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Target DNA Sequence (target + dna_sequence)
Selected AbstractsConjugated Polymers Combined with a Molecular Beacon for Label-Free and Self-Signal-Amplifying DNA MicroarraysADVANCED FUNCTIONAL MATERIALS, Issue 20 2009Kangwon Lee Abstract A conjugated polymer (CP) and molecular-beacon-based solid-state DNA sensing system is developed to achieve sensitive, label-free detection. A novel conjugated poly(oxadiazole) derivative exhibiting amine and thiol functional groups (POX-SH) is developed for unique chemical and photochemical stability and convenient solid-state on-chip DNA synthesis. POX-SH is soluble in most nonpolar organic solvents and exhibits intense blue fluorescence. POX-SH is covalently immobilized onto a maleimido-functionalized glass slide by means of its thiol group. Molecular beacons having a fluorescent dye or quencher molecule as the fluorescence resonance energy transfer (FRET) acceptor are synthesized on the immobilized POX-SH layer through direct on-chip oligonucleotide synthesis using the amine side chain of POX-SH. Selective hybridization of the molecular beacon probes with the target DNA sequence opens up the molecular beacon probes and affects the FRET between POX-SH and the dye or quencher, producing a sensitive and label-free fluorescence sensory signal. Various molecular design parameters, such as the size of the stem and loop of the molecular beacon, the choice of dye, and the number of quencher molecules are systematically controlled, and their effects on the sensitivity and selectivity are investigated. [source] Mutations that alter the regulation of the chb operon of Escherichia coli allow utilization of cellobioseMOLECULAR MICROBIOLOGY, Issue 6 2007Aashiq H. Kachroo Summary Wild-type strains of Escherichia coli are normally unable to metabolize cellobiose. However, cellobiose-positive (Cel+) mutants arise upon prolonged incubation on media containing cellobiose as the sole carbon source. We show that the Cel+ derivatives carry two classes of mutations that act concertedly to alter the regulation of the chb operon involved in the utilization of N,N,-diacetylchitobiose. These consist of mutations that abrogate negative regulation by the repressor NagC as well as single base-pair changes in the transcriptional regulator chbR that translate into single-amino-acid substitutions. Introduction of chbR from two Cel+ mutants resulted in activation of transcription from the chb promoter at a higher level in the presence of cellobiose, in reporter strains carrying disruptions of the chromosomal chbR and nagC. These transformants also showed a Cel+ phenotype on MacConkey cellobiose medium, suggesting that the wild-type permease and phospho-,-glucosidase, upon induction, could recognize, transport and cleave cellobiose respectively. This was confirmed by expressing the wild-type genes encoding the permease and phospho-,-glucosidase under a heterologous promoter. Biochemical characterization of one of the chbR mutants, chbRN238S, showed that the mutant regulator makes stronger contact with the target DNA sequence within the chb promoter and has enhanced recognition of cellobiose 6-phosphate as an inducer compared with the wild-type regulator. [source] Age-Related Mitochondrial DNA Mutations in the Human LarynxTHE LARYNGOSCOPE, Issue 12 2000Jose M. Manaligod MD Abstract Objective To determine whether age-related mitochondrial DNA mutations occur in the human larynx. Study Design Genetic study of cadaveric larynx specimens. Methods Vocal fold mucosa, thyroarytenoid muscle, and cricoarytenoid joint tissue were harvested from 13 fresh postmortem larynges (age range, 2 d,82 y). DNA was extracted from each sample, and the polymerase chain reaction (PCR) was used to amplify a target DNA sequence resulting from the common age-associated, 4977,base-pair (bp) mitochondrial DNA deletion. PCR products were visualized by agarose gel electrophoresis. Automated sequencing determined the sequence of identified PCR products. Subjects Thirteen cadaveric larynges were obtained through the University of Kentucky Medical Center (Lexington, KY). Specimens from patients with a history of head and neck cancer, previous laryngeal trauma, or surgery were excluded. Results Strongly positive bands were identified in samples from three individuals. Weaker bands were seen in samples from four other samples. No band was noted from the two pediatric larynges. Different band patterns were seen among the three different tissue sites in the larynges with positive PCR products, but no consistent pattern was seen. Sequencing of the identified PCR products from selected samples confirmed that they were products of the age-associated, 4977-bp mitochondrial DNA deletion. Conclusions An age-associated mitochondrial DNA deletion was detected in several postmortem human larynges. Its presence seemed to increase in appearance with age. In the larynges in which the deletion occurred, there were individual regional differences in the occurrence of the deletion, but no consistent pattern was noted