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Results for “Nucleic acid manipulation”

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At least 19 recordsLinked to original sources
PubMed2025

Hydrolytic endonucleolytic ribozyme (HYER): Systematic identification, characterization and potential application in nucleic acid manipulation.

Group II introns are transposable elements that can propagate in host genomes through the "copy and paste" mechanism. They usually comprise RNA and protein components for effective propagation. Recently, we found that some bacterial GII-C introns without protein components had multiple copies in their resident genomes, implicating their potential transposition activity. We demonstrated that some of these systems are active for hydrolytic DNA cleavage and proved their DNA manipulation capability in bacterial or mammalian cells. These introns are therefore named HYdrolytic Endonucleolytic Ribozymes (HYERs). Here, we provide a detailed protocol for the systematic identification and characterization of HYERs and present our perspectives on its potential application in nucleic acid manipulation.

RNA, Catalytic↗
PubMed2000

Peptide nucleic acid (PNA): its medical and biotechnical applications and promise for the future.

Synthetic molecules that can bind with high sequence specificity to a chosen target in a gene sequence are of major interest in medicinal and biotechnological contexts. They show promise for the development of gene therapeutic agents, diagnostic devices for genetic analysis, and as molecular tools for nucleic acid manipulations. Peptide nucleic acid (PNA) is a nucleic acid analog in which the sugar phosphate backbone of natural nucleic acid has been replaced by a synthetic peptide backbone usually formed from N-(2-amino-ethyl)-glycine units, resulting in an achiral and uncharged mimic. It is chemically stable and resistant to hydrolytic (enzymatic) cleavage and thus not expected to be degraded inside a living cell. PNA is capable of sequence-specific recognition of DNA and RNA obeying the Watson-Crick hydrogen bonding scheme, and the hybrid complexes exhibit extraordinary thermal stability and unique ionic strength effects. It may also recognize duplex homopurine sequences of DNA to which it binds by strand invasion, forming a stable PNA-DNA-PNA triplex with a looped-out DNA strand. Since its discovery, PNA has attracted major attention at the interface of chemistry and biology because of its interesting chemical, physical, and biological properties and its potential to act as an active component for diagnostic as well as pharmaceutical applications. In vitro studies indicate that PNA could inhibit both transcription and translation of genes to which it has been targeted, which holds promise for its use for antigene and antisense therapy. However, as with other high molecular mass drugs, the delivery of PNA, involving passage through the cell membrane, appears to be a general problem.

Animals↗
PubMed2004

Single-molecule manipulation of nucleic acids.

During the past decade, local force measurement techniques, such as atomic force microscopy and optical tweezers, were used to study the elastic properties and mechanically induced structural transitions of nucleic acids at the single-molecule level. Single-molecule manipulation has also increasingly been used to investigate DNA-dependent enzymatic processes, with implications for unfolding and modifying DNA, protein-DNA interactions, replication and transcription. Compared to classical techniques of molecular biology, single-molecule measurements avoid the need to average over a large number of events, and can thus potentially provide detailed and complementary information.

Biochemistry↗
PubMed2001

Molecular Biocomputing Suite: a word processor add-in for the analysis and manipulation of nucleic acid and protein sequence data.

In all fields of molecular biology, researchers are increasingly challenged by experiments planned and evaluated on the basis of nucleic acid and protein sequence data generally retrieved from public databases. Despite the wide spectrum of available Web-based software tools for sequence analysis, the routine use of these tools has disadvantages, particularly because of the elaborate and heterogeneous ways of data input, output, and storage. Here we present a Visual Basic-encoded Microsoft Word Add-In, the Molecular BioComputing Suite (MBCS), available at the BioTechniques Software Library (www.BioTechniques.com). The MBCS software aims to manage and expedite a wide range of sequence analyses and manipulations using an integrated text editor environment including menu-guided commands. Its independence of sequence formats enables MBCS to be used as a pivotal application between other software tools for sequence analysis, manipulation, annotation, and editing.

Amino Acid Sequence↗
PubMed1995

NUVIEW: software for display and interactive manipulation of nucleic acid models.

