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 271 records · Page 15Linked to original sources
PubMed1991

Analysis of ras DNA sequences in rat renal cell carcinoma.

The DNA sequences for Ha-, Ki-, and N-ras were determined in six cell lines derived from independent rat hereditary renal cell carcinomas (RCC). Genomic regions encompassing codons 12, 13, and 61 of Ha-ras, Ki-ras, and N-ras, and codon 117 of Ha-ras were PCR amplified and directly sequenced. The DNA sequences of Ha-ras and Ki-ras were normal in all lines tested, as were the codon 12 and 61 sequences of N-ras. However, DNA sequence variations that could code for amino acid substitutions were observed in codons 13, 14, and 18 of N-ras in all the lines. The codon 13 Gly----Val alteration observed was consistent with activating N-ras mutations previously reported. When normal kidney DNA from rats with the hereditary tumor syndrome was sequenced, the same N-ras sequence variations observed in the tumor lines were found. DNA from outbred Long-Evans and inbred Fischer rats also had the altered N-ras sequences. The variant N-ras sequence was not observed in PCR-amplified N-ras cDNA from the RCC lines. Thus, tumor-associated activation of ras oncogene appears to be an infrequent event in spontaneous rat RCC. In addition, these data indicate that rats contain an N-ras DNA polymorphism that appears to be a species-specific anomaly.

Animals↗
PubMed1993

Recovering phylogenetic signal from DNA sequences: relationships within the corvine assemblage (class aves) as inferred from complete sequences of the mitochondrial DNA cytochrome-b gene.

Phylogenetic analysis of cytochrome-b sequences and cranial osteological characters for nine genera of corvine passerine birds supports the hypothesis that the two major groups of birds of paradise, the manucodines and paradisaeinines, constitute a monophyletic group and that their postulated sister group is the Corvidae (crows, jays, and allies). The data are also consistent with the hypothesis that the bowerbirds are not closely related to the birds of paradise but instead lie near the base of the corvine assemblage. The corvine radiation exemplifies a case of multiple star phylogenies embedded within a major clade, with the branching pattern characterized by very short internodal divergence times. Such histories are difficult to resolve no matter what type of data is employed, because little change accumulates between branching events. With respect to sequence data, reconstructed tree topologies are sensitive to the choice of outgroup and to the method of analysis (e.g., transversion vs. global parsimony). In such cases, assessing the "reliability" of a best-fit or most-parsimonious tree inferred from any particular data set becomes problematic. Statistical tests of tree topologies that depend on random sampling of characters will generally be inconclusive in that all cladistic components will tend to be poorly supported because relatively few character-state changes will be recorded between branching events. It is suggested, on the other hand, that congruence in cladistic signal across different data sets may be a potentially more useful method for evaluating the reliability of the signal of any one data set. Resolution of star phylogenies will probably be possible only if DNA sequence and morphological characters are combined in a single analysis.

Animals↗
PubMed2000

Intermediate filaments reconstituted from vimentin, desmin, and glial fibrillary acidic protein selectively bind repetitive and mobile DNA sequences from a mixture of mouse genomic DNA fragments.