across all individuals who carried the deletion. [source] Design and Characterisation of an Artificial DNA-Binding CytochromeCHEMBIOCHEM, Issue 7 2004D. Dafydd Jones Dr. Abstract We aim to design novel proteins that link specific biochemical binding events, such as DNA recognition, with electron transfer functionality. We want these proteins to form the basis of new molecules that can be used for templated assembly of conducting cofactors or for thermodynamically linking DNA binding with cofactor chemistry for nanodevice applications. The first examples of our new proteins recruit the DNA-binding basic helix region of the leucine zipper protein GCN4. This basic helix region was attached to the N and C termini of cytochrome b562(cyt b562) to produce new, monomeric, multifunctional polypeptides. We have fully characterised the DNA and haem-binding properties of these proteins, which is a prerequisite for future application of the new molecules. Attachment of a single basic helix of GCN4 to either the N or C terminus of the cytochrome does not result in specific DNA binding but the presence of DNA-binding domains at both termini converts the cytochrome into a specific DNA-binding protein. Upon binding haem, this chimeric protein attains the spectral characteristics of wild-type cyt b562. The three forms of the protein, apo, oxidised holo and reduced holo, all bind the designed (ATGAcgATGA) target DNA sequence with a dissociation constant, KD, of approximately 90 nM. The protein has a lower affinity (KDca. 370 nM) for the wild-type GCN4 recognition sequence (ATGAcTCAT). The presence of only half the consensus DNA sequence (ATGAcgGGCC) shifts the KDvalue to more than 2500 nM and the chimera does not bind specifically to DNA sequences with no target recognition sites. Ultracentrifugation revealed that the holoprotein,DNA complex is formed with a 1:1 stoichiometry, which indicates that a higher-order protein aggregate is not responsible for DNA binding. Mutagenesis of a loop linking helices 2 and 3 of the cytochrome results in a chimera with a haem-dependent DNA binding affinity. This is the first demonstration that binding of a haem group to a designed monomeric protein can allosterically modulate the DNA binding affinity. [source] Control of Biocatalytic Transformations by Programmed DNA AssembliesCHEMISTRY - A EUROPEAN JOURNAL, Issue 12 2010Ronit Freeman Abstract This study demonstrates the self-assembly of inhibitor/enzyme-tethered nucleic acid fragments or enzyme I-, enzyme II-modified nucleic acids into functional nanostructures that lead to the controlled inhibition of the enzyme or the activation of an enzyme cascade. In one system, the anti-cocaine aptamer subunits are modified with monocarboxy methylene blue (MB+) as the inhibitor and with choline oxidase (ChOx). The cocaine-induced self-assembly of the aptamer subunits complex results in the inhibition of ChOx by MB+. In a further configuration, two nucleic acids of limited complementarity are functionalized at their 3, and 5, ends with glucose oxidase (GOx) and horseradish peroxidase (HRP), respectively, or with MB+ and ChOx. In the presence of a target DNA sequence, synergistic complementary base-pairing occurs, thus leading to stable supramolecular Y-shaped nanostructures of the nucleic acid units. A GOx/HRP bienzyme cascade or the programmed inhibition of ChOx by MB+ is demonstrated in the resulting nucleic acid nanostructures. A quantitative theoretical model that describes the nucleic acid assemblies and that results in the inhibition of ChOx by MB+ or in the activation of the GOx/HRP cascade, respectively, is provided. [source] Enhancement and rescue of target capture in Tn10 transposition by site-specific modifications in target DNAMOLECULAR MICROBIOLOGY, Issue 4 2004Patrick A. Pribil Summary The bacterial transposon Tn10 inserts preferentially into specific target sequences. This insertion specificity appears to be linked to the ability of target sites to adopt symmetrically positioned DNA bends after binding the transposition machinery. Target DNA bending is thought to permit the transposase protein to make additional contacts with the target DNA, thereby stabilizing the target complex so that the joining of transposon and target DNA sequences can occur efficiently. In the current work, we have asked whether the introduction of a discontinuity in a target DNA strand, a modification that is expected to make it easier for a DNA molecule to bend, can enhance or rescue target capture under otherwise suboptimal reaction conditions. We show that either a nick or a missing phosphate specifically at the site of reaction chemistry increases the ability of various target DNAs to form the target capture complex. The result suggests that the bends in the target DNA are highly localized and include the scissile phosphates. This raises the possibility that strand transfer is mechanistically linked to target capture. We have also identified specific residues in the target DNA and in transposase that appear to play an important role in target DNA bending. [source] | ||||||||||