Denatured Calf Thymus DNA Research Articles

A glassy carbon electrode modified with electrochemically reduced graphene for simultaneous determination of guanine and adenine

In this paper, the direct electrochemical oxidation and differential pulse voltammetry (DPV) determination of guanine and adenine are studied by using an electrochemically reduced graphene oxide (er-GO) modified glassy carbon electrode (er-GO/GCE). Compared to the GCE and the GO/GCE, the as-prepared er-GO/GCE shows excellent electrocatalytic activity towards guanine and adenine, with an increase of oxidation current along with a negative shift of oxidation potential. Using the as-prepared er-GO/GCE, the simultaneous determination of guanine and adenine is successfully carried out, and the detection limits (based on S/N of 3) for guanine and adenine are 1.5 × 10−7 mol L−1 and 2.0 × 10−7 mol L−1, respectively. In addition, this er-GO/GCE exhibits a high selectivity, reproducibility and stability, and also can be used for the detection of guanine and adenine in the thermally denatured calf thymus DNA and urine, showing its promising application in the analysis of real samples.

Voltammetric sensing of guanine and adenine using a glassy carbon electrode modified with a tetraoxocalix[2]arene[2]triazine Langmuir-Blodgett film

We have prepared a new voltammetric sensor for guanine and adenine. It is based on a glassy carbon electrode modified with a Langmuir-Blodgett film made from tetraoxocalix[2]arene[2]triazine. The direct electro-oxidation of adenine and guanine was investigated and the results indicat that in contrast to a bare glassy carbon electrode both guanine and adenine cause an increase in the oxidation peak currents along with a negative shift of the oxidation potentials. The electrode enables the simultaneous determination of guanine and adenine using square wave voltammetry. Analysis of acid denatured calf thymus DNA was carried out and the value of (G + C)/(A + T) was correctly found to be 0.75.

Myth and reality: practical test system for the measurement of anti‐DNA antibodies in the diagnosis of systemic lupus erythematosus (SLE)

The myth persists that only the labor intensive Farr radioimmunoassay and Crithidia luciliae immunofluorescence (CL-IFA) are systemic lupus erythematosus (SLE)-specific tests. We compared them to ELISA with bacteriophage lambda DNA (EL-dsDNA) and denatured calf thymus DNA (EL-ssDNA). By percentile ranking, the specificity cut-off level was set both out of clinical context (SOCC) on 100 blood bank donors, and in clinical context (SICC) on 100 patients with either rheumatoid arthritis or scleroderma (50/50). Clinical sensitivity was calculated on 100 random SLE patients. At 95% SICC, the sensitivity of Farr, CL-IFA, EL-dsDNA, and EL-ssDNA was similar (95%CI): 76% (66-84), 76% (66-84), 63% (53-72), and 75% (65-83), respectively; 87% of the patients were positive by at least one method and 55%by all methods. At 99% SICC, the sensitivity was also similar (95% CI): 57% (47-67), 47% (37-57), 58% (47-67), and 43% (33-53), respectively. The areas under ROC curve were similar (95% CI) when patients were used as controls for specificity. At 99% SOCC, EL-ssDNA identified 89% positive, 2 negative but positive by another method at 95% SICC, and 9 negative (i.e. 89/2/9), followed by CL-IFA (80/6/14), Farr (76/12/12), and EL-dsDNA (64/23/13). Thus, at relatively low cost and easy automation, under the same conditions of specificity, the two ELISA tests combined were at least as good, if not superior, to CL-IFA or Farr: they showed similar clinical sensitivity and also identified more patients with anti-DNA antibodies.

ESI-MS and thermal melting studies of nanoscale platinum(ii) metallomacrocycles with DNA

