SEARCH · PubMed Health

Results for “duplex sequencing”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

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

At least 1,081 records · Page 60Linked to original sources

PubMed1996

Structural prediction of A- and B-DNA duplexes based on coordinates of the phosphorus atoms.

The sequence-dependent structure of DNA double helices was studied extensively during the past 10 years. How the backbone structure correlates with the base structure in a duplex conformation is still an important yet open question. Using a set of reduced coordinates and a least-squares fitting procedure, we have developed a method to predict structures for B-DNA duplexes based on coordinates of the phosphorus atoms. This method can be used to predict all-atom structures for both bent and straight molecules. We estimated the accuracies of the predictions by studying a set of 10 oligonucleotides with their structures available from the Protein Data Bank. We used this method to construct a modeled structure for the bacteriophage lambda cro operator for which the phosphorus coordinates were known from 3.5-angstrum resolution crystal data (4CRO).

Bacteriophage lambda↗

PubMed2000

Duplex PCR for differential identification of Mycobacterium bovis, M. avium, and M. avium subsp. paratuberculosis in formalin- fixed paraffin-embedded tissues from cattle.

We previously isolated and sequenced two genomic segments of Mycobacterium avium subsp. paratuberculosis, namely, f57, a species-specific sequence, and the p34 gene, coding for a 34-kDa antigenic protein. Comparison of sequences upstream of the p34 open reading frame (us-p34) from M. avium subsp. paratuberculosis and M. tuberculosis showed a 79-base deletion in M. tuberculosis. Sequence analysis of the p34 genes in another two species, M. bovis (strain BCG) and M. avium (strain D4), confirmed the differences observed between tuberculous and nontuberculous species. A duplex diagnostic PCR strategy based on coamplification of nonhomologous us-p34 and species-specific f57 sequences was therefore developed. Duplex PCR yielded three different patterns, specific either for tuberculous bacilli (M. tuberculosis, M. bovis, and M. africanum), for both nontuberculous mycobacteria M. avium and M. intracellulare, or for M. avium subsp. paratuberculosis. The specificity of this single-step DNA-based assay was assessed on DNA from cultured mycobacterial strains, as well as on a panel of formalin-fixed and paraffin-embedded tissues from cattle. Molecular assay results from tissular DNA were compared to conventional bacteriological and histological test results, including those obtained by Ziehl-Neelsen staining on tissue biopsy specimens. Molecular discrimination was successful and confirmed the value of duplex us-p34 and f57 sequence amplification for differential diagnosis of tuberculosis, paratuberculosis, or infections caused by other members of the M. avium complex.

Animals↗

PubMed2000

Sequence-dependent bending of DNA induced by cisplatin: NMR structures of an A.T-rich 14-mer duplex.

The NMR solution structure of the A.T rich DNA 14-mer duplex d(ATACATGGTACATA).d(TATGTACCATGTAT) is reported. This is compared with the NMR structure of the same duplex intrastrand cross-linked at the d(G*pG*) site by cis-(Pt(NH3)2¿2+, derived from the anticancer drug cisplatin. The unmodified duplex has B-DNA geometry, but there is a large positive base-pair roll (roll angle 24 +/- 2 degrees) at the T9-A10 step on the 3' side of the central GG site. Platination of the DNA duplex causes the adjacent guanine bases to roll toward one another (roll angle 44 +/- 4 degrees), leading to an overall helix bend of 52 +/- 9 degrees. The platinum atom is displaced from the planes of the coordinated G7* and G8* by 0.8 A and 0.3 A, respectively. The minor groove opposite the platinum lesion is widened and flattened, with geometric parameters similar to those of A-form DNA. The unwinding of the helix at the platination site is 26 degrees. Platination causes the DNA duplex to bend toward the 3'-end (with respect to the G*G* strand), in contrast to G C-rich structures reported previously, which bend toward the 5'-end. This difference can be attributed to the predisposition of the A.T rich duplex toward bending in this region. Protein recognition of bent platinated G*G* lesions may therefore exhibit a strong dependence on the local DNA structure.

