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 1,369 records · Page 76Linked to original sources

PubMed2005

Periodic DNA nanotemplates synthesized by rolling circle amplification.

Rolling circle amplification (RCA) is an elegant biochemical method by which long single-stranded DNA molecules with a repeating sequence motif can be readily synthesized. In RCA, small circular single-stranded oligonucleotides serve as templates for the polymerization of the complementary strand. A DNA polymerase with an efficient strand displacement activity can copy the circular template without stopping. This results in a long DNA strand with periodic sequence. We here demonstrate that this method, using DNA recognition and biotin-streptavidin binding, provides a simple procedure for DNA-directed nanoscale organization of matter. As an example, a 74 nucleotide (nt) long circular DNA molecule is amplified into a sequence-periodic single strand with a length up to several micrometers. Hybridization of this long periodic DNA template to the biotinylated complement of the sequence motif results in a long DNA duplex with a periodic arrangement of biotin binding sites. On this duplex, streptavidin-coated particles can be organized into one-dimensional arrays. The resulting DNA constructs are characterized by gel electrophoresis and atomic force microscopy.

Biotin↗

PubMed2002

Fluorescence characteristics of site-specific and stereochemically distinct benzo[a]pyrene diol epoxide-DNA adducts as probes of adduct conformation.

Spectroscopic fluorescence quenching techniques are described for distinguishing the conformational characteristics of adducts derived from the binding of the benzo[a]pyrene metabolite anti-BPDE (the diol epoxide r7,t8-dihydroxy-t9,10epoxy-7,8,9,10-tetrahydrobenz[a]pyrene) to the exocyclic amino groups of guanine ([BP]-N(2)-dG) and adenine ([BP]-N(6)-dA) in double stranded oligonucleotides. These methods are calibrated by comparing the fluorescence quenching and UV absorbance characteristics of different, stereoisomeric anti-[BP]-N(2)-dG adducts of known adduct conformations, previously established by high-resolution NMR techniques. It is shown that intercalative adduct conformations can be distinguished from solvent-exposed adduct conformations, e.g., adducts in which the pyrenyl residues are positioned in the minor groove. These low resolution fluorescence methods are at least 4 orders of magnitude more sensitive than the high-resolution NMR techniques; the fluorescence methods are useful for distinguishing adduct conformations when either small amounts of material are available or the NMR signals are of such poor quality that high-resolution structures cannot be determined. This methodology is illustrated using a variety of anti-BPDE-modified oligonucleotides of varying adduct conformations. It is shown that the 10S (+)-trans-anti-[BP]-N(6)-dA adduct in an oligonucleotide duplex containing an N-ras protooncogene sequence, believed to be conformationally heterogeneous and disordered, is significantly more exposed to the solvent environment than the stereoisomeric, intercalated 10R adduct [Zegar et al. (1996) Biochemistry 35, 6212]. These differences suggest an explanation for the greater efficiencies of excision of the 10S adduct (relative to the 10R adduct) by human nucleotide excision repair enzymes [Buterin et al. (2000) Cancer Res. 60, 1849].

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed2000

On the kinetics of distamycin binding to its target sites on duplex DNA.

Distamycin A is a well known polyamide antibiotic that can bind in the minor groove of duplex DNA primarily at AT-rich sequences both as a monomer or as a side-by-side antiparallel dimer. The association phase of the distamycin binding reaction has not been studied in either of its binding modes, because of the lack of an adequate UV or CD signal at the low concentrations needed to monitor the fast bimolecular reaction. We report a significant increase in fluorescence amplitude, accompanied by a small red shift, on binding distamycin to its specific target sites. This signal can be used to monitor drug binding in steady-state and time-resolved processes. Distamycin shows extremely fast association with the 1:1 binding site, with a bimolecular rate of 7 x 10(7) M(-1) small middle dots(-1) and also fairly rapid dissociation ( approximately 3 s(-1)). When DNA is in excess, there is a slow component in the association reaction whose rate decreases strongly with increasing DNA concentration. Binding of the drug to the 2:1 site occurs in two distinct steps: fast, sequential binding of each drug molecule to the DNA with a bimolecular rate comparable to that at the 1:1 site, followed by a slow ( approximately 4 s(-1)) equilibration to the final population. Dissociation from the 2:1 site is approximately 40-fold slower than from the 1:1 site. This study provides the groundwork for analysis of the binding kinetics of longer polyamides and covalently linked polyamides that have recently been shown to inhibit transcription in vivo.

