Determination Of Calf Thymus DNA Research Articles

Development and Validation of A Spectrofluorimetric Determination of Calf Thymus DNA Using a Terbium-Danofloxacin Probe

Background: Analysis of biomolecules is required in many biomedical research areas. A spectrofluorimetric method is proposed for determination of calf thymus DNA (ctDNA) based on the fluorescence enhancement of terbium-danofloxacin (Tb3+-Dano) in the presence of ctDNA. Methods: A probe with maximum excitation and emission wavelengths of 347 nm and 545 nm, respectively, was developed. The enhanced fluorescence intensity of Tb3+-Dano system was proportional to the concentration of ctDNA. The effective factors and the optimum conditions for the determination of ctDNA were studied. Under the optimum conditions of [Tris buffer]= 0.01 mol L-1 (pH 7.8), [ Tb3+]= 1×10-5 mol L-1 and [Dano]= 5×10-5 mol L-1, the maximum response was achieved. The developed method was evaluated in terms of accuracy, precision and limit of detection. Results: The linear concentration range for quantification of ctDNA was 36-3289 ng mL-1 and the detection limit (S/N=3) was 8 ng mL-1. The concentration of DNA extracted from Escherichia coli as an extracted sample was also determined using the developed probe. The concentration of DNA in extracted sample was determined using UV assay and developed method, the results were satisfactory. Conclusion: The proposed method is a simple, practical and relatively interference free method to follow up the concentrations of ctDNA.

Resonance light scattering spectroscopy of procyanidin–CPB–DNA ternary system and its potential application

A new method for the determination of calf thymus DNA at nanogram level was proposed based on the enhanced resonance light scattering (RLS) signals of DNA in the presence of procyanidin and cetylpyridinium bromide dihydrate (CPB). Under the experimental conditions, the RLS intensity of DNA at 291.0nm was greatly enhanced by procyanidin–CPB at pH 7.0. There was a good linear relationship (r=0.9993) between the enhanced RLS intensity (ΔIRLS) and DNA concentration of 0.0084–3.36μgmL−1. The limit of detection (LOD) was 2.27ngmL−1 (3S0/S). Three synthetic DNA samples were measured with satisfactory, and the recovery was 102.3–107.2%.

Study on interactions of aminoglycoside antibiotics with calf thymus DNA and determination of calf thymus DNA via the resonance Rayleigh scattering technique.

A simple and sensitive resonance Rayleigh scattering (RRS) spectra method was developed for the determination of calf thymus DNA (ctDNA). The enhanced RRS signals were based on the interactions between ctDNA and aminoglycoside antibiotics (AGs) including kanamycin (KANA), tobramycin (TOB), gentamicin (GEN) and neomycin (NEO) in a weakly acidic medium (pH 3.3-5.7). Parameters influencing the method were investigated. Under optimum conditions, increments in the scattering intensity (∆I) were directly proportional to the concentration of ctDNA over certain ranges. The detection limit ranged from 12.2 to 16.9 ng/mL. Spectroscopic methods, including RRS spectra, absorption spectra and circular dichroism (CD) spectroscopy, coupled with thermo-denaturation experiments were used to study the interactions, indicating that the interaction between AGs with ctDNA was electrostatic binding mode.

Electrochemical determination of calf thymus DNA on Zr(IV) immobilized on gold–mercaptopropionic-acid self-assembled monolayer

An electrochemical biosensor, constructed by immobilization of Zr(IV) on the topside of gold–mercaptopropionic acid self-assembled monolayer (Au–MPA–Zr SAM), is developed for the sensitive quantification of calf thymus DNA (ct-DNA). The sensor is based on ionic adsorption of ct-DNA from its phosphate backbone onto the Au–MPA–Zr(IV) SAM electrode. Preparation, characterization, and application of the sensor for determination of ct-DNA are described by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and Osteryoung square wave voltammetry (OSWV) in the presence of an appropriate redox reaction probe. Parameters influencing the method have been tested. A linear range calibration curve from 1.0 × 10 − 4 to 5.0 × 10 − 7 g mL − 1 ct-DNA with a detection limit of 9.5 × 10 − 8 g mL − 1 and mean of relative standard deviations (R.S.D) of 2.5% for n = 4 at each point was observed in the best conditions by EIS. Regeneration of the surface was carried out successfully by 5 min sonication in 0.1 M KOH solution and then 1 min incubation in 1.0 × 10 − 3 M Zr(IV) with a good reproducibility, R.S.D = 1.5% for n = 4 as detected by EIS. The long-term storage stability of the electrode was also studied.

