. alpinus with S. malma as the most recently derived species, and pairs S. confluentus with S. leucomaenis. Three other possible topologies favor the pairing of S. namaycush and S. fontinalis, with one tree placing them on separate branches, and vary the branching order of the interior groups. These results agree with previous studies based on comparisons of morphologies, isozymes, karyotypes, and restriction sites showing a close genetic relationship and possible hybridization between the members of this genus.

Animals↗
PubMed1996

Postelectrophoresis capillary scanning method for DNA sequencing.

A postelectrophoresis capillary scanning method for increasing the throughput of DNA sequencing has been developed. The method features a spatially and temporally separated arrangement between capillary gel electrophoresis separation of DNA sequencing fragments and the visualization of the separation pattern. Fluroescently labeled DNA sequencing fragments of pBluescript SK(-) were produced in enzymatic chain-termination reactions and separated by capillary gel electrophoresis using a 110 cm fused silica capillary with a transparent polymer coating. A partial sequence from the DNA was then determined by repeated scanning of the capillary at a rate of 8.39 cm/s, with changing excitation wavelengths and spectral filter sets.

Base Sequence↗
PubMed2005

Phylogenetic relationships of salangid fishes (Osmeridae, Salanginae) with comments on phylogenetic placement of the salangids based on mitochondrial DNA sequences.

We used partial DNA sequences of cytochrome b and 16S mitochondrial genes to determine the phylogenetic placement of salangid fishes and the generic relationships within the salangids. Our molecular data strongly support the monophyly of salangid fishes, the inclusion of salangids in the Osmeridae, and the sister group relationship between salangids and osmerids. Our analyses suggest that Plecoglossus can be separated from all the other salangids and osmerids. Mallotus and Hypomesus are clustered within Osmerinae, rather than allied with Salanginae. As regards the relationships within the salangids, our analyses are incongruent with all previous classification hypotheses. Our phylogenetic analyses support the sister group relationships between Protosalanx and Neosalanx, and between Salanx and Hemisalanx. More evidences show that Leucosoma is more closely related to the Salanx-Hemisalanx clade, while Salangichthys forms part of an unresolved basal polytomy.

Animals↗
PubMed2005

Nucleotide correlations and electronic transport of DNA sequences.

We use a tight-binding formulation to investigate the transmissivity and wave-packet dynamics of sequences of single-strand DNA molecules made up from the nucleotides guanine G , adenine A , cytosine C , and thymine T . In order to reveal the relevance of the underlying correlations in the nucleotides distribution, we compare the results for the genomic DNA sequence with those of two artificial sequences: (i) the Rudin-Shapiro one, which has long-range correlations; (ii) a random sequence, which is a kind of prototype of a short-range correlated system, presented here with the same first-neighbor pair correlations of the human DNA sequence. We found that the long-range character of the correlations is important to the persistence of resonances of finite segments. On the other hand, the wave-packet dynamics seems to be mostly influenced by the short-range correlations.

Base Sequence↗
PubMed2026

esloco: simulation-based estimation of local coverage in long-read DNA sequencing.

SUMMARY: Long-read DNA sequencing is increasingly applied for whole-genome studies, yet experimental planning often lacks reliable estimates of target region coverage, leading to costly and time-consuming pilot studies and replicates. We present esloco, a Monte Carlo-based simulation framework for estimating local coverage in long-read sequencing experiments, including scenarios with unknown target regions (e.g. viral integration, CRISPR-Cas9) or PCR-free designs (e.g. base modifications). By modeling coverage as a function of sequencing depth and read length distribution, esloco enables informed predictions of local sequencing outcomes. Benchmarking across a 45-gene panel demonstrated close agreement with empirical data, underscoring the framework's reliability. AVAILABILITY AND IMPLEMENTATION: esloco is a Python package available on PyPI (https://pypi.org/project/esloco/), GitHub (https://github.com/aweich/esloco), and Zenodo (https://doi.org/10.5281/zenodo.17776161).