The hydrophilic, long-chain diamine PEGda (O,O'-bis(2-aminoethyl)octadeca(ethylene glycol)), when complexed with cis-protected Pt(II) ions afforded water-soluble complexes of the type [Pt(N,N)(PEGda)](NO(3))(2) (N,N = N,N,N',N'-tetramethyl-1,2-diaminoethane (tmeda), 1,2-diaminoethane (en), and 2,2'-bipyridine (2,2'-bipy)) featuring unusual 62-membered chelate rings. Equimolar mixtures containing either the 16-mer duplex DNA D2 or the single-stranded D2a and [Pt(N,N)(PEGda)](2+) were analyzed by negative-ion ESI-MS. Analysis of D2-Pt(II) mixtures showed the formation of 1 : 1 adducts of [Pt(en)(PEGda)](2+), [Pt(tmeda)(PEGda)](2+) and the previously-described metallomacrocycle [Pt(2)(2,2'-bipy)(2){4,4'-bipy(CH(2))(4)4,4'-bipy}(2)](8+) with D2; the dinuclear species bound to D2 most strongly, consistent with its greater charge and aromatic surface area. D2 formed 1 : 2 complexes with the acyclic species [Pt(2,2'-bipy)(Mebipy)(2)](4+) and [Pt(2,2'-bipy)(NH(3))(2)](2+). Analyses of D2a-Pt(II) mixtures gave results similar to those obtained with D2, although fragmentation was more pronounced, indicating that the nucleobases in D2a play more significant roles in mediating the decomposition of complexes than those in D2, in which they are paired in a complementary manner. Investigations were also conducted into the effects of selected platinum(II) complexes on the thermal denaturation of calf thymus DNA (CT-DNA) in buffered solution. Both [Pt(2)(2,2'-bipy)(2){4,4'-bipy(CH(2))(6)4,4'-bipy}(2)](8+) and [Pt(2,2'-bipy)(Mebipy)(2)](4+) stabilized CT-DNA. In contrast, [Pt(tmeda)(PEGda)](2+) and [Pt(en)(PEGda)](2+) (as well as free PEGda) caused negligible changes in melting temperature (ΔT(m)), suggesting that these species interact weakly with CT-DNA.

Direct electrochemistry of thermally denatured calf thymus DNA on a poly(methyl methacrylate)–graphite microcomposite electrode

Remarkable direct electrochemical behaviors of thermally denatured calf thymus DNA (ssDNA) on the poly(methyl methacrylate)-graphite powder microcomposite electrode (PMMA/GME) were observed and investigated. The result indicated that the PMMA/GME showed great promotion of the electrochemical response towards the ssDNA oxidation due to the specific characteristics of the nanostructured interface on the resultant composite electrode. Two irreversible oxidation peaks at +1.04 and +0.76 V with obvious negative movement of oxidation potential and increase of the oxidation current (vs. Ag/AgCl) were observed on the PMMA/GME, which corresponded to the oxidation of adenine and guanine residues in the ssDNA molecules. Therefore, detection of ssDNA was then performed at the PMMA/GME. Under the optimal conditions, a good linear relationship was obtained between the oxidation peak currents of guanine or adenine residues and the ssDNA concentration in the range of 5.9 × 10(-3) to ca. 1.1 mg mL(-1) with a detection limit of 1.0 μg mL(-1). This PMMA/GME exhibits some outstanding advantages, such as ease of fabrication, high stability, renewable surface, excellent stability, mechanical rigidity and high electrochemical reactivity, which holds huge promise for further DNA biosensor design.

DNA Denaturation Under Freezing in Alkaline Medium

It is generally accepted that DNA conserves its secondary structure after a freeze-thaw cycle. A negligible amount of degradation occurs after this procedure. Degradation becomes appreciable only after multiple cycles of freezing and thawing. In this study, we have found that a single freeze-thaw cycle in alkaline medium (pH ≥ 10.8) gives rise to denaturation of calf thymus DNA, although the melting temperature of intact DNA in the solution used for the freeze-thaw experiments is higher than 60 °C. The degree of denaturation is almost independent of the regime of freezing. The melting curve obtained after DNA is frozen at −2 °C and then thawed is almost the same as after a freezing carried out in liquid nitrogen (-196 °C). However, incubation in the same solution at 0 °C for 24 hours without freezing does not give rise to any denaturation. The degree of denaturation caused by freezing increases with pH (if pH ≥ 10.8) and decreases with Na2CO3 concentration at fixed pH and [Na+], although Na2CO3 decreases the melting temperature of intact DNA. A preliminary treatment of DNA with cisplatin or transplatin (0.01 Pt atoms per nucleotide) gives rise to a full recovery of the DNA secondary structure after freezing and thawing similar to what occurs after heating DNA to 100 °C and quick cooling. Possible mechanisms that may cause DNA denaturation during a freeze-thaw cycle in alkaline medium are discussed.