Base Sequence↗

PubMed1996

Effect of third strand composition on the triple helix formation: purine versus pyrimidine oligodeoxynucleotides.

Exon 5 of the human aprt gene contains an oligo-purine-oligopyrimidine stretch of 17 bp (5'-CCCTCTTCTCTCTCCT-3') within the coding region. (T,C)-, (G,T)- and (G,A)-containing oligonucleotides were compared for their ability to form stable triple helices with their DNA target. (G,T) oligodeoxynucleotides, whether parallel or antiparallel, were unable to bind to this sequence. This is in contrast to (G,A) (purine) and (T,C) (pyrimidine) oligonucleotides, which bind to the duplex at near neutral pH. Binding was highly sequence specific, as unrelated competitors were unable to interfere with target recognition. A major difference between the purine and pyrimidine oligodeoxynucleotides was observed in the kinetics of binding: the (G,A) oligonucleotide binds to its target much faster than the (T,C) oligomer. With the purine oligonucleotide, complete binding was achieved in a matter of minutes at micromolar concentrations, whereas several hours were required with the pyrimidine oligomer. Thus, the general observation that triplex formation is slow with pyrimidine oligodeoxynucleotides does not hold for (G,A) oligodeoxynucleotides. Purine and pyrimidine oligodeoxynucleotides covalently linked to a psoralen group were able to induce crosslinks on the double-stranded DNA target upon UV irradiation. This study provides a detailed comparison of the different types of DNA triplexes under the same experimental conditions.

Acridines↗

PubMed1994

Sequence-selective metal ion binding to DNA hexamers.

Base-selective interaction between divalent metal ions and DNA oligomers has been studied by 1D and 2D NMR spectroscopy. Titration with paramagnetic metal ions induces selective line broadening of resonances from protons close to the binding site. Also the intensities of 2D NOESY cross-peaks involving paramagnetic affected protons will be quenched. Two hexamers, 5'-d(CGTACG)2 (I) and 5' (GCATGC)2 (II) have been titrated with Mn(II) ions. Manganese binds selectively to the terminal guanine, G1, in sequence II as manifested through pronounced paramagnetic line broadening and loss of intensities of NOESY cross-peaks involving G-H8 protons. The second guanine, G5, and the non-guanine residues are appreciably less affected. In sequence I both guanines, G2 and G6, are the targets for selective metal binding as judged from G-H8 line broadening. The extent of interaction is almost identical for the two G-residues and comparable to that observed for G1 in sequence II. The metal binding site in the duplexes is most likely nitrogen G-N7. Selective metal binding to oligonucleotides may be related to sequence-dependent variation in molecular electrostatic potentials (MEP) along the chain.

Base Sequence↗

PubMed2003

Cloned plasmid DNA fragments as calibrators for controlling GMOs: different real-time duplex quantitative PCR methods.

Analytical real-time PCR technology is a powerful tool for implementation of the GMO labeling regulations enforced in the EU. The quality of analytical measurement data obtained by quantitative real-time PCR depends on the correct use of calibrator and reference materials (RMs). For GMO methods of analysis, the choice of appropriate RMs is currently under debate. So far, genomic DNA solutions from certified reference materials (CRMs) are most often used as calibrators for GMO quantification by means of real-time PCR. However, due to some intrinsic features of these CRMs, errors may be expected in the estimations of DNA sequence quantities. In this paper, two new real-time PCR methods are presented for Roundup Ready soybean, in which two types of plasmid DNA fragments are used as calibrators. Single-target plasmids (STPs) diluted in a background of genomic DNA were used in the first method. Multiple-target plasmids (MTPs) containing both sequences in one molecule were used as calibrators for the second method. Both methods simultaneously detect a promoter 35S sequence as GMO-specific target and a lectin gene sequence as endogenous reference target in a duplex PCR. For the estimation of relative GMO percentages both "delta C(T)" and "standard curve" approaches are tested. Delta C(T) methods are based on direct comparison of measured C(T) values of both the GMO-specific target and the endogenous target. Standard curve methods measure absolute amounts of target copies or haploid genome equivalents. A duplex delta C(T) method with STP calibrators performed at least as well as a similar method with genomic DNA calibrators from commercial CRMs. Besides this, high quality results were obtained with a standard curve method using MTP calibrators. This paper demonstrates that plasmid DNA molecules containing either one or multiple target sequences form perfect alternative calibrators for GMO quantification and are especially suitable for duplex PCR reactions.

