DNA Thermostability Research Articles

Antibody free ELISA-like assay for the detection of transcription factors based on double-stranded DNA thermostability.

Transcription factors (TFs) play critical roles in gene expression regulation and disease development. In this paper, we report an antibody free ELISA-like assay which could be used to analyze transcription factor NF-κB p50 with comparatively low cost and high throughput. This assay is based on the stabilization of a duplex DNA probe by binding with a transcription factor. The double-stranded DNA (dsDNA) probe immobilized on a 96-well plate was too short in length to stabilize its duplex structure at a relatively high temperature and would unwind into a single strand. In the presence of a target TF, the protein bound to a specific TF-binding site and prevented the specific dsDNA probe from being unzipped at the washing temperature, thus holding the chemiluminescence signal. This method could sensitively detect NF-κB p50 with a detection limit of 0.5 nM. The proposed strategy provides a convenient, cheap and high throughput detection method of TFs, which can potentially help the development of drug discovery and disease diagnosis.

Genome Compositional Organization in Gars Shows More Similarities to Mammals than to Other Ray-Finned Fish.

Genomic GC content can vary locally, and GC-rich regions are usually associated with increased DNA thermostability in thermophilic prokaryotes and warm-blooded eukaryotes. Among vertebrates, fish and amphibians appeared to possess a distinctly less heterogeneous AT/GC organization in their genomes, whereas cytogenetically detectable GC heterogeneity has so far only been documented in mammals and birds. The subject of our study is the gar, an ancient "living fossil" of a basal ray-finned fish lineage, known from the Cretaceous period. We carried out cytogenomic analysis in two gar genera (Atractosteus and Lepisosteus) uncovering a GC chromosomal pattern uncharacteristic for fish. Bioinformatic analysis of the spotted gar (Lepisosteus oculatus) confirmed a GC compartmentalization on GC profiles of linkage groups. This indicates a rather mammalian mode of compositional organization on gar chromosomes. Gars are thus the only analyzed extant ray-finned fishes with a GC compartmentalized genome. Since gars are cold-blooded anamniotes, our results contradict the generally accepted hypothesis that the phylogenomic onset of GC compartmentalization occurred near the origin of amniotes. Ecophysiological findings of other authors indicate a metabolic similarity of gars with mammals. We hypothesize that gars might have undergone convergent evolution with the tetrapod lineages leading to mammals on both metabolic and genomic levels. Their metabolic adaptations might have left footprints in their compositional genome evolution, as proposed by the metabolic rate hypothesis. The genome organization described here in gars sheds new light on the compositional genome evolution in vertebrates generally and contributes to better understanding of the complexities of the mechanisms involved in this process.

Differential Effect of Three Base Modifications on DNA Thermostability Revealed by High Resolution Melting

High resolution melting (HRM) can detect and quantify the presence of 5-methylcytosine (5mC) in DNA samples, but the ability of HRM to diagnose other DNA modifications remains unexplored. The DNA bases N6-methyladenine and 5-hydroxymethylcytosine occur across almost all phyla. While their function remains controversial, their presence perturbs DNA structure. Such modifications could affect gene regulation, chromatin condensation and DNA packaging. Here, we reveal that DNA containing N6-methyladenine or 5-hydroxymethylcytosine exhibits reduced thermal stability compared to cytosine-methylated DNA. These thermostability changes are sufficiently divergent to allow detection and quantification by HRM analysis. Thus, we report that HRM distinguishes between sequence-identical DNA differing only in the modification type of one base. This approach is also able to distinguish between two DNA fragments carrying both N6-methyladenine and 5-methylcytosine but differing only in the distance separating the modified bases. This finding provides scope for the development of new methods to characterize DNA chemically and to allow for low cost screening of mutant populations of genes involved in base modification. More fundamentally, contrast between the thermostabilizing effects of 5mC on dsDNA compared with the destabilizing effects of N6-methyladenine (m6A) and 5-hydroxymethylcytosine (5hmC) raises the intriguing possibility of an antagonistic relationship between modification types with functional significance.

INFLUENCE OF LOW INTENSITY COHERENT ELECTROMAGNETIC MILLIMETER RADIATION (EMR) ON AQUA SOLUTION OF DNA

The thermostability and density of water-salt solutions of DNA, irradiated by non thermal coherent millimeter electromagnetic waves with frequency 64.5 GHz have been investigated using the methods of spectrophotometry and densitometry. It is shown that the thermostability of DNA and density of its solutions are increased, depending on time of irradiation. It is expected that under the influence of millimeter electromagnetic radiation the hydration of DNA and ions of Na+ that are present in solution decrease. As a result, the physicochemical characteristics of DNA are changed.

Interaction of fragmented double-stranded DNA with carbon nanotubes in aqueous solution

Aqueous suspensions of ultrasonically fragmented double-stranded (fds-) DNA and single-walled carbon nanotubes (SWNTs) have been investigated by UV- and IR-absorption, NIR-emission and Raman spectroscopy. According to gel-electrophoresis, the lengths of the polymer fragments were 100–500 base pairs. Analysis of IR and UV data indicates the presence of both double-stranded (ds) and single-stranded (ss)-regions in the fragments. SWNT complex with DNA was revealed by NIR-emission and Raman spectroscopy. It turned out that fds-DNA is less efficient in holding nanotubes in the aqueous solution than ss-DNA. From the UV-data, the character of the helix-coil transition is seen to be like that for fds-DNA off and on nanotube, however, DNA thermostability increased in this latter case. The effective charge density on the DNA sugar-phosphate backbone of the fds-DNA:SWNT hybrid was less than that of DNA alone. Spectroscopic data can be explained by a model in which the formation of hybrids starts due to the interaction between untwisted ss-regions of DNA and the nanotube: the strands wrap on the tube and thus create an ‘anchor’ for the whole polymer. The ds-part of the polymer is located close to the nanotube.