The NUVIEW software package allows skeletal models of any double helical nucleic acid molecule to be displayed on a graphics monitor and to apply various rotations, translations and scaling transformations interactively, through the keyboard. The skeletal model is generated by connecting any pair of representative points, one from each of the bases in the basepair. In addition to the above mentioned manipulations, the base residues can be identified by using a locator and the distance between any pair of residues can be obtained. A sequence based color coded display allows easy identification of sequence repeats, such as runs of Adenines. The real time interactive manipulation of such skeletal models for large DNA/RNA double helices, can be used to trace the path of the nucleic acid chain in three dimensions and hence get a better idea of its topology, location of linear or curved regions, distances between far off regions in the sequence etc. A physical picture of these features will assist in understanding the relationship between base sequence, structure and biological function in nucleic acids.

Animals↗
PubMed1997

In vivo footprinting of the interaction of proteins with DNA and RNA.

Analysis of the interaction of proteins with either DNA or RNA sequences by in vivo footprinting involves two steps: (i) the in situ modification of nucleic acids by the footprinting reagent and (ii) the visualization of the footprints. Ligation-mediated PCR (LM-PCR) procedures provide a level of sensitivity and specificity that is suitable for visualization of footprints of single-copy genes or low-abundance mRNAs in higher eukaryotes. In this article, we discuss several of the technical aspects of these multistep procedures that contribute to the quality of the results, particularly the parameters that affect the specificity and fidelity of the reactions: (i) the design of the primers, which is important to achieve optimal specificity; (ii) the choice of polymerases so that the amplified material represents faithfully the initial nucleic acid population; and (iii) the impact of the plateau effect within the PCR on the interpretation of the data. We then discuss aspects of in vivo nucleic acid manipulation that may affect the quality of the footprinting image, in particular the choice of the footprinting reagent and its condition of use (e.g., on intact or permeabilized cells or prepared nuclei) and the extent of nucleic acid modification. Finally, we provide detailed experimental procedures corresponding to the techniques we have developed or modified: LM-PCR, reverse ligation-mediated PCR, and nuclease treatment of RNAs in vivo.

Base Sequence↗
PubMed1999

Identification of the essential EPE1 gene involved in retention of secreted proteins on the cell surface of Saccharomyces cerevisiae cells.

Saccharomyces cerevisiae yeast cells secrete extracellularly low amounts of a few proteins. The reasons for retardation of secreted proteins on the cell surface remain obscure. We describe here a mutant able to export enhanced amount of proteins. Classical genetic methods, nucleic acids manipulations and cloning procedures were used to isolate and characterize the mutant and to clone and sequence the corresponding wild type gene. The isolated Saccharomyces cerevisiae mutant MW11, is temperature sensitive and exports on average twenty-fold more proteins at 37 degrees C than parental wild type strain (80 micrograms of proteins/1 x 10(8) mutant cells, SEM +/- 5, n22; versus 3 micrograms of proteins/1 x 10(8) parental cells, SEM +/- 1, n22). Protein overexport in the mutant requires a functional SEC1 pathway and is independent of cell lysis. Cloning and sequencing of the corresponding wild type gene identified an open reading frame of 786 bp coding for a hydrophilic protein with predicted molecular mass of 30 kDa and cytosolic localization. The newly identified gene, designated EPE1, is an essential gene. Its DNA and amino acids sequence showed no homology with other yeast genes and proteins. It is concluded that the function of unknown yet genes, such as EPE1 is needed for retention of secreted proteins on the surface of Saccharomyces cerevisiae cells.

Amino Acid Sequence↗
PubMed1997

Temperature-dependent formation of a conjugate between tris(hydroxymethyl)aminomethane buffer and the malondialdehyde-DNA adduct pyrimidopurinone.

The stability of the major adduct formed between the endogenous product malondialdehyde (MDA) and deoxyguanosine, a pyrimidopurinone termed M1G-dR, was tested under a variety of conditions required for nucleic acid manipulation. M1G-dR was found to be stable at neutral pH and 37 degrees C but to be unstable when stored at -20 degrees C in the presence of Tris buffers. A new product with a characteristic absorption band at 350 nm was identified by 1H-NMR as an enamino-imine comprised of one molecule of Tris, one molecule of MDA, and deoxyguanosine. The formation of the conjugate was observed on reaction of Tris with M1G-dR or its ring-opened derivative N2-(3-oxo-1-propenyl)deoxyguanosine. The Tris-M1G-dR conjugate was unstable in aqueous solutions at room temperature, undergoing hydrolysis. However, the Tris conjugate of M1G base remained stable at room temperature in organic solvent. The isolation and properties of a conjugate between M1G-dR and Tris suggest that cross-links may form by reaction of MDA with DNA but they are likely to be unstable to hydrolysis.