Calibration↗

PubMed2006

Metal-salen-base-pair complexes inside DNA: complexation overrides sequence information.

Two isomeric salicylic aldehyde nucleobases have been prepared and incorporated into various DNA duplexes. Reaction with ethylenediamine leads to formation of the well-known salen ligand inside the DNA double helix. Addition of transition-metal ions such as Cu(2+), Mn(2+), Ni(2+), Fe(2+), or VO(2+) results in the formation of metal-salen-base-pair complexes, which were studied by using UV and circular dichroism (CD) spectroscopy. HPLC and ESI mass spectrometric measurements reveal an unusually high stability of the DNA-metal system. These metal-salen complexes act as interstrand cross-links and thereby lead to a strong stabilization of the DNA duplexes, as studied by thermal de- and renaturing experiments. Complex formation is strong enough to override sequence information even when the preorganization of the ligand precursors is unfavorable and the DNA duplex is distorted by the metal complexation. Furthermore, melting-point studies show that the salen complex derived from ligand 2 fits better into the DNA duplex, in accordance with results obtained from the crystal structure of the corresponding copper-salen complex 8.

Base Pairing↗

PubMed1998

Interaction of human DNA topoisomerase I with specific sequence oligodeoxynucleotides.

The interaction of human DNA topoisomerase I (topo I) with specific sequence oligodeoxynucleotides (ODNs) of different length and structure has been investigated. All the ODNs used were shown to be effective enzyme inhibitors and to inhibit the topo I catalyzed relaxation of scDNA in a competitive manner. Among two DNA regions (A and B) required for topo I-mediated DNA cleavage, the former was found to display the higher affinity for the enzyme. The enzyme's affinity for ODNs corresponding to the scissile strand (five and nine nucleotide units in length) is about 2-4 orders of magnitude higher than that for non-specific ODNs of the same length. Topo I can efficiently recognize even extremely short specific ODNs containing only two or three bases (AGA and pAG, Ki = 15 and 60 microM, respectively): the sequence AAGA (Ki = 10 microM) is essential for tight DNA binding to topo I. The affinities of ODNs corresponding to the non-scissile strand are significantly lower. The ligand's affinity increases with its length. Additionally, about a ten-fold enhancement of specific sequence affinity occurs due to stable duplex formation during enzyme preincubation with ligands before addition of scDNA. We believe the possibility of using the short specific oligonucleotides and its derivatives as topoisomerase I-targeting drugs could not be excluded.

Base Sequence↗

PubMed1992

Gilvocarcin V exhibits both equilibrium DNA binding and UV light induced DNA adduct formation which is sequence context dependent.

The relative degree of both equilibrium binding and of ultraviolet light induced adduct formation for the antitumor antibiotic gilvocarin V with two hexaecamer DNA sequence isomers, d[ATATATAGCTATATAT]2 and d[AAAAAAAGCTTTTTTT]2, was assessed. The experiments reveal that gilvocarin V binds, under equilibrium conditions, and reacts, in the presence of exogenously applied UV light, more efficiently with the alternating purine:pyrimidine sequence hexadecamer than the homopurine:homopyrimidine duplex at identical gilvocarcin V to DNA duplex ratios. DNAse I digests of adduct containing duplexes derived from the d[AAAAAAAGCTTTTTTT]2 duplex, identified and isolated using gel shift assays employing denaturing polyacrylamide gels, confirm that gilvocarcin V adducts can be formed with thymine residues but suggest that adduct formation with either adenine or guanine residues is also possible.