Thermostability of DNA from wheat in heated food products

To assess the stability of DNA from heated food products, the PCR of the 18S rDNA gene was utilized. The results show as follows; 1. DNA extracted from heated wheat flour was available for PCR amplification when heat temperature was less than 130°C. 2. DNA was directly decomposed by heat over 130°C. 3. DNA in food products was stable for heat compare with DNA or DNA of wheat flour. As a result, the thermal history of the center of the food product is important for DNA thermostability.

DNA helix: the importance of being GC-rich.

A new explanation for the emergence of heavy (GC-rich) isochores is proposed, based on the study of thermostability, bendability, ability to B-Z transition and curvature of the DNA helix. The absolute values of thermostability, bendability and ability to B-Z transition correlated positively with GC content, whereas curvature correlated negatively. The relative values of these parameters were determined as compared to randomized sequences. In genes and intergenic spacers of warm-blooded animals, both the relative bendability and ability to B-Z transition increased with elevation of GC content, whereas the relative thermostability and curvature decreased. The usage of synonymous codons in GC-rich genes was also found to augment bendability and ability to B-Z transition and to reduce thermostability of DNA (as compared to synonymous codons with the same GC content). The analysis of transposable elements (Alu and B2 repeats in the human and mouse) showed that the level of their divergence from the consensus sequence positively correlated with relative bendability and ability to B-Z transition and negatively with relative thermostability. The bendability and ability to B-Z transition are known to relate to open chromatin and active transcription, whereas curvature facilitates chromatin condensation. Because heavy isochores are known to be gene-rich and show a high level of transcription, it is suggested here that isochores arose not as an adaptation to elevated temperature but because of a certain grade of general organization and correspondingly advanced level of genomic organization, reflected in genome structuring, with physical properties of DNA in the gene-rich regions being optimized for active transcription and in the gene-poor regions for chromatin condensation ('transcription/grade' concept).

Effects of radical scavengers on radiation-induced DNA double strand breaks

Purpose: To investigate possible effects of Tris and phenol on the dynamic properties of gamma-irradiated DNA molecules in addition to their well known scavenging capacity. Materials and methods: Native and fragmented calf thymus DNA molecules were exposed to various doses of 60 Co gamma-rays at ~4.5Gy/min. Using thermal transition spectrophotometry, pulsed field gel electrophoresis and standard agarose gel electrophoresis, the effects of Tris, phenol and NaCl on the double helix to single coil thermal transition temperature, Tm, and the yield of the double-strand breaks (G dsb) of the irradiated DNA molecules have been studied. Results: DNA molecules exposed to gamma-rays showed a decreased T m and a corresponding increase of the G dsb yield. Tris, as well as phenol, exhibited a strong protection against preventing these radiation-induced alterations. In addition, both substances strongly affected the thermal stability of the non-irradiated DNA samples. These results, compared with data obtained by NaCl and its effects on DNA thermostability and G dsb, revealed that in the presence of both scavengers the observed dsb decrease was correlated to an increased molecular stability of DNA. Conclusions: This work suggests that the total protective effect of Tris and phenol against radiation-induced dsb is mainly attributed to their well-known radical scavenging properties, while relatively minor protective effects arise from their contribution to an increased molecular stability of DNA.

The inhibition of nuclear DNA polymerizing activity by captan

Abstract A study was conducted concerning the inhibition of calf thymus nuclear DNA synthesis by captan. Captan was shown to be toxic to the in vitro incorporation of [ 3 H]dTTP into calf thymus DNA, with an ID 50 value of 0.16 m M being measured. This inhibition was determined to be independent of Mg 2+ concentration. Although intact nuclear activities were affected, the soluble DNA polymerizing activity isolated from calf thymus nuclei exhibited no inhibition when exposed to captan. Treatment of purified calf thymus DNA with 10 −5 and 10 −4 M captan caused an elevation of the T m by 2 and 6°C, respectively. The inhibitory characteristic of captan on DNA polymerizing activities and the influence of this compound on the thermostability of DNA indicate a mechanism of inhibition which is located in the nucleus and is possibly related to the template function of DNA and/or with the nuclear DNA polymerizing enzymes.

Increased DNA thermostability produced by substances in mammalian cells and plasma

Abstract Some mammalian DNA prepared in this laboratory became hyperchromic at temperatures much above that expected from base ratios. Thermal denaturation ( Marmur and Doty method) of these pure, polymerized, undenaturated DNA preparations resulted in curves showing increased thermostability. Indirect evidence suggested that these unusual DNA's had been altered during preparation by some tissue substance. Addition of the saline-soluble supernatant of cell homogenates to standard DNA markedly shifted the thermal denaturation curves to higher temperatures. A standardized system to assay this tissue activity on calf-thymus DNA was developed. The active cell substance was non-dialyzable, heat-labile and removed by deproteinization. A mouse-liver fraction containing activity was separated which, in minute amounts, altered DNA quantitatively. A similar activity increasing DNA thermostability was demonstrated in rabbit plasma. Analyses of the altered, thermostable DNA showed no unusual bases or base ratios and no change in viscosity or reactivity with DNAase. The data indicate substances in mammalian cells and plasma which alter interstrand linkages of intact DNA.