Gapped DNA and cyclization of short DNA fragments

We use the cyclization of small DNA molecules, approximately 200 bp in length, to study conformational properties of DNA fragments with single-stranded gaps. The approach is extremely sensitive to DNA conformational properties and, being complemented by computations, allows a very accurate determination of the fragment's conformational parameters. Sequence-specific nicking endonucleases are used to create the 4-nt-long gap. We determined the bending rigidity of the single-stranded region in the gapped DNA. We found that the gap of 4 nt in length makes all torsional orientations of DNA ends equally probable. Our results also show that the gap has isotropic bending rigidity. This makes it very attractive to use gapped DNA in the cyclization experiments to determine DNA conformational properties, since the gap eliminates oscillations of the cyclization efficiency with the DNA length. As a result, the number of measurements is greatly reduced in the approach, and the analysis of the data is greatly simplified. We have verified our approach on DNA fragments containing well-characterized intrinsic bends caused by A-tracts. The obtained experimental results and theoretical analysis demonstrate that gapped-DNA cyclization is an exceedingly sensitive and accurate approach for the determination of DNA bending.     

Accurate phylogenetic classification of variable-length DNA fragments

Metagenome studies have retrieved vast amounts of sequence data from a variety of environments leading to new discoveries and insights into the uncultured microbial world. Except for very simple communities, the encountered diversity has made fragment assembly and the subsequent analysis a challenging problem. A taxonomic characterization of metagenomic fragments is required for a deeper understanding of shotgun-sequenced microbial communities, but success has mostly been limited to sequences containing phylogenetic marker genes. Here we present PhyloPythia, a composition-based classifier that combines higher-level generic clades from a set of 340 completed genomes with sample-derived population models. Extensive analyses on synthetic and real metagenome data sets showed that PhyloPythia allows the accurate classification of most sequence fragments across all considered taxonomic ranks, even for unknown organisms. The method requires no more than 100 kb of training sequence for the creation of accurate models of sample-specific populations and can assign fragments >or=1 kb with high specificity.     

Aggregate formation from short fragments of plant DNA

Large aggregates have been observed after partial reassociation of pea (Pisum sativum L.) DNA preparations sheared to mean single strand fragment lengths as short as 350 nucleotides. At high DNA concentrations and conditions of salt and temperature which require only moderate precision of base pairing, aggregates pelletable by brief centrifugation account for 30 to 40% of the total DNA from peas, while calf thymus DNA reassociated under similar conditions forms less than 10% pelletable structures. In contrast to networks formed during the reassociation of long DNA fragments containing interspersed repetitive sequences, these aggregates contain a high percentage of double-stranded DNA and are enriched in repetitive sequences.     

Rapid phylogenetic and functional classification of short genomic fragments with signature peptides

ABSTRACT: BACKGROUND: Classification is difficult for shotgun metagenomics data from environments such as soils, where the diversity of sequences is high and where reference sequences from close relatives may not exist. Approaches based on sequence-similarity scores must deal with the confounding effects that inheritance and functional pressures exert on the relation between scores and phylogenetic distance, while approaches based on sequence alignment and tree-building are typically limited to a small fraction of gene families. We describe an approach based on finding one or more exact matches between a read and a precomputed set of peptide 10-mers. RESULTS: At even the largest phylogenetic distances, thousands of 10-mer peptide exact matches can be found between pairs of bacterial genomes. Genes that share one or more peptide 10-mers typically have high reciprocal BLAST scores. Among a set of 403 representative bacterial genomes, some 20 million 10-mer peptides were found to be shared. We assign each of these peptides as a signature of a particular node in a phylogenetic reference tree based on the RNA polymerase genes. We classify the phylogeny of a genomic fragment (e.g., read) at the most specific node on the reference tree that is consistent with the phylogeny of observed signature peptides it contains. Using both synthetic data from four newly-sequenced soil-bacterium genomes and ten real soil metagenomics data sets, we demonstrate a sensitivity and specificity comparable to that of the MEGAN metagenomics analysis package using BLASTX against the NR database. Phylogenetic and functional similarity metrics applied to real metagenomics data indicates a signal-to-noise ratio of approximately 400 for distinguishing among environments. Our method assigns ~6.6 Gbp/hr on a single CPU, compared with 25 kbp/hr for methods based on BLASTX against the NR database. CONCLUSIONS: Classification by exact matching against a precomputed list of signature peptides provides comparable results to existing techniques for reads longer than about 300 bp and does not degrade severely with shorter reads. Orders of magnitude faster than existing methods, the approach is suitable now for inclusion in analysis pipelines and appears to be extensible in several different directions.     