Anti-Bacterial Agents↗

PubMed2000

Purification and characterization of OF-1, a host factor implicated in herpes simplex replication.

A human cellular factor (OF-1) has been previously implicated in replication of herpes simplex virus, type 1. This protein binds to three conserved regions (Boxes I, II, and III) in the viral replication origin and appears to be required for viral DNA synthesis (Dabrowski, C. C., Carmillo, P. J., and Schaffer, P. A. (1994) Mol. Cell. Biol. 14, 2545-2555). In the present study, we have partially purified and characterized OF-1 from human cells. This protein appears to consist of a tetramer composed of two heterodimers with subunits of 73 and 90 kDa. The smaller subunit contains the DNA binding activity. We have investigated the binding specificity of OF-1 using a mobility shift analysis. These studies reveal that binding is specific for both duplex and single-stranded Box I sequences and that the strongest preference is for the bottom strand of Box I. We present evidence suggesting that the binding of OF-1 to Box I DNA is enhanced in the presence of the herpes simplex-encoded UL9 protein, which also binds to Box I in oriS and is required for viral replication. Implications of these findings for the initiation step in viral replication are discussed.

Base Sequence↗

PubMed2001

Bacteriophage T4 Dam DNA-[N6-adenine]methyltransferase. Kinetic evidence for a catalytically essential conformational change in the ternary complex.

We carried out a steady state kinetic analysis of the bacteriophage T4 DNA-[N6-adenine]methyltransferase (T4 Dam) mediated methyl group transfer reaction from S-adenosyl-l-methionine (AdoMet) to Ade in the palindromic recognition sequence, GATC, of a 20-mer oligonucleotide duplex. Product inhibition patterns were consistent with a steady state-ordered bi-bi mechanism in which the order of substrate binding and product (methylated DNA, DNA(Me) and S-adenosyl-l-homocysteine, AdoHcy) release was AdoMet downward arrow DNA downward arrow DNA(Me) upward arrow AdoHcy upward arrow. A strong reduction in the rate of methylation was observed at high concentrations of the substrate 20-mer DNA duplex. In contrast, increasing substrate AdoMet concentration led to stimulation in the reaction rate with no evidence of saturation. We propose the following model. Free T4 Dam (initially in conformational form E) randomly interacts with substrates AdoMet and DNA to form a ternary T4 Dam-AdoMet-DNA complex in which T4 Dam has isomerized to conformational state F, which is specifically adapted for catalysis. After the chemical step of methyl group transfer from AdoMet to DNA, product DNA(Me) dissociates relatively rapidly (k(off) = 1.7 x s(-1)) from the complex. In contrast, dissociation of product AdoHcy proceeds relatively slowly (k(off) = 0.018 x s(-1)), indicating that its release is the rate-limiting step, consistent with kcat = 0.015 x s(-1). After AdoHcy release, the enzyme remains in the F conformational form and is able to preferentially bind AdoMet (unlike form E, which randomly binds AdoMet and DNA), and the AdoMet-F binary complex then binds DNA to start another methylation cycle. We also propose an alternative pathway in which the release of AdoHcy is coordinated with the binding of AdoMet in a single concerted event, while T4 Dam remains in the isomerized form F. The resulting AdoMet-F binary complex then binds DNA, and another methylation reaction ensues. This route is preferred at high AdoMet concentrations.