Employing the whole-genome PCR technique, intermediate filaments (IFs) reconstituted from vimentin, desmin, and glial fibrillary acidic protein were shown to select repetitive and mobile DNA sequence elements from a mixture of mouse genomic DNA fragments. The bound fragments included major and minor satellite DNA, telomere DNA, minisatellites, microsatellites, short and long interspersed nucleotide elements (SINEs and LINEs), A-type particle elements, members of the mammalian retrotransposon-like (MaLR) family, and a series of repeats not assignable to major repetitive DNA families. The latter sequences were either similar to flanking regions of genes; possessed recombinogenic elements such as polypurine/polypyrimidine stretches, GT-rich arrays, or GGNNGG signals; or were characterized by the distribution of oligopurine and pyrimidine motifs whose sequential and vertical alignment resulted in patterns indicative of high recombination potentials of the respective sequences. The different IF species exhibited distinct quantitative differences in DNA selectivities. Complexes consisting of vimentin IFs and DNA fragments containing LINE, (GT)(n) microsatellite, and major satellite DNA sequences were saturable and dynamic and were formed with high efficiency only when the DNAs were partially denatured. The major-groove binder methyl green exerted a stronger inhibitory effect on the binding reaction than did the minor-groove binder distamycin A; the effects of the two compounds were additive. In addition, DNA footprinting studies revealed significant configurational changes in the DNA fragments on interaction with vimentin IFs. In the case of major satellite DNA, vimentin IFs provided protection of the T-rich strand from cleavage by DNase I, whereas the A-rich strand was totally degraded. Taken together, these observations suggest that IF protein(s) bind to double-stranded DNAs at existing single-stranded sites and, taking advantage of their helix-destabilizing potential, further unwind them via a cooperative effort of their N-terminal DNA-binding regions. A comparison of the present results with literature data, as well as a search in the NCBI database, showed that IF proteins are related to nuclear matrix attachment region (MAR)-binding proteins, and the DNA sequences they interact with are very similar or even identical to those involved in a plethora of DNA recombination and related repair events. On the basis of these comparisons, IF proteins are proposed to contribute in a global fashion, not only to genetic diversity, but also to genomic integrity, in addition to their role in gene expression.

Animals↗
PubMed1980

The nucleotide sequence of the ubiquitous repetitive DNA sequence B1 complementary to the most abundant class of mouse fold-back RNA.

Three copies of a highly repetitive DNA sequence B1 which is complementary to the most abundant class of mouse fold-back RNA have been cloned in pBR322 plasmid and sequenced by the method of Maxam and Gilbert. All the three have a length of about 130 base pairs and are very similar in their base sequence. The deviation from the average sequence is equal to 4% and the overall mismatch between each two is not higher than 8%. One of the recombinant clones used contained two copies of B1 oriented in the same direction. All of the B1 copies are flanked with sequences which possess nonidentical but very similar structure. They consist of a number of AmCn blocks (where m varies from 2 to 8 and n equals 1-2). These peculiar sequences in all cases are separated from B1 by non-homologous DNA stretches of 2-8 residues. In one case, a long polypurine stretch is located next to such a block. It consists of 74 residues most of which represent a reiteration of the basic sequence AAAAG. We have found two regions within the B1 sequence which are homologous to the intron-exon junctions, especially to those present in the large intron of the mouse beta-globin gene. It may indicate the involvement of the B1 sequence in pre-mRNA splicing.

Animals↗
PubMed2000

Metric representation of DNA sequences.

A metric representation of DNA sequences is borrowed from symbolic dynamics. In view of this method, the pattern seen in the chaos game representation of DNA sequences is explained as the suppression of certain nucleotide strings in the DNA sequences. Frequencies of short nucleotide strings and suppression of the shortest ones in the DNA sequences can be determined by using the metric representation.

Globins↗
PubMedDate not supplied

A gel retardation assay system for studying protein binding to simple repetitive DNA sequences.

Simple repetitive DNA sequences have been regarded as mere "junk" present in all eukaryotic genomes. In fact, mixed simple repeat (gt)n(ga)m sequences are present in major histocompatibility complex MHC-DRB genes for long evolutionary times, including such distant animals as artiodactyla and man. We describe herein an unsophisticated method which reveals that at least certain simple repetitive (gt)n(ga)m sequences bind nuclear proteins and show characteristics of a specific DNA-protein interaction via gel retardation.

Animals↗
PubMed1988

The sequence specificity of bleomycin damage in three cloned DNA sequences that differ by a small number of base substitutions.