Synthesis, characterization, and biological activity studies of copper(II) mixed compound with histamine and nalidixic acid.

A mixed copper complex with deprotonated nalidixic acid (nal) and histamine (hsm) was synthesized and characterized by FTIR, UV-Vis, elemental analysis, and conductivity. The crystal structure of [Cu(hsm)(nal)H2O]Cl·3H2O (chn) showed a pentacoordinated cooper(II) in a square pyramidal geometry surrounded by two N atoms from hsm, two O atoms from the quinolone, and one apical water oxygen. Alteration of bacterial DNA structure and/or associated functions in vivo by [Cu(hsm)(nal)H2O]Cl·3H2O was demonstrated by the induction of a recA-lacZ fusion integrated at the amyE locus of a recombinant Bacillus subtilis strain. Results from circular dichroism and denaturation of calf thymus DNA (CT-DNA) suggested that increased amounts of copper complex were able to stabilize the double helix of DNA in vitro mainly by formation of hydrogen bonds between chn and the sugars of DNA minor groove. In vivo and in vitro biological activities of the chn complex were compared with the chemical nuclease [Cu(phen)(nal)H2O]NO3 · 3H2O (cpn) where phen is phenanthroline.

Stability of DNA upon interaction with dimethyltin dichloride

Chemical and thermal denaturation of calf thymus DNA (as a multidomain macromolecule) have been investigated in the presence of high concentrations of dimethyltin dichloride (Me2SnCl2) over the temperature range (55–95°C) in 50.0 mM phosphate buffer at pH 7.6 using temperature scanning spectroscopy and calorimetry methods. Results showed that over the concentration range of 6–16 mM, Me2SnCl2 is a chemical denaturant and denatures the double-strand DNA in a three-state manner. The denaturation data are analyzed based on the effective Gibbs free energy (ΔG°eff) approach and the chemical denaturation parameters including ΔG°eff, m value and equilibrium unfolding constant (K U) were obtained. Ultraviolet (UV) melting curves of the DNA at 260 nm as well as the calorimetric measurements were used to estimate the binding constants (K), melting enthalpy (ΔH°m) and binding enthalpy (ΔH°b). Furthermore, at low concentrations (up to 5 mM), Me2SnCl2 binds to the phosphate groups of DNA in an exothermic step and had no significant effect on double-strand DNA stability, confirmed by the fact that the T m value did not change. However, high (denaturing) concentrations of Me2SnCl2 (more than 9 mM) caused considerable destabilization of DNA associated with the formation of a partially unfolded intermediate at 13.6 mM of Me2SnCl2. The formed intermediate showed a lower thermal transition temperature (T m) by a magnitude of 10°C in relation to the native DNA. Finally, a new correlation is introduced for interpretation of thermal denaturation behavior of calf thymus DNA over the whole range of ligand (Me2SnCl2) concentration (0–16 mM).

Creation of Mammalian Single- and Double-Stranded DNA Surfaces: A Real-Time QCM-D Study

The quartz crystal microbalance with dissipation monitoring (QCM-D) is an excellent method for studying the creation of DNA-based surfaces and films. Previous studies have used QCM-D to focus on the construction of DNA surfaces composed of short synthetic DNA oligomers or plasmid DNA. Here, we have used QCM-D to monitor the creation of genomic single- and double-stranded calf thymus DNA surfaces on a polycation adsorbed to a SiO2 support. We have successfully monitored the hybridization between the ssDNA surfaces and their complementary strands in solution and have also shown that DNA multilayer formation can be observed using denatured calf thymus DNA. We have furthermore established that the ssDNA and dsDNA surfaces show different binding characteristics to ethidium bromide, a common dsDNA intercalator, demonstrating the potential use of such surfaces to identify possible DNA ligands.