Bacteriophage T4↗

PubMed1992

Sequence effects on the propeller twist of base pairs in DNA helices.

Molecular mechanics calculations are performed on all the ten base pair steps (duplex dimers) and also a number of trimer and tetramer duplexes comprising them, in an attempt to systematically examine the possible base sequence effects on the magnitudes of propeller twists of base pairs at a given step. The analysis reveals that though propeller twist is a base pair property, it behaves very much like other base step parameters such as slide, roll, twist etc., Hence, it may be necessary to monitor the nature and variation of magnitudes of pt at a step. Calculations performed on 45 out of the 136 unique tetramer combinations involving all the ten unique base steps show that the difference in magnitudes of propeller twists of the base pairs of a given step has been found to be either steep or moderate depending on base pairs that flank the base step. These observations compare very well with the available experimental data. Tetramer sequences, wherein a base pair of a base step repeats in the same direction, exhibit a relatively steep difference in propeller twist at the step. Tetramers other than these exhibit moderate difference in propeller twist. Such sequences are broadly classified as type-I and type-II respectively. Practically all the tetrads considered in the study, excepting those with GT step and a few involving CG and GC steps, conform to the above classification.

Base Composition↗

PubMed1988

Joining of nonhomologous DNA double strand breaks in vitro.

Extracts of Xenopus laevis eggs can efficiently join ends of duplex DNA that differ in structure and sequence. This was analysed by recircularisation of linear plasmid DNA molecules with dissimilar termini, generated by successive cuts with two different restriction enzymes within the pSP65 polylinker. Use of various enzymes provided blunt ended or 4 nucleotides long 3' and 5' protruding single strand (PSS) termini which were successfully joined in vitro in any tested combination. Sequence analysis of numerous junctions from cloned reaction products of 7 terminus combinations reveal: apart from very rare base exchanges and single nucleotide insertions less than 10% deletions (1 to 18 nucleotides long) were detected. Blunt/PSS or 3'PSS/5'PSS terminus pairs undergo simple "blunt end" joining which preserves PSS ends by fill-in. In contrast, equally polar 3'PSS/3'PSS or 5'PSS/5'PSS terminus pairs are joined by a complex mode: PSS ends overlap by a defined number of nucleotides, set by matching basepairs. Even one basematch suffices to define the setting. This then determines the final mismatch repair and fill-in pattern. We propose that yet unknown terminal DNA-binding proteins stabilize the energetically highly unfavorable configuration of single matching basepairs and help to support defined overlap structures.

Animals↗

PubMed1997

Strong, specific, monodentate G-C base pair recognition by N7-inosine derivatives in the pyrimidine.purine-pyrimidine triple-helical binding motif.

The nucleoside analogs 7-(2'-deoxy-alpha-D-ribofuranosyl)hypoxanthine (alpha7H,1), 7-(2'-deoxy-beta-D-ribofuranosyl)hypoxanthine (beta7H,2) and 7-7-(2'-O-methyl-beta-D- ribofuranosyl)hypoxanthine (beta7HOMe,3) were prepared and incorporated into triplex forming oligodeoxynucleotides, designed to bind to DNA in the parallel (pyrimidine.purine-pyrimidine) motif. By DNase I footprinting techniques and UV-melting curve analysis it was found that, at pH 7. 0, the 15mer oligonucleotides d(TTTTTMeCTXTMeCTMeCTMeCT) (MeC = 5-methyl-deoxycytidine, X =beta7H,beta7HOMe) bind to a DNA target duplex forming a H.G-C base triple with equal to slightly increased (10-fold) stability compared to a control oligodeoxynucleotide in which the hypoxanthine residue is replaced by MeC. Remarkably, triple-helix formation is specific to G-C base pairs and up to 40 microM third strand concentration, no stable triplex exhibiting H.A-T, H.T-A or H.C-G base arrangements could be found (target duplex concentration approximately 0.1 nM). Multiply substituted sequences containing beta7H residues either in an isolated [d(TTTTTbeta7HTbeta7HTbeta7HTbeta7HTbeta7HT)] or in a contiguous [d(TTTbeta7Hbeta7Hbeta7Hbeta7HTTTTbeta7HTTT)] manner still form triplexes with their targets of comparable stability as the control (MeC-containing) sequences at pH 7.0 and high salt or spermine containing buffers. General considerations lead to a structural model in which the recognition of the G-C base pair by hypoxanthine takes place via only one H-bond of the N-H of hypoxanthine to N7 of guanine. This model is supported by a molecular dynamics simulation. A general comparison of the triplex forming properties of oligonucleotides containing beta7H with those containing MeC or N7-2'-deoxyguanosine (N7G) reveals that monodentate recognition in the former case can energetically compete with bidentate recognition in the latter two cases.