Aminoglycosides↗

PubMed2003

Independent combinatorial effect of antisense oligonucleotides and RNAi-mediated specific inhibition of the recombinant rat P2X3 receptor.

Synthetic 21-bp-long short interfering RNAs (siRNA) can stimulate sequence-specific mRNA degradation in mammalian cell cultures, a process referred to as RNA interference (RNAi). In the present study, the potential of RNAi was compared to the traditional antisense approach, acting mainly via RnaseH, for targeting the recombinant rat pain-related cation-channel P2X3 expressed in CHO-K1 and a rat brain tumour-derived cell line, 33B. Downregulation of the P2X3 receptor was evaluated at the mRNA, protein, and functional levels. In this study, four siRNA duplexes induced up to 95% sequence-specific inhibition of the P2X3 mRNA, independent of the type of 2 nt 3'-overhang modification and the location of the targeted sequences. Furthermore, we detected and characterised an independent combinatorial effect of antisense oligonucleotides (ASOs) and RNAi-mediated specific inhibition of the P2X3 receptor. Enhanced downregulation was observed only when siRNA was combined with nonhomologous ASO, targeting distant regions on the common P2X3 mRNA. The two reagents resulted in more efficient downregulation of P2X3 mRNA when administered in combination rather than separately. To our knowledge, this is the first investigation at the molecular level of the potential benefits of mixed antisense and RNAi-mediated treatment for inhibiting expression of a medically relevant pain-related gene.

Animals↗

PubMed1996

Creating RNA bulges: cleavage of RNA in RNA/DNA duplexes by metal ion catalysis.

The manipulation of a single-stranded RNA target by forming different RNA/antisense hybrids demonstrates the possibility of cleaving the RNA strand within duplexes. This was achieved using the sequence composition of the antisense oligonucleotide, an approach that results in various bulges [unpaired base(s)] in the RNA target, which is then cleavable at these specific bulge sites under free metal ion or metal complex catalysis. RNA cleavages promoted by metal ions were performed under mild conditions and characterized by separating the RNA fragments carrying end label. The observed products result from intramolecular transesterification causing RNA strand scission. No detectable cleavage of the RNA was observed with either a fully complementary RNA/antisense hybrid or a bulged base in the antisense strand. A molecular modeling study of the RNA backbone suggests that the local conformation of the RNA backbone at a bulge in such hybrid duplexes greatly facilitates the metal-assisted catalytic cleavage. Endonucleolytic RNA cleavage within an RNA/antisense hybrid by metal complexes attached to the antisense oligonucleotide might lead to a new approach in antisense technology with artificial ribonucleases which operate with catalytic turnover.

Base Sequence↗

PubMed1991

Synthesis of a wild-type and three mutant Cucurbita maxima trypsin inhibitor-encoding genes by a single-strand approach.

A single-strand approach to gene assembly, based on a modification of an in vitro complementary oligodeoxyribonucleotide template-directed ligation of the desired sequence to a linearized vector [Chen et al., Nucleic Acids Res. 18 (1990) 871-878], is described. The gene coding for the wild-type Cucurbita maxima trypsin inhibitor of 29 amino acid residues [Bode et al., FEBS Lett. 242 (1989) 285-292], as well as three mutant forms of the gene, in which two of the three disulfide bonds have been replaced singly or as a pair, have been synthesized in a single synthesis run with minimal manual intervention. Subsequent to ligation to pUC9 and in vivo gapped duplex repair by Escherichia coli, their sequences have been verified.

Amino Acid Sequence↗

PubMed1996

The Ku-like protein from Saccharomyces cerevisiae is required in vitro for the assembly of a stable multiprotein complex at a eukaryotic origin of replication.