Preparation of silica encapsulated quantum dot encoded beads for multiplex assay and its properties

Novel –COOH modified polystyrene beads were prepared by sulfonation grafting, and the surface area and pore volume are greatly improved in comparison with the swelling-treated beads. The optimization coating time is 4 h, and the corresponding –COOH content is approximately 2.1 mmol/g. The scanning electron microscope results show that the silica particles deposited on the beads and formed a silica shell that decreases the leakage of quantum dots (QDs) preferably and improves the bar code stability greatly. The anti-photobleaching of silica-coated beads was studied systemically, and the results show that the half-decay time ( t 1/2) of the coated beads increases to 537 s—seven times longer than that of the uncoated ones. Further DNA probe hybridization experiments indicated that the coding signal and target signal can be detected simultaneously and that the assays based on these probe-conjugated silica/QD/polystyrene beads have good specificity and sensitivity that can detect a concentration as low as 0.01 μg/ml target DNA in denatured calf thymus DNA solution, indicating that it is feasible to use this kind of bead for multiplex analysis.

Determination of DNA with Imidacloprid by a Resonance Light Scattering Technique at Nanogram Levels and Its Application

In this paper, we have set up a determination method of nucleic acids at nanogram levels by using a common spectrofluorometer to detect intensity of resonance light scattering (RLS). We have also studied the characteristics of RLS spectra of Imidacloprid with nucleic acids, the effective factors, and the optimum conditions. The RLS of imidacloprid was greatly enhanced by DNA in the range of pH 1.5–1.7. A RLS peak at 310 nm was found, and the enhanced intensity of RLS at this wavelength was proportional to the concentration of DNA. The linear range of the calibration curve was 0–5.0 µg mL−1, 5.0–20.0 µg mL−1 for calf thymus DNA, 0–1.0 µg mL−1 for thermally denatured calf thymus DNA, 0–14.6 µg mL−1 for salmon sperm DNA (ssDNA), and 4.0–7.0 µg mL−1 for yeast RNA (yRNA). The limits of detection (LOD, 3σ) were 12.8, 8.7, 29.5, and 35.5 ng mL−1, respectively. The nucleic acids in six synthetic samples and in three real samples were determined. The results were satisfactory, and the recovery rates were in the range of 99–106%, 91–109%, respectively. Compared with other methods, this method has larger detective range, which can reduce the interference of background and increase the intensity of RLS.

Square wave and elimination voltammetric analysis of azidothymidine in the presence of oligonucleotides and chromosomal DNA

Azidothymidine (AZT, 3′-azido-3′-deoxythymidine, Zidovudine, Retrovir) is an approved and widely used antiretroviral drug for the treatment of human immunodeficiency virus (HIV) infection. Dynamic electrochemical methods have been employed for the fast and inexpensive determination of this drug in natural samples. The electrochemical signal of AZT, resulting from the reduction of azido group, was studied by square wave voltammetry (SWV), linear sweep voltammetry (LSV) and elimination voltammetry with linear scan (EVLS) using a hanging mercury drop electrode (HMDE). This paper explores the possibility of determining AZT in the presence of native (dsDNA) or denatured calf thymus DNA (ssDNA), and/or some synthetic oligodeoxynucleotides (ODNs). The detection limit of AZT in the absence and in the presence of ssDNA (10 μg/ml) is 1 and 250 nM, respectively. It was found that the signal of AZT is not substantially affected by the presence of DNA. We can therefore assume that the electrons are transferred through the adsorption layer of nucleic acids. By using the elimination procedure, both irreversible reduction signals of AZT and DNA are augmented. Moreover, the elimination signal in the peak–counterpeak form may indicate the adsorption of the analytes on the electrode surface preceding an electron transfer.

Electrochemical behavior of DNA at a silver electrode studied by cyclic and elimination voltammetry

Electrochemical characteristics of native and denatured calf thymus DNA have been studied by voltammetry on a silver electrode (AgE). Experimental results obtained from linear sweep or cyclic voltammetry (LSV or CV) have been employed in elimination voltammetry. The elimination voltammetry with linear scan (EVLS), using the linear combination of the total currents measured at different scan rates, enables one or two selected particular currents to be eliminated. The best results have been obtained by using a function eliminating the kinetic and charging currents ( I k, I c), and conserving the diffusion current ( I d). This function makes it possible to increase significantly voltammetric signals of native and denatured DNAs, and to reveal processes not detectable by conventional electrochemical methods. The influence of electrochemical pretreatment of silver electrode surfaces and of starting and switching potentials on DNA voltammetric signals have been discussed. Silver electrodes coupled with elimination voltammetry represent promising tools for developing new nucleic acids biosensors.