Cross-Linking Reagents↗
PubMed1989

Molecular and cellular biology of the inner ear. The next frontier.

Molecular and cellular biology is the study of cellular ultrastructure and function. Current research in immune regulation, delineation of receptor control function (signal translation at the cellular level), and nucleic acid manipulation (genetic engineering) illustrates fundamental relationships among biochemistry, immunology, and molecular and cellular biology. Our knowledge already includes significant discoveries in membrane permeability, ionic gradients, and mediators of homeostasis. To these are added newer discoveries in bone metabolism, vascular anatomy and physiology, trace element deficiency, and the broader field of nutrition. Biochemical research gradually is unlocking the mysteries of otosclerosis, Paget's disease, endolymphatic hydrops, ototoxicity, acoustic trauma, presbycusis, and other forms of sensorineural hearing loss. This paper presents some of the research topics of current interest. Relationships of molecular and cellular biology, immunology, and nutrition are discussed. Molecular and cellular biology of the inner ear is an exciting "new" frontier.

Adolescent↗
PubMed1995

Gene therapy in acute critical illness.

The development of techniques for manipulating nucleic acids and strategies for delivering DNA to humans has made gene therapy a reality. Although mostly focused on genetically based diseases so far, there is every reason to expand the concept to include acquired diseases. Critical illness may be a good target for gene therapy because of the high mortality and need for only transient treatment. Genes can be delivered in vivo using viral vectors (replication-deficient adenovirus and adeno-associated virus most often). Viral vectors have some negatives, mainly the triggering of an inflammatory and an immune response. Nonviral DNA delivery systems include liposomes (cationic or anionic), direct DNA injection, and polycation-DNA-glycoconjugates. Combining liposomes with viral components to deliver plasmids with a transgene may improve efficiency of delivery without causing toxicity. In a model of acute lung injury, in vivo delivery of a vector hyperexpressing the prostaglandin synthase gene using cationic liposomes resulted in increased production of prostaglandin E2 and prostacyclin in the lungs, and protected the lungs from the effects of endotoxin. This end-result demonstrates the feasibility of this approach. A similar rationale for the treatment of sepsis could be used. Other promising therapeutic genes would include those encoding antioxidant enzymes or antiproteases. The logistics for moving to initial studies of gene therapy in critically ill humans have been worked out for other diseases; such steps should expedite the exploration of this new category of therapies.

Acute Disease↗
PubMedDate not supplied

Performing nucleic acid reactions using predispensed lyophilized reaction mixtures.

A system is described in which manipulations of nucleic acids are performed in wells containing predispensed lyophilized reaction mixtures requiring addition of only nucleic acid. This allows increased reproducibility for single-step reactions (e.g., restrictions and ligations), as well as improved productivity for complex reactions (e.g., sequencing). Enzymes, co-factors, nucleotides and buffers can be dried and stored at room temperature without loss of essential function. When used for DNA sequencing, hundreds of templates a day can be sequenced with the potential to determine megabase amounts of sequence per week.

Base Sequence↗
PubMed1997

A universal procedure for primer labelling of amplicons.

Detection and visualisation of nucleic acids is integral to genome analyses. Exponential amplification procedures have provided the means for the manipulation of nucleic acid sequences, which were otherwise inaccessible. We describe the development and application of a universal method for the labelling of any PCR product using a single end-labelled primer. Amplification was performed in a single reaction with the resulting amplicon labelled to a high specific activity. The method was adapted to a wide range of PCRs and significantly reduced the expense of such analyses.

DNA Primers↗
PubMed1980

Steps toward computer analysis of nucleotide sequences.

Advances in recombinant DNA technology have allowed the isolation of large numbers of biologically interesting fragments of DNA. Concomitant improvements in methods for nucleic acid sequencing have led many investigators to characterize their clones by sequencing them. This has resulted in the accumulation of such large amounts of sequence data that computer-assisted methods, with programs directed toward the manipulation of nucleic acid sequences, have become indispensable during the collection and analysis of that data.

Autoanalysis↗