Sequence Analysis, DNA↗
PubMed1994

1,N6-etheno deoxy and ribo adenosine and 3,N4-etheno deoxy and ribo cytidine phosphoramidites. Strongly fluorescent structures for selective introduction in defined sequence DNA and RNA molecules.

Synthesis of 1,N6-etheno-2'-deoxyadenosine, 3,N4-etheno-2'-deoxycytidine, and further chemistry on both deoxy and ribo series etheno nucleosides produces the corresponding phosphoramidites. These novel phosphoramidites are introduced selectively, quantitatively, and at specific positions at single or multiple sites into DNA or RNA sequences. The purification and chemistry involved in the synthesis of these products has been optimized to achieve the purity in excess of 99%. The resulting phosphoramidites were tested for their ability to couple and produce poly deoxy and ribonucleotides by solid phase chemistry. The coupling efficiency achieved was greater than 99% per step. Due to the instability of these etheno compounds in acidic and basic medium, various criteria to obtain pure oligomers have been established. The selective introduction of these fluorescent nucleosides into defined sequence DNA and RNA molecule will greatly facilitate the structure-function studies of various RNAs, protein-RNA structures, and DNA-RNA based diagnostics applications. The characteristic and high fluorescent intensity (detection below 1 x 10(-9) M for adenosine sites and below 1 x 10(-7) M for cytidine sites) is particularly suited for the biochemical and biological research and product development applications. The usefulness of these etheno containing modified sequences as sequencing and amplification primers is demonstrated by their full participation in polymerase chain reaction experiments.

Base Sequence↗
PubMed1994

Variations in base pair composition and associated long-range correlations in DNA sequences--computer simulation results.

Recently, the possible occurrence of long-range correlations between nucleotides in DNA sequences of living organisms has excited considerable interest. Of particular importance is the claim that only intron-containing sequences exhibit these correlations. Different investigations, however, have disproved the claimed difference between intron-containing and intron-less DNA sequences. Moreover, very recent investigations pointed out that the long-range correlations appear only if relatively large variations in nucleotide composition along the DNA sequence are present. Furthermore, some examples demonstrated that these variations may have clear biological reasons. In this paper we investigate in detail, with the aid of computer simulations, the connection between compositional heterogeneity of a DNA sequence and the appearance of long-range correlations. As an explicit example, the DNA sequence of the lambda-phage is compared with different artificial sequences of similar compositional heterogeneity. The results demonstrate that the variations of the nucleotide composition along the sequence can fully account for all properties of the claimed long-range correlations. New results of extensive computer simulations are presented which clearly demonstrate how the apparent 'fractal' (or 'long-range-correlated') character of a sequence gradually evaporates, as the frequency of the compositional variations of a simulated sequence continuously increases.

Bacteriophage lambda↗
PubMed2001

Recognition of specific DNA sequences.

Proteins that recognize specific DNA sequences play a central role in the regulation of transcription. The tremendous increase in structural information on protein-DNA complexes has uncovered a remarkable structural diversity in DNA binding folds, while at the same time revealing common themes in binding to target sites in the genome.

Amino Acid Motifs↗
PubMed1989

Automated DNA sequencing methods involving polymerase chain reaction.

Polymerase chain reaction (PCR) as a method for preparing DNA templates has been used for several DNA sequencing applications. An in situ procedure for directly sequencing PCR products by the dideoxy-termination method has been developed by using fluorophore-labeled sequencing primers. Completed sequence reactions were combined and loaded into a single electrophoretic lane of a fluorescence-based DNA sequence analyzer. DNA targets devoid of a universal primer sequence could be sequenced with labeled universal primers by incorporating a universal primer sequence into the PCR product. With this method, the sequence of a 351-bp region in the bacteriophage lambda genome was fully analyzed in a single lane with automatic base identification accuracy of greater than 99%. An unknown sequence, 1.7 kb long, also was sequenced by this procedure, in combination with a "PCR gene walking" strategy. Comparison of the 1110 bases in overlapping sequence data from both strands yielded only two single-base ambiguities. Automated DNA sequence analysis of the highly polymorphic HLA-DQA-1 (alpha) region in the human genome can be performed with this simple methodology. Use of this PCR-sequencing method to analyze DNA extracted from a one-month-old blood sample from an individual who is heterozygous at this locus allowed unambiguous assignment of genotype.