High-speed electrophoretic separation of DNA fragments using a short capillary

Capillary electrophoresis using a replaceable gel buffer was applied to the separation of DNA fragments. A short effective length capillary (1–2 cm) at low electric field allowed the separation of a 20–1000 bp ladder in 1 min. Although similar separation speed was achieved with a longer capillary at high field, the resolution of larger fragments was degraded. The short effective length capillaries were able to separate the wildtype and mutant PCR products of the TGF-β₁ gene in under 45 s.     

Affinity capture of specific DNA fragments with short synthetic sequences

The ability of short peptide nucleic acid (PNA) oligomers and oligonucleotides containing modified residues of 5-methylcitidine, 2-aminoadenosine, and 5-propynyl-2′-deoxyuridine (strong binding oligonucleotides, SBO) to affinity capture the target double-stranded DNA fragment from mixture by means of the end invasion was compared. Both types of probes were highly effective at the conditions used. The SBO-based probes may represent a handy and easily prepared alternative to PNA for selection of target DNA fragments in mixtures.     

Anisotropic overall and internal motions of short DNA fragments.

Anisotropic motions of DNA fragments in the size range 6-118 base pairs are studied by the steady-state fluorescence polarization of different excitation transitions in the intercalated ethidium cation. Calculated effective tumbling and twisting times are found to be shorter than predicted for overall motions of rigid DNA, indicating that internal motions and/or dye wobbling contribute to the depolarization. The data are consistent with a model where the DNA fragments are considered to be rigid against bending but torsionally flexible, and where the dye can wobble within the intercalated site. We also discuss the possibility of correlated out-of-plane motions of the dye and the DNA bases.     

Visualizing ion relaxation in the transport of short DNA fragments

Ion relaxation plays an important role in a wide range of phenomena involving the transport of charged biomolecules. Ion relaxation is responsible for reducing sedimentation and diffusion constants, reducing electrophoretic mobilities, increasing intrinsic viscosities, and, for biomolecules that lack a permanent electric dipole moment, provides a mechanism for orienting them in an external electric field. Recently, a numerical boundary element method was developed to solve the coupled Navier-Stokes, Poisson, and ion transport equations for a polyion modeled as a rigid body of arbitrary size, shape, and charge distribution. This method has subsequently been used to compute the electrophoretic mobilities and intrinsic viscosities of a number of model proteins and DNA fragments. The primary purpose of the present work is to examine the effect of ion relaxation on the ion density and fluid velocity fields around short DNA fragments (20 and 40 bp). Contour density as well as vector field diagrams of the various scalar and vector fields are presented and discussed at monovalent salt concentrations of 0.03 and 0.11 M. In addition, the net charge current fluxes in the vicinity of the DNA fragments at low and high salt concentrations are briefly examined and discussed.     

Accumulation of short DNA fragments in hydroxyurea treated mouse L-cells

Treatment of L-cells with hydroxyurea markedly inhibits the incorporation of [³H]thymidine into DNA. The ³H incorporation that persists during hydroxyurea inhibition is largely into 7S DNA chains. The labelled fragments can be chased into higher MW DNA, suggesting that they are intermediates in the replication process. This interpretation concurs with that of earlier reports which describe a similar effect of hydroxyurea on the replication of viral DNA.     

Modeling the free solution electrophoretic mobility of short DNA fragments

Boundary element methods are used to model the free solution electrophoretic mobility of short DNA fragments. The Stern surfaces of the DNA fragments are modeled as plated cylinders that reproduce translational and rotational diffusion constants. The solvent-accessible and ion-accessible surfaces are taken to be coincident with the Stern surface. The mobilities are computed by solving simultaneously the coupled Navier–Stokes, Poisson, and ion-transport equations. The equilibrium electrostatics are treated at the level of the full Poisson–Boltzmann equation and ion relaxation is included. For polyions as highly charged as short DNA fragments, ion relaxation is substantial. At .11 M KCl, the simulated mobilities of a 20 base pair DNA fragment are in excellent agreement with experiment. At .04 M Tris acetate, pH = 8.0, the simulated mobilities are about 10–15% higher than experimental values and this discrepancy is attributed to the relatively large size of the Tris counterion. The length dependence of the mobility at .11 M KCl is also investigated. Earlier mobility studies on lysozyme are reexamined in view of the present findings. In addition to electrophoretic mobilities, the effective polyion charge measured in steady state electrophoresis and its relationship to the preferential interaction parameter γgG is briefly considered. © 1998 John Wiley & Sons, Inc. Biopoly 46: 359–373, 1998     