Base Composition↗

PubMed2002

Binding analysis between naphthyridine dimers and GG mismatch DNA.

Kinetics and thermodynamics studies of the binding between naphthyridine dimer and DNA duplexes having a G-G mismatch sequence were performed by using Surface Plasmon Resonance (SPR) assay. Naphthyridine dimers possessing a different length of PEG linker were synthesized and immobilized to the dextran surface of the sensor chip to obtain an improved sensitivity of the SPR sensor toward a G-G mismatch. The effect of the directly neighboring base pair to the G-G mismatch was estimated from the thermodynamic parameters of the complex. These results indicate that stacking with adjacent base pair and naphthyridine dimer is the key roll for the formation of the complex between mismatched base pair and naphthyridine dimer.

Base Pair Mismatch↗

PubMed1997

A histone octamer blocks branch migration of a Holliday junction.

The Holliday junction is a key intermediate in genetic recombination. Here, we examine the effect of a nucleosome core on movement of the Holliday junction in vitro by spontaneous branch migration. Histone octamers consisting of H2A, H2B, H3, and H4 are reconstituted onto DNA duplexes containing an artificial nucleosome-positioning sequence consisting of a tandem array of an alternating AT-GC sequence motif. Characterization of the reconstituted branch migration substrates by micrococcal nuclease mapping and exonuclease III and hydroxyl radical footprinting reveal that 70% of the reconstituted octamers are positioned near the center of the substrate and the remaining 30% are located at the distal end, although in both cases some translational degeneracy is observed. Branch migration assays with the octamer-containing substrates reveal that the Holliday junction cannot migrate spontaneously through DNA organized into a nucleosomal core unless DNA-histone interactions are completely disrupted. Similar results are obtained with branch migration substrates containing an octamer positioned on a naturally occurring sequence derived from the yeast GLN3 locus. Digestion of Holliday junctions with T7 endonuclease I establishes that the junction is not trapped by the octamer but can branch migrate in regions free of histone octamers. Our findings suggest that migration of Holliday junctions during recombination and the recombinational repair of DNA damage requires proteins not only to accelerate the intrinsic rate of branch migration but also to facilitate the passage of the Holliday junction through a nucleosome.

Animals↗

PubMed2005

Recognition of chromosomal DNA in human cells by peptide nucleic acids and small duplex RNAs.

Inhibition of translation by duplex RNA (siRNA) complementary to mRNA is a powerful approach to silencing genes in mammalian cells and RNA interference (RNAi) is an important natural biological mechanism for controlling gene expression. Anti-mRNA duplexes are widely used for laboratory studies, target validation, and therapeutic development. Endogenously expressed duplex RNAs (microRNAs, miRNAs) have been shown to target mRNA and be natural regulators of expression. Recently, we have shown that peptide nucleic acids (PNAs) or duplex antigene RNAs (agRNAs) that target DNA sequences can also inhibit gene transcription. These findings extend gene silencing to targets within chromosomal DNA.