Fluorescence Interaction and Determination of Calf Thymus DNA with Two Ethidium Derivatives

In this paper, we reported the syntheses and investigation of the modes of binding to DNA of the two new ethidium derivatives containing benzoyl and phenylacetyl groups of both amines at 3-and 8- positions. The interactions between calf thymus DNA (ct-DNA) and the two derivatives, 3,8-dibenzoylamino-5-ethyl-6-phenylphenantridinium cloride (E2) and 3,8-diphenylacetylamino-5-ethyl-6-phenylphenantridinium chloride (E3), were investigated by fluorescence quenching spectra and UV-vis absorption spectra. The Stern-Volmer quenching constants, binding constants, binding sites and the corresponding thermodynamic parameters DeltaH, DeltaS and DeltaG were calculated at different temperatures. The results indicated the formation of E2 and E3-DNA complexes and van der Waals interactions as the predominant intermolecular forces in stabilizing for each complex. In addition, increasing nucleophilicity of the functional groups at 3- and 8- positions exhibited the respectable increment the DNA binding affinities of derivatives. The results of absorption, ionic strength and iodide ion quenching suggested that the interaction mode of E2 and E3 with ct-DNA was intercalative binding. The limit of detection (LOD) of ct-DNA were 7.49 x 10(-8) (n = 4) and 4.18 x 10(-8) mol/l (n = 7) in presence of E2 and E3, respectively.

Determination of calf thymus DNA using resonance light-scattering quenching method based on the terbium (Ш) (Tb 3+)/europium (Ш) (Eu 3+)–quercetin system

It is found that at pH 7.5 Tris-HCl buffer solution, calf thymus DNA (ctDNA) can quench the resonance light-scattering (RLS) of terbium (Ш) (Tb 3+)/europium (Ш) (Eu 3+)–quercetin system. Based on this, a sensitive method for the determination of ctDNA is proposed. The characteristics of RLS spectra, the effective factors and optimal experimental conditions were investigated. Under the optimal conditions, the quenched RLS intensity is in proportion to the concentration of ctDNA. For Tb 3+–quercetin–ctDNA system, the linear concentration ranges for determining ctDNA are 0.03–0.2 and 0.5–4 μg/ml, respectively, while for Eu 3+–quercetin–ctDNA system, the linear concentration range is 0.05–2.5 μg/ml.

Enhanced fluorescence of the Eu 3+–naphthalenediimide derivative–phenanthroline ternary complex and the determination of DNA

The naphthalenediimide derivative carrying a single bis(β-diketone)- o-terphenyl moiety (NDI–BHHCT) and its chelates formed with europium(III) were studied using absorption and fluorescence spectroscopy. The effect of solvent on the spectral properties and solubility of the system was investigated. Enhancement of the europium fluorescence as a function of 1,10-phenanthroline (phen) and yttrium(III) ions was examined in the presence and in the absence of DNA. On the basis of these studies, a system Eu 3+/Y 3+/NDI–BHHCT/phen was applied for the determination of calf thymus DNA. In the presence of DNA, the Eu 3+ and Y 3+ ternary complexes intercalate to DNA that results in their proper arrangement on the DNA helix, thus enabling energy transfer between the non-fluorescent Y 3+ and fluorescent Eu 3+ chelates. As the result, the enhancement of Eu emission due to the co-fluorescence effect is observed in the presence of DNA. The calibration graph was linear up to 1.3 μg ml −1 with a detection limit of 12 ng ml −1.

Chemiluminescence determination of ultramicro DNA with a flow-injection method

A high sensitive flow-injection chemiluminescence method for determination of calf thymus DNA and herring sperm DNA has been developed. The method is based on the chemiluminescence reaction of Rhodamine B–cerium(IV)-thermally denatured DNAs in sulfuric acid media. The proposed procedure allows quantitation of DNAs in the range 2.6×10 −5 to 0.26 μg ml −1 for calf thymus DNA and 5.0×10 −8 to 5.0×10 −5 μg ml −1 for herring sperm DNA with correlation coefficients 0.9998 and 0.9996 (both n=11), respectively. The detection limits (3 σ) are 6.5×10 −6 μg ml −1 for calf thymus DNA and 4.3×10 −8 μg ml −1 for herring sperm DNA. The possible mechanism of chemiluminescence in the system is discussed.

Determination of nucleic acids based on shifting the association equilibrium between tetrasulfonated aluminium phthalocyanine and acridine orange.

Based on the ability of nucleic acids to shift the association equilibrium of the ion-association complex of Acridine Orange and tetrasulfonated aluminium phthalocyanine, thus leading to an increase in the phthalocyanine fluorescence, a method is suggested for the fluorimetric determination of nucleic acids. Investigations were carried out on the spectral characteristics, order of addition of reagents, selection of the buffer system, effect of pH, influence of reaction time, effect of salt, the usage of reagents, interference of foreign substances and the effect of different acridine derivatives. Under the optimum conditions, the calibration graphs for the determination of calf thymus DNA (CT DNA), salmon DNA (SM DNA) and yeast RNA were linear over the ranges 0.04-1.2, 0.04-1.2 and 0.1-1.2 micrograms cm-1, respectively. The detection limits for CT DNA, SM DNA and RNA were 17, 24 and 98 micrograms cm-3, respectively. The relative standard deviation (n = 6) was within 4.6% for the detection of samples. The method was applied to the determination of Staphylococcus aureus DNA and the result was in agreement with that achieved by a UV method.