Autoanalysis↗
PubMed1989

The study of DNA sequences by their sequences of twist angles.

To study the properties of DNA sequences we have transformed the sequences of bases into the sequences of twist angles along the chain of DNA double helix by using the Dickerson sum function. The Fourier transform and the auto-correlation function of the twist angles sequences have been used to study the periodicity and randomness of the original DNA sequences. Basing on the correlation coefficient, a "distance" between two DNA fragments has been defined and used to compare some realistic DNA sequences. It is hoped that the techniques developed here could be used to analyze more realistic DNA sequences.

Base Sequence↗
PubMed1996

Gains and losses of DNA sequences in liposarcomas evaluated by comparative genomic hybridization.

Comparative genomic hybridization (CGH) was used to detect and map the regions of gain, high-level amplification, and loss of DNA sequences in 14 liposarcomas. Thirteen tumors showed DNA sequence copy number changes of one or more genomic regions (mean, six aberrations/tumor; range, 0-17). These aberrations were observed in almost every chromosome but some chromosomal regions were affected more often than others. DNA sequence gains were more frequent than losses. The most common gain was seen at 12q14-21 (50% of tumors). Other frequent gains (29%) were of 1q21-24, 8cen-q21.2, 19q, and 20q. High-level amplification was observed in six (43%) tumors and included as minimal common segments bands 12q15, 1q22, and 1q24. In five (36%) tumors, sequences at 1q21-24 and 1q32 were found to be gained simultaneously with 12q14-21, which means that in 71% of the tumors with gain at 12q, an increase of DNA sequence copy number at 1q was also observed. The most common losses of DNA sequences (21%) occurred from regions 9p21-pter and 13q21-qter. Most of the aforementioned regions have not previously been reported to be altered in liposarcomas. The detection of a novel recurring amplicon at 1q21-24 with high-level amplification at 1q22 and frequent simultaneous DNA sequence gain at 12q14-21 (high-level amplification at 12q15) suggests that genes linked to both these regions may play a significant role in the development and progression of liposarcomas.

Adult↗
PubMed1993

Pattern recognition for automated DNA sequencing: I. On-line signal conditioning and feature extraction for basecalling.

The massive scale of DNA sequencing for the Human Genome Initiative compels efforts to reduce the cost and increase the throughput of DNA sequencing technology. Contemporary automated DNA sequencing systems do not yet meet estimated performance requirements for cost-effective and timely completion of this project. Greater accuracy of basecalling software would minimize manual review and editing of basecalling results, and facilitate assembly of primary sequences to large contig(uous) arrays. In this report we describe a neural network model for photometric signal conditioning during raw data acquisition with an automated DNA sequencer. This network supports on-line extraction and evaluation of informative arrays of oligomer separations and yields, as a feature table for accurate, real-time basecalling.

Automation↗
PubMed2000

Automated parallel DNA sequencing on multiple channel microchips.

We report automated DNA sequencing in 16-channel microchips. A microchip prefilled with sieving matrix is aligned on a heating plate affixed to a movable platform. Samples are loaded into sample reservoirs by using an eight-tip pipetting device, and the chip is docked with an array of electrodes in the focal plane of a four-color scanning detection system. Under computer control, high voltage is applied to the appropriate reservoirs in a programmed sequence that injects and separates the DNA samples. An integrated four-color confocal fluorescent detector automatically scans all 16 channels. The system routinely yields more than 450 bases in 15 min in all 16 channels. In the best case using an automated base-calling program, 543 bases have been called at an accuracy of >99%. Separations, including automated chip loading and sample injection, normally are completed in less than 18 min. The advantages of DNA sequencing on capillary electrophoresis chips include uniform signal intensity and tolerance of high DNA template concentration. To understand the fundamentals of these unique features we developed a theoretical treatment of cross-channel chip injection that we call the differential concentration effect. We present experimental evidence consistent with the predictions of the theory.

Automation↗