In-situ FTIR study on adsorption and oxidation of native and thermally denatured calf thymus DNA at glassy carbon electrodes

In-situ Fourier transform infra-red (FTIR) spectra of native and thermally denatured calf thymus DNA (CT DNA) adsorbed and/or oxidized at a glassy carbon (GC) electrode surface are reported. The adsorption of native DNA occurs throughout the potential range (−0.2∼1.3 V) studied, and the adsorbing state of DNA at electrode surface is changed from through the CO band of bases and pyrimidine rings to through the CO of cytosine and imidazole rings while the potential shifts negatively from 1.3 V to −0.2 V. An in-situ FTIR spectrum of native CT DNA adsorbed at GC electrode surface is similar to that of the dissolved DNA, indicating that the structure of CT DNA is not distorted while it is adsorbed at the GC electrode surface. In the potential range of −0.2∼1.30 V, the temperature-denatured CT DNA is adsorbed at the electrode surface first, then undergoes electrochemical oxidation reaction and following that, diffuses away from the electrode surface.

Quantitative reactions of anti 5,9-dimethylchrysene dihydrodiol epoxide with DNA and deoxyribonucleotides

Native as well as denatured calf thymus DNA, deoxyguanylic and deoxyadenylic acid, respectively, were reacted with the racemic anti 5,9-dimethylchrysene dihydrodiol epoxide (5,9-DMCDE). The deoxyribonucleoside adducts were separated by HPLC and characterized by CD and NMR. Approximately 17% of the epoxide was trapped by native DNA and 76% of the adducts were derived from the RSSR enantiomer. The ratios of dAdo/dGuo modification in DNA were 14/86 and 19/81 for RSSR and SRRS enantiomers, respectively. By monitoring the product yields of anti 5,9-DMCDE with DNA and deoxyribonucleotides, we hoped to gain further insight into the factors responsible for deoxyguanosine adduct formation by 5-methylchrysene dihydrodiol epoxide (5-MCDE) compared to 5,6-dimethylchrysene dihydrodiol epoxide (5,6-DMCDE). The adduct yields in deoxyribonucleotide reactions of 5,9-DMCDE were slightly higher than those from 5-MCDE. However, the reaction yields of 5,9-DMCDE with DNA were lower than those with 5-MCDE in most cases, particularly for the cis and trans deoxyadenosine adducts. It seems that the 9-methyl group of 5,9-DMCDE significantly influences adduct formation with the deoxyadenosine residue in DNA in contrast to the 6-methyl group of 5,6-DMCDE. The 9-methyl group sterically decreases deoxyadenosine adduct yields more in reaction with native DNA than denatured DNA, but it has little effect on deoxyribonucleotide reactions. Adduct formation with deoxyguanosine residues in DNA by all three dihydrodiol epoxides correlate with their respective tumorigenic and mutagenic activities.

Ornithine Decarboxylase of Stagonospora (Septoria) nodorum Is Required for Virulence toward Wheat

A knockout strain of Stagonospora (Septoria) nodorum lacking the single ornithine decarboxylase (ODC) allele has been created by targeted gene replacement. A central region of the S. nodorum ODC gene was isolated by polymerase chain reaction using degenerate oligonucleotides and used to probe a lambda genomic library. The gene was sequenced and the encoded ODC protein sequence was shown to be similar to those from other fungi. The functionality of the S. nodorum ODC was confirmed by complementation of an Aspergillus nidulans mutant (puA) strain devoid of ODC activity, restoring growth in the absence of exogenous polyamines. Sporulation of the transformants was reduced suggesting abberant regulation of the S. nodorum gene in A. nidulans. Transformation-mediated gene replacement was used to create strains which were auxotrophic for putrescine and lack ODC coding sequences. Pathogenicity studies on these mutants showed that they are greatly reduced in virulence compared with non-disrupted transformants. This confirms that the strains carrying an ODC disruption cannot obtain sufficient polyamines from the host plant for normal growth and, thus, that fungal ODC may be a suitable target for chemical intervention.