Alternative Splicing↗

PubMed1997

Influence of Substrate and Ribozyme Sequences on the Functions of Ribozyme.

The mono-component ribozyme RZ1, RZ3 and RZ4 were ligated in different orders into two tri-component ribozymes RZ134 and RZ413.Both tri-component ribozymes could cleave individual specific substrate sequences from different regions of the tobacco mosaic virus (TMV) RNA. The cleavage efficiency and specificity of each unit-ribozymes in the tri-component ribozymes to its substrate was similar to its mono-ribozyme counterpart. When the three substrates were mixed and used as substrates for the tri-component ribozymes, only the activity of the unit-ribozymes RZ4 was clearly observed, the activities of the other two unit-ribozymes were greatly reduced. The reduction was mainly due to the fact that the two substrates BT2(+) and BT2(-) for RZ1 and RZ3 were complementary in sequence to each other and therefore formed duplex in the target region, thus must have strongly prevented the two ribozymes from forming the correct structures required for cleavage activity. In addition to its activity on the substrate BT2(+), the ribozyme RZ1 was found unexpectedly to be able to cleave BT4(-) specifically into various unique products.

Journal Article↗

PubMed1977

Hemoglobin switching in sheep and goats. Preparation and characterization of complementary DNAs specific for the alpha-, beta-, and gamma-globin messenger RNAs of sheep.

Specific complementary DNAs (cDNAs) for the messenger RNAs coding for sheep alpha-, betaA-, betaB-, betaC-, and gamma-globins were prepared by thermal denaturation of heterologous hybrids (e.g. alphabetaB-cDNA-alphagamma-mRNA) followed by hydroxylapatite chromatography. Each cDNA represented a nearly full-length copy of its globin mRNA complement as determined by electrophoretic analysis in polyacrylamide gels containing 98% formamide. The purity of each cDNA fraction was estimated by hybridization analysis and thermal denaturation. The beta- and gamma-cDNAs contained 5 to 20% contaminating alpha-cDNA while the alpha-cDNA was 25 to 30% contaminated with non-alpha-cDNA. The melting temperatures (Tm) of homologous duplexes between each non-alpha chain cDNA and its mRNA complement ranged from 69.5-71.5 degrees in 50% formamide while alpha-alpha duplexes melted with a Tm of 75-76 degrees. The Tm values of heterologous duplexes formed between each non-alpha-cDNA and the various globin mRNAs (e.g. betaB-cDNA-Hb C mRNA) ranged between 64.5 degrees and 68 degrees and thus were only 1.5-5.0degrees below that of homologous duplexes. These results suggest that the nucleotide sequence divergence among the various non-alpha-mRNAs (or cDNAs) is not greatly different from the minimum predicted from the amino acid sequence differences of the corresponding globins. When annealing reactions were performed above the Tm of the heterologous hybrids (68 degrees), each non-alpha-cDNA hybridized only to its own complementary mRNA. Thus the purified cDNAs provide molecular probes for the quantitation of alpha-, beta-, and gamma-globin-specific nucleotide sequences.

Animals↗

PubMed1984

Isolation and visualization of active presynaptic filaments of recA protein and single-stranded DNA.

In the presence of ATP, recA protein forms a presynaptic complex with single-stranded DNA that is an obligatory intermediate in homologous pairing. Presynaptic complexes of recA protein and circular single strands that are active in forming joint molecules can be isolated by gel filtration. These isolated active complexes are nucleoprotein filaments with the following characteristics: (i) a contour length that is at least 1.5 times that of the corresponding duplex DNA molecule, (ii) an ordered structure visualized by negative staining as a striated filament with a repeat distance of 9.0 nm and a width of 9.3 nm, (iii) approximately 8 molecules of recA protein and 20 nucleotide residues per striation. The widened spacing between bases in the nucleoprotein filament means that the initial matching of complementary sequences must involve intertwining of the filament and duplex DNA, unwinding of the latter, or some combination of both to equalize the spacing between nascent base pairs. These experiments support the concept that recA protein first forms a filament with single-stranded DNA, which in turn binds to duplex DNA to mediate both homologous pairing and subsequent strand exchange.