Association of short DNA fragments: Steady state fluorescence polarization study

We have studied aggregation/association of monodisperse DNA fragments (ranging from 30–90 base pairs) by steady-state fluorescence polarization of intercalculated ethidium. The method of excitation at different wavelengths in the ethidium absorption spectrum provides information about anisotropic twisting and tumbling mobility of the fragments. We find that end-over-end tumbling rather than axial spinning and internal twisting motions are affected by aggregation/association. The critical concentration for observing the effects of intermolecular interactions is approximately 5 mg DNA/mL at room temperature, independent of fragment length. Association is favored by low temperature and high (> 10 mM) concentration of Mg²⁺. From temperature-and salt-dependence experiments we infer that the “aggregates” are similar to those observed in a recently discovered DNA sol–gel transition [M. G. Fried and V. A. Bloomfield (1984) Biopolymers 23 , 2141–2155]. We also discuss possible arrangements of the fragments within the aggregates and their possible relation to formation of DNA liquid crystals.     

95 Orientational ordering of short DNA fragments in the polymer matrix

As a vital part of modern nanotechnology, nanofabrication aims to develop nanoscale components and nanomaterials in large quantities at relatively low cost. The promising strategy is the bottom-up self-assembly techniques of chemical assembly and molecular recognition to bring together individual atoms, molecules, or supramolecular building blocks to form useful constructs. The DNA-DNA self-assembly seems to be the key point regulating the polymer composites formation. We address the mixture of a flexible polymer with short double-strand DNA fragments, where the persistence length is in comparable with the contour length of the molecule. We investigate the conditions affecting the orientational order formation of short double-strand DNA fragments, immersed in the flexible polymer. It is shown that short double-strand DNA fragments exhibit the formation of a liquid crystalline ordered phase, in dependence on the value of the Flory–Huggins parameter, aspect ratio , and the attraction energy (Mamasakhlisov et al., 2009; Todd et al., 2008) of the double strand DNA molecules and volume fraction of polymer.     

DNA gyrase can cleave short DNA fragments in the presence of quinolone drugs.

We have analysed the DNA cleavage reaction of DNA gyrase using oligonucleotides annealed to a single-stranded M13 derivative containing a preferred gyrase cleavage site. We find that gyrase can cleave duplexes down to approximately 20 bp in size in the presence of the quinolone drugs ciprofloxacin and oxolinic acid. Ciprofloxacin shows a variation in its site specificity with an apparent preference for G bases adjacent to the cleavage sites, whereas oxolinic acid stimulates cleavage predominantly at the previously determined site. With either drug, cleavage will not occur within 6 bases from the end of a DNA duplex or a nick. We suggest that cleavage site specificity with short DNA duplexes is determined by drug-DNA interactions whereas with longer fragments the positioning effect of the DNA wrap around gyrase prescribes the site of cleavage.     

A simple method to make better probes from short DNA fragments

A detailed method is presented tor the creation of head-to-tail multimers of short blunt restriction fragments, ligaled into a plasmid vector in a singletube: reaction. Random priming of the concatemer insert readily yields hybridization probes of high specificity, unattainable from the short monomer fragments.     

A self-probing primer PCR method for detection of very short DNA fragments

A novel self-probing primer method that based on the fluorescence resonance energy transfer principle is designed to detect DNA fragments of approximately 40 bp. Four self-probing primer reaction systems were developed to target a maize endogenous reference gene (HMG), a soybean endogenous reference gene (Lectin), a rapeseed endogenous reference gene (CruA) and an exogenous gene 5-enolpyruvylshikimate-3-phosphate synthase (ctp2-cp4epsps). These four primer systems were confirmed to have a high level of inter-species specificity and good intra-species stability. The limit of detection was estimated to be 10 copies of haploid genomes for all four assays. The validation results demonstrated that the self-probing primer methods are able to quantify the DNA amount in the different samples with good sensitivity and precision. When highly processed food products were assayed, the self-probing primer method produced better results than the TaqMan probe method. Overall, the self-probing primer method is suitable for qualitative and quantitative detection of very short DNA targets in samples of different sources.     