We have previously shown that three distinct DNA-binding activities, in crude form, are necessary for the ATP-dependent assembly of a specific and stable multiprotein complex at a yeast origin of replication. Here we show the purification of one of these DNA binding activities, referred to as origin binding factor 2 (OBF2). The purified protein is a heterodimer composed of two polypeptides with molecular mass values of 65 and 80 kDa as determined by SDS/PAGE. Purified OBF2 not only binds DNA but also supports the formation of a protein complex at essential sequences within the ARS121 origin of replication. Interestingly, OBF2 binds tightly and nonspecifically to both duplex DNA and single-stranded DNA. The interaction with duplex DNA occurs at the termini. N-terminal sequencing of the 65-kDa subunit has revealed that this polypeptide is identical to the previously identified HDF1 peptide, a yeast homolog of the small subunit of the mammalian Ku autoantigen. Although the potential involvement of Ku in DNA metabolic events has been proposed, this is the first requirement for a Ku-like protein in the assembly of a protein complex at essential sequences within a eukaryotic origin of replication.

Animals↗

PubMed1998

Base orientation of second DNA in RecA.DNA filaments. Analysis by combination of linear dichroism and small angle neutron scattering in flow-oriented solution.

To gain insight into the mechanism of pairing two complementary DNA strands by the RecA protein, we have determined the nucleobase orientation of the first and the second bound DNA strands in the RecA.DNA filament by combined measurements of linear dichroism and small angle neutron scattering on flow-oriented samples. An etheno-modified DNA, poly(depsilonA) was adapted as the first DNA and an oligo(dT) as the second DNA, making it possible to distinguish between the linear dichroism signals of the two DNA strands. The results indicate that binding of the second DNA does not alter the nucleobase orientation of the first bound strand and that the bases of the second DNA are almost coplanar to the bases of the first strand although somewhat more tilted (60 degrees relative to the fiber axis compared with 70 degrees for the first DNA strand). Similar results were obtained for the RecA.DNA complex formed with unmodified poly(dA) and oligo(dT). An almost coplanar orientation of nucleobases of two DNA strands in a RecA-DNA filament would facilitate scanning for, and recognition of, complementary base sequences. The slight deviation from co-planarity could increase the free energy of the duplex to facilitate dissociation in case of mismatching base sequences.

DNA↗

PubMed1999

How NF-kappaB can be attracted by its cognate DNA.

NF-kappaB is involved in the transcriptional regulation of a large number of genes, in particular those of human immunodeficiency virus (HIV). Recently, we used NMR spectroscopy and molecular modelling to study the solution structure of a native duplex related to the HIV-1 kappaB site, together with a mutated duplex for which a three base-pair change abolishes NF-kappaB binding. The native duplex shows unusual dynamics of the four steps surrounding the kappaB site. Here, we explore the intrinsic properties of the NMR-refined structures of both duplexes in order to understand why the native sequence is recognised by NF-kappaB among other DNA sequences. We establish that only the native kappaB site can adopt a conformation where its structure (curvature and base displacement), the accessibility and the electrostatic potentials of key atoms become very favourable for binding the large loops of NF-kappaB, in contrast to the mutated duplex. Finally, we show that the neutralisation of phosphate groups contacted by NF-kappaB favours a more canonical DNA structure. These findings lead to a new hypothesis for specific recognition through the phosphodiester backbone dynamics of the sequences flanking a binding site. Such unusual behaviour confers upon the overall duplex properties that can be used by NF-kappaB to select its binding site. Thus, the selectivity determinants for NF-kappaB binding appear to depend on deformability of an "extended" consensus sequence.

Binding Sites↗

PubMed2001

The Bloom's and Werner's syndrome proteins are DNA structure-specific helicases.