Studies on the Fluorescence Reaction between Nucleic Acid and the Complex of Cobalt(II) with 5-(3-Fluoro-4-chlorophenylazo)-8-sulfonamidoquinoline and Its Applications

Based on the enhancement of the fluorescence intensity of the complex of 5-(3-fluoro-4-chlorophenylazo)-8-sulfonamidoquinoline (FCPBSQ) with Co2+ by nucleic acid in the presence of Tween 80 and in the weakly basic medium, a fluorescence method for the determination of nucleic acid was proposed. The calibration graphs for the determination of denatured calf thymus DNA (ct DNA), fish sperm DNA (fs DNA), and yeast RNA (yt RNA) were obtained in concentration ranges 0.050–4.0, 0.10–3.0, and 0.050–3.0 μg/ml with limits of detection of 0.010, 0.030, and 0.020 μg/ml, respectively. The method has been satisfactorily used for the determination of DNA in wheat cell extraction and ct DNA, fs DNA, and yt RNA in synthetic samples. Investigations on the binding mode by the Scatchard plots method suggested that both intercalation and electrostatic binding modes existed in this system.

The effects of polyols on the thermal stability of calf thymus DNA

The effects on thermal denaturation of calf thymus DNA (ct-DNA) and its conformational changes induced by the presence in solution of different polyols, namely glycerol, i-erytritol, l(−) and d(+) arabitol, d-mannitol, d-sorbitol and myo-inositol, have been investigated by means of differential scanning calorimetry (DSC) and circular dichroism (CD). By increasing the concentration of these additives a decrease in both the denaturation enthalpy (Δ d H) and temperature of the maximum of the denaturation peak ( T max) of DNA is observed. The values of these thermodynamic parameters depend on both the nature and concentration of the solute. The overall destabilization of DNA molecule has been related to the different capability of polyhydric alcohols to interact with the polynucleotide solvation sites replacing water and to the modification of the electrostatic interactions between the polynucleotide and its surrounding atmosphere of counterions. The particular behaviour of l(−) arabitol, which showed a much greater destabilizing ability compared to the other polyols, was further investigated and attributed to a direct more effective interaction with the double helix of DNA. CD spectra showed only a slight alteration of DNA-B structure in the presence of all the molecules here studied, except for l(−) arabitol where the DNA molecule seems to undergo a meaningful conformational change. The salt concentration dependence of DNA thermal stability in the presence of l(−) arabitol indicates a conformational change of polynucleotide towards a more extended conformation.

Studies of an Alkaline Activated Glassy Carbon Electrode on the Determination of Purines and DNA

Performance of glassy carbon electrode on the determination of purines and DNA was found to be improved dramatically by activating the GCE with a simple but effective electrochemical pretreatment. Characteristics such as lowering of oxidation potential, enhancement of peak current and elimination of fouling effect were found for the activated GCE. By flow injection analysis, good reproducibility with relative standard deviations of 0.59 and 2.01% (n = 11) and rather low detection limits of 0.6 + 0.1 and 3.0 ± 0.4 nM can be obtained for the analysis of guanine and adenine. Solutions of denatured calf thymus DNA were analyzed by differential pulse voltammetry with the activated GCE. Good agreement between the obtained results and the known values confirms the feasibility of the activated GCE for DNA analysis.

Quercetin–La(III) complex for the fluorimetric determination of nucleic acids

The ternary fluorescent complexes of nucleic acids–quercetin–lanthanum(III) were studied. Nucleic acids in this study involve natural and thermally denatured calf thymus DNA (ctDNA), yeast DNA (yDNA) and yeast RNA (yRNA). In the pH range 7.8–8.3 (controlled by NH 3–NH 4Cl buffer) ternary fluorescent complexes are formed which give maximum emission at 470 nm when excited at 280 nm. Based on the fluorescence reactions sensitive fluorimetric methods for nucleic acids were proposed. Using optimal conditions, the calibration curves were linear in the range of 0.5–3.0 μg ml −1 for ctDNA, 0.5–4.0 μg ml −1 for yDNA and yRNA. The limits of determination (3σ) were 0.072 μg ml −1 for ctDNA, 0.142 μg ml −1 for yDNA and 0.307 μg ml −1 for yRNA, respectively. Six synthetic samples were determined with satisfaction.