The DNA sequence specificity of the cancer chemotherapeutic agent, bleomycin, has been investigated in three clones of human alpha RI-DNA. The three 340-base pair alpha RI-DNA sequences were almost identical in their nucleotide sequence enabling the study of subtle effects of base substitutions on bleomycin cleavage. By utilizing densitometer scanning and statistical analysis of the degree of bleomycin DNA cleavage, we found 17 significant differences between the three DNA sequences. Eleven of these differences could be attributed to base substitutions close to the dinucleotide cleavage site. However, six of the differences were at positions two or more base pairs from the base substitution sites. The significant differences were up to 12 base pairs from base substitutions. It is proposed that these long range effects are due to base substitutions causing microvariation in the DNA structure to which bleomycin cleavage is sensitive.

Base Sequence↗
PubMed2001

Determination of bias in the relative abundance of oligonucleotides in DNA sequences.

Different statistical measures of bias of oligonucleotide sequences in DNA sequences were compared, both by theoretical analysis and according to their abilities to predict the relative abundances of oligonucleotides in the genome of Escherichia coli. The expected frequency of an oligonucleotide calculated from a maximal order Markov model was shown to be a degenerate case of the expected frequency calculated from biases of all subwords arising when noncontiguous subwords exhibit no bias. Since (at least in E. coli) noncontiguous sequences exhibit significant bias, the total compositional bias approach is expected to represent biases in genomic sequences more faithfully than Markov approaches. In fact, the efficacy of statistics based on Markov analysis even at the highest order were inferior in predicting actual frequencies of oligonucleotides to methods that factored out biases of internal subwords with gaps. Using total compositional bias as a measure of relative abundance, tetranucleotide and hexanucleotide palindromes were found to be distributed differently from nonpalindromic sequences, with their means shifted somewhat towards underrepresentation. A subpopulation of palindromic hexanucleotides, however, was highly underrepresented, and this group consisted almost entirely of targets for Type II restriction enzymes found within strains of E. coli. Sites recognized by Type I endonucleases from related strains were not markedly biased, and with pentanucleotides, palindromic and nonpalindromic sequences had nearly identical distributions. The loss of restriction sites may be explained by the free transfer of plasmids encoding restriction enzymes and episodic selection for the presence of the enzymes.

Base Composition↗
PubMed1996

Strategy for distinguishing a new DQB1 allele (DQB1*0611) from the closely related DQB1*0602 allele via sequence specific PCR or direct DNA sequencing.

A novel DQ6 allele (DQB1*0611) was identified via direct DNA sequencing in an African-American donor for bone marrow transplantation. The allele was not suspected on the basis of a sequence specific PCR assay which instead indicated the presence of DQB1*0602. DQB1*0602 and DQB1*0611 differ in exon 2 only at codon 9 resulting in a tyrosine substitution for phenylalanine. A modification of current DQB1 sequence specific PCR assays was devised which allows distinction between the closely related DQB1*0602 and DQB1*0611 alleles. Preliminary allele frequency studies suggest that DQB1*0611 is rare both in a non-African American sample and in American of African descent carrying DR11, DQ6 haplotypes. The selection of various DQB1*0611 detection methods is discussed.

Alleles↗
PubMed2003

Multiple group-specific sequencing primers for reliable and rapid DNA sequencing.

Pyrosequencing technology is a bioluminometric DNA sequencing method that employs a cascade of four enzymes to deliver sequence signals. To date this technology has been limited to the sequencing of short stretches of DNA. As an improvement to this technique, we have introduced a bacterial group-specific, multiple sequencing primer approach that circumvents sequencing of less informative semi-conservative regions of the 16S rRNA gene. This new approach is suitable for challenging templates, improving sequence data quality, avoiding sequencing of non-specific amplification products, lessening sequencing time, and moreover, this strategy should open the way for many new applications in the future. The group-specific, multiple sequencing primers can be applied in the Sanger dideoxy sequencing method as well. In addition, we have improved the chemistry of the Pyrosequencing system enabling sequencing of longer stretches of DNA, which allows numerous new applications.