Functional correction of episomal mutations with short DNA fragments and RNA-DNA oligonucleotides

Background

Gene correction is an alternative approach to replacement gene therapy. By correcting mutations within the genome, some of the barriers to effective gene therapy are avoided. Homologous nucleic acid sequences can correct mutations by inducing recombination or mismatch repair. Recently, encouraging data have been presented using both short DNAfragments (SDFs) and RNA–DNA oligonucleotides (RDOs) in experimental strategies to realize clinical gene correction.

Methods

The delivery of labelled SDFs and RDOs to a variety of cell lines was tested using both FACS analysis and confocal microscopy. A GFP‐based reporter system was constructed, containing a nonsense mutation, to allow quantitation of gene correction in living cells. This reporter was used to compare efficiencies of functional gene correction using SDFs and RDOs in arange of mammalian cell lines.

Results

The delivery experiments highlight the inefficient delivery of SDFs and RDOs to the nucleus using polyethylenimine (PEI) transfection. This study compared the episomal correction efficiency of the reporter plasmid mediated by SDFs and RDOs within different cell types; low levels of functional correction were detected in cell culture.

Conclusions

Whilst delivery of PEI‐complexed SDFs or RDOs to the cell is highly effective, nuclear entry appears to be a limiting factor. SDFs elicited episomal GFP correction across a range of cell lines, whereas RDOs only corrected the reporter in a cell line that overexpresses RAD51. Copyright © 2002 John Wiley & Sons, Ltd.

     

In situ monitoring of DNA: the plant nuclear envelope allows passage of short DNA fragments

We monitored the cellular localization of fluorescently labeled foreign DNA in living plant cells. After physical delivery of labeled DNA fragments to the cytoplasm, short fragments up to 1.5 kb in length were found equally distributed between the cytoplasm and nucleus after 60 min. In contrast, 2.5 kb DNA fragments did not appear inside the nucleus. Thus, foreign DNA can enter plant nuclei through the intact nuclear envelope, but the efficiency of this process declines with increasing size of fragment.     

Analysis of 5'-ends of short DNA fragments excreted by phytohemagglutinin stimulated lymphocytes

Human peripheral blood lymphocytes were stimulated with phytohemagglutinin and the excreted DNA was isolated from the medium after four days of incubation of cells. The excreted DNA was labeled at the 5'-end with [gamma-32P]ATP and polynucleotide kinase. Analysis of the end-labeled material revealed a size distribution with a chain length of 6 - 60 nucleotides. These short DNA fragments did not contain ribo-nucleotides at their 5'-termini. P1 nuclease digestion did not release specific deoxyribonucleoside monophosphates from the 5'-end of the excreted DNA fragments. These results point to the non-specific degradation of DNA excreted by stimulated lymphocytes.     

Transient electric birefringence study of the length and stiffness of short DNA restriction fragments

Transient electric birefringence measurements of the rotational diffusion constant of five short restriction fragments of the plasmid pBR322 show that the hydrodynamic length is independent of sodium ion concentration in the range of 0.2 to 2.5 mM. The fragments are too stiff to be modeled as wormlike molecules. The rotational relaxation times of the fragments, which range from 64 to 124 base pairs, have been used to calculate the rise per base pair using six different theoretical expressions for the length dependence of the rotational diffusion coefficient of straight cylinder. The best estimate for the rise per base pair of Na-DNA in solution is 3.3 ± 0.1 Å.     

Enzymatic conversion of long DNA to small DNA fragments for the construction of short hairpin RNA expression libraries

Several techniques to enzymatically construct a short hairpin RNA (shRNA) expression library have been reported as tools for comprehensive genetic analyses by RNA interference. Our technique constructs an shRNA expression library from 25- to 35-bp DNA fragments by fragmenting given double-stranded DNA (dsDNA). We compared the following two procedures to efficiently prepare such small DNA fragments: one is the cleavage of dsDNA with deoxyribonuclease I (DNase I) in the presence of Mn²⁺ followed by blunting with T4 DNA polymerase, and the other is the introduction of nicks with DNase I in the presence of Mg²⁺ followed by blunting with the Klenow fragment. Consequently, the latter yielded the DNA fragments more efficiently. However, these DNA fragments were contaminated with fused DNA fragments that had originated from two regions of original dsDNA. Therefore, we used single-strand-specific exonucleases and succeeded in suppressing the production of such fused DNA fragments. Our technique allows the efficient conversion of given dsDNA to small DNA fragments.