Adenosine Triphosphate↗

PubMed2002

Intercalation of an acridine-peptide drug in an AA/TT base step in the crystal structure of [d(CGCGAATTCGCG)](2) with six duplexes and seven Mg(2+) ions in the asymmetric unit.

We present the crystal structure of an acridine drug derivatized at carbon 9, [N(alpha)-(9-acridinoyl)-tetraarginine], intercalated within the dodecamer [d(CGCGAATTCGCG)](2). The presence of a lateral chain at the central carbon 9 atom differentiates this compound from most acridine drugs hitherto studied, which are usually derivatized at carbon 4. The DNA:drug interaction we observe differs from that observed in previous studies, which primarily involves shorter, mainly hexameric sequences, in two important regards: the acridine intercalates within an AA/TT base step, rather than within a CG/CG base step; and the binding site is located at the center of the sequence, rather than at one end of the duplex. In addition, we observe a novel crystal packing arrangement, with six dodecamer duplexes and seven hydrated magnesium ions in the asymmetric unit of a large (66.5 x 68.4 x 77.4 A(3)) unit cell in space group P2(1)2(1)2(1). The duplexes are organized in layers parallel to the ab plane, with consecutive layers crossing each other at right angles.

Acridines↗

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↗

PubMed1985

The influence of DNA sequence on terbium (III) fluorescence enhancement by DNA.

The synthetic DNA duplexes, poly(dA-dC):poly(dG-dT), poly(dG):poly(dC), poly(dG-dC):poly(dG-dC), and poly(dG-m5dC):poly(dG-m5dC), were analyzed as double- and single-strand polymers for the ability to enhance terbium fluorescence. Using conditions which limited the enhancement of Tb3+ fluorescence to that from DNA-guanosines, our results showed that (a) guanosines in single-strand DNA enhanced terbium fluorescence equally well irrespective of the primary sequence surrounding them, and (b) guanosines in either left- (Z-form) or right- (B-form) handed double helixes failed to enhance terbium fluorescence.

DNA↗

PubMed1999

IS1631 occurrence in Bradyrhizobium japonicum highly reiterated sequence-possessing strains with high copy numbers of repeated sequences RSalpha and RSbeta.

From Bradyrhizobium japonicum highly reiterated sequence-possessing (HRS) strains indigenous to Niigata and Tokachi in Japan with high copy numbers of the repeated sequences RSalpha and RSbeta (K. Minamisawa, T. Isawa, Y. Nakatsuka, and N. Ichikawa, Appl. Environ. Microbiol. 64:1845-1851, 1998), several insertion sequence (IS)-like elements were isolated by using the formation of DNA duplexes by denaturation and renaturation of total DNA, followed by treatment with S1 nuclease. Most of these sequences showed structural features of bacterial IS elements, terminal inverted repeats, and homology with known IS elements and transposase genes. HRS and non-HRS strains of B. japonicum differed markedly in the profiles obtained after hybridization with all the elements tested. In particular, HRS strains of B. japonicum contained many copies of IS1631, whereas non-HRS strains completely lacked this element. This association remained true even when many field isolates of B. japonicum were examined. Consequently, IS1631 occurrence was well correlated with B. japonicum HRS strains possessing high copy numbers of the repeated sequence RSalpha or RSbeta. DNA sequence analysis indicated that IS1631 is 2,712 bp long. In addition, IS1631 belongs to the IS21 family, as evidenced by its two open reading frames, which encode putative proteins homologous to IstA and IstB of IS21, and its terminal inverted repeat sequences with multiple short repeats.

Amino Acid Sequence↗