Bacteria↗
PubMed2004

Microfluidic PicoArray synthesis of oligodeoxynucleotides and simultaneous assembling of multiple DNA sequences.

Large DNA constructs of arbitrary sequences can currently be assembled with relative ease by joining short synthetic oligodeoxynucleotides (oligonucleotides). The ability to mass produce these synthetic genes readily will have a significant impact on research in biology and medicine. Presently, high-throughput gene synthesis is unlikely, due to the limits of oligonucleotide synthesis. We describe a microfluidic PicoArray method for the simultaneous synthesis and purification of oligonucleotides that are designed for multiplex gene synthesis. Given the demand for highly pure oligonucleotides in gene synthesis processes, we used a model to improve key reaction steps in DNA synthesis. The oligonucleotides obtained were successfully used in ligation under thermal cycling conditions to generate DNA constructs of several hundreds of base pairs. Protein expression using the gene thus synthesized was demonstrated. We used a DNA assembly strategy, i.e. ligation followed by fusion PCR, and achieved effective assembling of up to 10 kb DNA constructs. These results illustrate the potential of microfluidics-based ultra-fast oligonucleotide parallel synthesis as an enabling tool for modern synthetic biology applications, such as the construction of genome-scale molecular clones and cell-free large scale protein expression.

Base Sequence↗
PubMed2001

Standard deviations and correlations of GC levels in DNA sequences.

In a DNA sequence that exhibits long-range correlations, standard deviations among the GC levels of its segments can be up to an order of magnitude higher than in a sequence consisting of independent, identically distributed nucleotides. Conversely, plots of inter-segment standard deviations vs. segment length reveal quantitative information about the correlations present in a sequence. We present and discuss formulae that relate long-range (power-law) correlations between the nucleotides of a sequence to the expected standard deviations of the GC levels of its segments, and to the correlations between them.

Animals↗
PubMed1993

A quality control algorithm for DNA sequencing projects.

Heterologous DNA sequences from rearrangements with the genomes of host cells, genomic fragments from hybrid cells, or impure tissue sources can threaten the purity of libraries that are derived from RNA or DNA. Hybridization methods can only detect contaminants from known or suspected heterologous sources, and whole library screening is technically very difficult. Detection of contaminating heterologous clones by sequence alignment is only possible when related sequences are present in a known database. We have developed a statistical test to identify heterologous sequences that is based on the differences in hexamer composition of DNA from different organisms. This test does not require that sequences similar to potential heterologous contaminants are present in the database, and can in principle detect contamination by previously unknown organisms. We have applied this test to the major public expressed sequence tag (EST) data sets to evaluate its utility as a quality control measure and a peer evaluation tool. There is detectable heterogeneity in most human and C.elegans EST data sets but it is not apparently associated with cross-species contamination. However, there is direct evidence for both yeast and bacterial sequence contamination in some public database sequences annotated as human. Results obtained with the hexamer test have been confirmed with similarity searches using sequences from the relevant data sets.

Algorithms↗
PubMed1989

Single amino acid changes that alter the DNA sequence specificity of the DNA-[N6-adenine] methyltransferase (Dam) of bacteriophage T4.

Bacteriophage T4 codes for a DNA-[N6-adenine] methyltransferase (Dam) which recognizes primarily the sequence GATC in both cytosine- and hydroxymethylcytosine-containing DNA. Hypermethylating mutants, damh, exhibit a relaxation in sequence specificity, that is, they are readily able to methylate non-canonical sites. We have determined that the damh mutation produces a single amino acid change (Pro126 to Ser126) in a region of homology (III) shared by three DNA-adenine methyltransferases; viz, T4 Dam, Escherichia coli Dam, and the DpnII modification enzyme of Streptococcus pneumoniae. We also describe another mutant, damc, which methylates GATC in cytosine-containing DNA, but not in hydroxymethylcytosine-containing DNA. This mutation also alters a single amino acid (Phe127 to Val127). These results implicate homology region III as a domain involved in DNA sequence recognition. The effect of several different amino acids at residue 126 was examined by creating a polypeptide chain terminating codon at that position and comparing the methylation capability of partially purified enzymes produced in the presence of various suppressors. No enzyme activity is detected when phenylalanine, glutamic acid, or histidine is inserted at position 126. However, insertion of alanine, cysteine, or glycine at residue 126 produces enzymatic activity similar to Damh.