BLM and WRN, the products of the Bloom's and Werner's syndrome genes, are members of the RecQ family of DNA helicases. Although both have been shown previously to unwind simple, partial duplex DNA substrates with 3'-->5' polarity, little is known about the structural features of DNA that determine the substrate specificities of these enzymes. We have compared the substrate specificities of the BLM and WRN proteins using a variety of partial duplex DNA molecules, which are based upon a common core nucleotide sequence. We show that neither BLM nor WRN is capable of unwinding duplex DNA from a blunt-ended terminus or from an internal nick. However, both enzymes efficiently unwind the same blunt-ended duplex containing a centrally located 12 nt single-stranded 'bubble', as well as a synthetic X-structure (a model for the Holliday junction recombination intermediate) in which each 'arm' of the 4-way junction is blunt-ended. Surprisingly, a 3'-tailed duplex, a standard substrate for 3'-->5' helicases, is unwound much less efficiently by BLM and WRN than are the bubble and X-structure substrates. These data show conclusively that a single-stranded 3'-tail is not a structural requirement for unwinding of standard B-form DNA by these helicases. BLM and WRN also both unwind a variety of different forms of G-quadruplex DNA, a structure that can form at guanine-rich sequences present at several genomic loci. Our data indicate that BLM and WRN are atypical helicases that are highly DNA structure specific and have similar substrate specificities. We interpret these data in the light of the genomic instability and hyper-recombination characteristics of cells from individuals with Bloom's or Werner's syndrome.

Base Sequence↗

PubMed1989

Solution structure of the EcoRI DNA sequence: refinement of NMR-derived distance geometry structures by NOESY spectrum back-calculations.

The solution structure of the self-complementary DNA duplex [d(CGCGAATTCGCG)]2, which contains the EcoRI restriction site sequence GAATTC at the center, has been studied by two-dimensional nuclear magnetic resonance spectroscopy. Time-dependent nuclear Overhauser effect spectra were used to obtain the initial cross-relaxation rates between 155 pairs of protons. These initial cross-relaxation rates were converted into interproton distances and entered into a distance (bounds) matrix. A distance geometry algorithm (DSPACE) was used to create embedded starting structures and to refine these structures until they showed good agreement with the distance matrix; symmetry constraints were included in the refinement procedure, making the two strands in the refined distance geometry structures virtually identical and significantly improving the agreement with the distance matrix. The NOESY spectrum for one of these distance geometry structures was then calculated from the explicit coordinates by numerically integrating all the z-magnetization transfer pathways among neighboring protons within a specified radius. Distances in this distance geometry structure that did not agree with the experimental NOESY time course were then adjusted accordingly. This process was iterated until a good agreement between calculated and experimental NOESY spectra was reached. The final structure, which generates good agreement with the experimental NOESY spectrum, displays kinks at the C3-G4 base step and at the A6-T7 base step that appear to be similar to those reported for the EcoRI restriction site DNA bound to its endonuclease. The solution structure is not the same as the crystal structure of this DNA duplex.

Base Sequence↗

PubMed1979

An analysis of cytoplasmic RNA populations in Drosophila melanogaster, Oregon R.

Nucleic acid reassociation methods were used to estimate the number of different polyadenylated RNA [poly (A) + RNA] sequences in the cytoplasm of whole Drosophila melanogaster at different stages of development and in the cytoplasm of cells of the L3 cell line. The number-average length (LN) of poly (A) + RNA from L3 cells is 1.4 kb, and the steady-state LN of the poly (A) tracts is 70 nucleotides. Analysis of RNA-driven reassociation with copy DNA shows that the poly (A) + RNA from L3 cells contains 5200 different sequences distributed in three abundance classes. The RNA forms hybrid duplexes with about 4.5% of single-copy Drosophila DNA, corresponding to 6500 sequences of 1.4 kb. The LN of poly (A) tracts present in whole embryos, larvae, pupae, and imagos is in each case close to 70 nucleotides. RNA-driven reassociation experiments show that poly (A) + RNA from these sources contains, respectively, 3500, greater than or equal to 4900, 6900, and greater than or equal to 4900 sequences. Cross-hybridization reactions show extensive homology between these RNA populations. All five poly (A) + RNA preparations contain a prominent component with a sedimentation coefficient of 13 and a size of 1.78 kb, which is shown to be the larger species of mitochondrial rRNA. This rRNA binds to oligo (dT) cellulose and serves as an efficient template for cDNA synthesis by reverse transcriptase. In RNA-driven reassociation experiments it behaves like an abundant mRNA.

Animals↗