A direct chemiluminescence method for the determination of nucleic acids using Ru(phen) 3 2+ -Ce(IV) system

A direct chemiluminescence method for the determination of nucleic acids has been developed based on the enhancement of nucleic acids on the chemiluminescence light emission of the reaction between Ru(phen)3 2+(phen = 1,10-phenanthroline) and Ce(IV). Under the optimum conditions, the calibration graphs are linear in the range of 5.0 × 10–8–5.0 × 10–5 g/mL for calf thymus DNA, 8.0 × 10–8–5.0 × 10–5 g/mL for fish sperm DNA and 1.0 × 10–7–3.0 × 10–5 g/mL for yeast RNA, respectively. The limits of detection are 1.0 × 10–8 g/mL for calf thymus DNA, 3.1 × 10–8 g/mL for fish sperm DNA and 5.2 × 10–8 g/mL for yeast RNA, respectively. The final procedure allows the successful determination of calf thymus DNA, fish sperm DNA and yeast RNA in six synthetic samples. This method is simple, rapid and specific.

Fluorescence energy transfer between Acridine Orange and Safranine T and its application in the determination of DNA

The fluorescence energy transfer (FET) between Acridine Orange and Safranine T, two intercalators of DNA, was studied in this paper. The FET efficiency between Acridine Orange and Safranine T is higher and the critical distance, R 0, is longer in the intercalated state than in the free one. A new method for the determination of calf thymus DNA (ctDNA) was presented. The linear range of the calibration curve is (0∼1.1)×10 −5 mol l −1 in bases for ctDNA, and the limit of detection is 2.6×10 −7 mol l −1.

Interaction of a novel red-region fluorescent probe, Nile blue, with DNA and its application to nucleic acids assay.

A novel fluorimetric method was developed for the rapid determination of DNA and RNA based on their quenching effect on the cationic red-region fluorescent dye Nile Blue (NB). In the investigation of the interaction of NB with DNA by steady-state polarization measurements, thermal denaturing study, determination of absorption and fluorescence characteristics, salt effect study and electrophoresis experiments, the results supported the suggestion that NB served as an intercalator to the stack base pairs of nucleic acids. Further evidence showed that the quenching could be ascribed to the static quenching mode. A binding constant of about 10(6) M-1 and a binding site size of about three base pairs were obtained by spectral methods. Under optimum conditions, the calibration curves for the determination of calf thymus DNA (CT DNA) and yeast RNA were linear over the ranges 3.0 ng mL-1-2.0 micrograms mL-1 and 27 ng mL-1-10 micrograms mL-1, respectively. The detection limits were 3.0 ng mL-1 for CT DNA and 27 ng mL-1 for RNA. The relative standard deviation (n = 6) was within 2.1% in the middle of the linear range. Interferences from some interesting co-existing substances in the determination of DNA were also examined.

Development of a Direct Chemiluminescence Method for the Determination of Nucleic Acids Based upon Their Reaction with Cerium(IV) in the Presence of Rutheniumtrisdipyridine.

A direct chemiluminescent method for the determination of nucleic acids has been developed. The method is based on the enhancement of nucleic acids on the chemiluminescence light emission of the reaction between Ru(bipy)2+3 and Ce(IV). Under the optimum conditions, the calibration graphs are linear over the ranges 7.0×10–7–5.0×10–5 g/ml for calf thymus DNA, 4.0×10–7–5.0×10–5 g/ml for fish sperm DNA and 5.0×10–7–5.0×10–5 g/ml for yeast RNA, respectively. The limits of detection are 1.8×10–7 g/ml for calf thymus DNA, 1.1×10–7 g/ml for fish sperm DNA and 1.2×10–7 g/ml for yeast RNA, respectively. The final procedue allows the sucessful determination of calf thymus DNA, fish sperm DNA and yeast RNA in six synthetic samples. This method is simple, rapid and specific. A possible reaction mechanism is discussed.

Fluorescence Investigation of the Interaction of Safranine T with DNA

A fluorescence probe, safranine T (ST), was proposed for the investigation of DNA. ST is a fluorescent cationic dye. Its fluorescence intensity decreased significantly in the presence of DNA. Evidence showed that the fluorescence quenching was due to the formation of a complex between dye and DNA which is less fluorescent than the free dye. Steady-state anisotropy measurements demonstrated that ST bound to DNA and resulted in an increase in anisotropy. The calibration graphs for the determination of Calf thymus DNA (CT DNA) and fish sperm DNA (FS DNA) were obtained. Good linear dependency of F0/F on the concentration of DNA was observed with the concentration ranging from 8.3 ng /ml to 15.0 μg / ml for CT DNA and from 14.0 ng / ml to 16.0 μg / ml for FS DNA. The LOD were 8.3 ng /ml and 14.0 ng / ml for CT DNA and FS DNA, respectively. The relative standard deviations were 1.44 % and 0.92 % for 4.0 μg / ml and 0.40 μg / ml of CT DNA, respectively, and 1.52% and 1.12% for 4.0 μg / ml and 0.50 μg / ml of FS DNA, respectively.