Amino Acid Sequence↗
PubMed1997

Ligation mediated fluorescent labeling of DNA sequencing primers.

Automated DNA sequencing utilizing fluorescently labeled primers is a proven methodology for generating quality sequence data. However, for directed primer walking strategies this necessitates synthesis and labeling of a unique primer for each sequencing reaction. Here, we describe a rapid ligation-based method of generating labeled sequencing primers. An unlabeled 5'-phosphorylated sequencing primer is ligated to a fluorescent oligonucleotide by use of a bridge primer which is complementary to portions of the previous two oligonucleotides, thus aligning them properly for ligation. The resulting fluorescent hybrid primer can be utilized directly in cycle sequencing reactions without any prior purification.

Chromosome Walking↗
PubMed2001

A novel class of plant-specific zinc-dependent DNA-binding protein that binds to A/T-rich DNA sequences.

Complementary DNA encoding a DNA-binding protein, designated PLATZ1 (plant AT-rich sequence- and zinc-binding protein 1), was isolated from peas. The amino acid sequence of the protein is similar to those of other uncharacterized proteins predicted from the genome sequences of higher plants. However, no paralogous sequences have been found outside the plant kingdom. Multiple alignments among these paralogous proteins show that several cysteine and histidine residues are invariant, suggesting that these proteins are a novel class of zinc-dependent DNA-binding proteins with two distantly located regions, C-x(2)-H-x(11)-C-x(2)-C-x((4-5))-C-x(2)-C-x((3-7))-H-x(2)-H and C-x(2)-C-x((10-11))-C-x(3)-C. In an electrophoretic mobility shift assay, the zinc chelator 1,10-o-phenanthroline inhibited DNA binding, and two distant zinc-binding regions were required for DNA binding. A protein blot with (65)ZnCl(2) showed that both regions are required for zinc-binding activity. The PLATZ1 protein non-specifically binds to A/T-rich sequences, including the upstream region of the pea GTPase pra2 and plastocyanin petE genes. Expression of the PLATZ1 repressed those of the reporter constructs containing the coding sequence of luciferase gene driven by the cauliflower mosaic virus (CaMV) 35S90 promoter fused to the tandem repeat of the A/T-rich sequences. These results indicate that PLATZ1 is a novel class of plant-specific zinc-dependent DNA-binding protein responsible for A/T-rich sequence-mediated transcriptional repression.

AT Rich Sequence↗
PubMed1989

How different DNA sequences are recognized by a DNA-binding protein: effects of partial proteolysis.

MDBP is a sequence-specific DNA-binding protein from mammals that recognizes a variety of DNA sequences, all of which show much homology to a partially palindromic 14 base-pair consensus sequence. MDBP subjected to limited proteolysis and then incubated with various specific oligonucleotide duplexes yielded two types of complexes. The relative concentrations of these complexes varied greatly depending on how closely the MDBP site matched the consensus sequence. No such DNA sequence-specific differences in the types of complexes formed were seen with intact MDBP. Partial proteolysis also changed the relative affinity of MDBP for several of its binding sites. The nature of the two types of complexes formed from fragmented MDBP and DNA was studied by DNA competition assays, protein titration, site-directed mutagenesis, and dimethyl sulfate and missing base interference assays. The results suggest that, for some specific DNA sequences, half-site interactions with one MDBP subunit predominate and for others, strong interaction of two subunits with both half-sites readily occur.

Animals↗