Size-exclusion chromatography of DNA restriction fragments

Ellegren, Hans; Låås, Torgny

doi:10.1016/s0021-9673(01)93966-4

Cited by 39 publications

(9 citation statements)

References 22 publications

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“…In exclusion chromatography, the shorter is the strand of a DNA molecule, the longer is the retention time of its components. 12,13 After electrolysis, some new peaks in the chromatogram appeared at long retention times, which revealed that the strand of partial DNA molecules was broken from longer to shorter strands. Undoubtedly, the components of the retention time at 16.26 and 17.94 min were much smaller DNA molecular fragments.…”

Section: Resultsmentioning

confidence: 99%

The Interaction of Copper-Bipyridyl Complex with DNA and Cleavage to DNA

2004

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Deoxyribonucleic acid (DNA) is the primary target molecule for most anticancer and antiviral therapies according to cell biology. Investigations of the interaction of DNA with small molecules are basic work in the design of new types of pharmaceutical molecules. Since the chemical nuclease activity of the copper-phenanthroline complex was discovered in the 1980's, 1-3 studying the interaction model and the mechanism of transition-metal complexes with DNA, and exploring the application of metal complexes in antineoplastic medication, molecular biology and bioengineering were hotspots in recent years. Especially interesting are metal complexes of the bipyridyl ligands. [4][5][6][7] When some kinds of metal complexes interacted with DNA, they could induce the breakage of DNA strands by appropriate methods. Thus, to cancer genes, after DNA strand are cleaved, the DNA double strands break. The replication ability of cancer gene is destroyed. Ueda et al. 8 investigated that the Cu(phen)2 2+ complex could cleave DNA in the presence of ascorbate or hydroquinone, and the Cu(en)2 2+ complex could also cleave DNA in the presence of ascorbate. It was suggested that the reductive capability of the reductants had a critical influence on DNA cleavage.Hettich et al. 9investigated the dinucleotide TpT reacted with 5 mM EuCl3 at 70˚C for up to 14 days, which allowed the reaction to be followed up to 25% completion. The electrochemical method can modulate the redox reaction under physiological conditions. DNA cleavage can be performed by oxygen reduction at an electrode in the presence of transition metals during electrolysis of an aerobic DNA solution. This method has been paid much attention because its system of DNA cleavage reaction is simple, and the cleavage efficiency is very high.Here, based on the interaction of the Cu(bpy)2 2+ complex with DNA, we used an electrochemical method to cleave DNA in the presence of the Cu(bpy)2 2+ complex. DNA was cleaved by electrochemically inducing the Cu(bpy)2 2+ complex.The cleaved DNA fragments were separated and detected by highperformance liquid chromatography. The experimental results revealed that the proposed method for DNA cleavage is highly efficient. ExperimentalApparatus and reagents CHI660A electrochemical analyzer (Shanghai Chenhua Apparatus Corporation, China) and Model 363 potentiostat/galvanostat (EG&G Princeton Applied Reseatch) were applied. Absorbance spectra were measured on a UV-265 spectrophotometer (Shimadzu, Japan), and the fluorescence spectra on a RF-540 spectrofluorimeter (Shimadzu, Japan). Gold disk electrodes (diameter 1 mm and 5 mm) were used as working electrodes. A platinum auxiliary electrode and an Ag/AgCl reference electrode were used for measurements. A L-7100 high-performance liquid chromatograph (Hitachi HighTechnologies Corporation, Tokyo Japan) equipped with a L-7420 UV-vis spectrophotometric detector was used. A ShimPack chromatographic column Diol-300 (7.9 mm Φ × 25 cm) packed with 5 µm porous silica micropheres (pore diameter 300 Å), which h...

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Section: Resultsmentioning

confidence: 99%

The Interaction of Copper-Bipyridyl Complex with DNA and Cleavage to DNA

2004

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“…For the Shim-pack Diol-300 column this was attributed to the size-exclusion chromatographic column, and its separation was based on the size of the molecules separated. In exclusion chromatography, the shorter strand of DNA is, the longer the retention time of components is [32,33]. For new peaks appearing at long retention time on the chromatogram, indicating the structure of DNA molecules was destroyed and the strand of partial DNA molecules was broken from longer to shorter.…”

Section: Resultsmentioning

confidence: 99%

Electrochemically Induced DNA Cleavage by Copper-Phenanthroline Complex

Yang

Chen

2002

Electroanalysis

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The interaction of copper‐1,10‐phenanthroline (phen) complex with calf thymus DNA has been investigated by cyclic voltammetry and differential pulse voltammetry at gold electrode. DNA can be efficiently cleaved by electrochemically reduced Cu(phen)$\rm{ {_{2}^{2+}}}$ complex at −0.5 V (vs. Ag/AgCl) in the presence of oxygen without addition of extra reductive agent. Oxygen is required for electrochemically induced DNA cleavage by Cu(phen)$\rm{ {_{2}^{2+}}}$ complex because it serve as a precursor formation of hydrogen peroxide. DNA cleavage is inhibited by bathocuproine and hydroxyl radical scavenges. These results reveal Cu(I) complex and hydroxyl radical are involved in DNA cleavage reaction. The percentage of DNA cleavage is enhanced with increasing electrolysis time and is also dependent on the ratio of Cu(phen)$\rm{ {_{2}^{2+}}}$ to DNA concentration. The cleaved DNA fragments are separated by high performance liquid chromatography (HPLC). The experimental results indicate that the method for electrochemically induced DNA cleavage by Cu(phen)$\rm{ {_{2}^{2+}}}$ is simple and efficient.

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“…Die Trennung von Nukleinsäuren kann mittels Hochdruckflüss-gkeitschromatographie (HPLC) in sechs unterschiedlichen Modii erfolgen: Größenaus-schluss-Chromatographie [1], Anionenaustausch-HPLC (AEHPLC) [2,3], HPLC im gemischten Modus [4], Ionenpaar-Umkehrphasen-Hochdruckflüssgkeitschromatographie (IP-RP-HPLC) [5] und Affinitäts-Chromatographie [6]. In den meisten Fällen wird Anionenaustausch-HPLC und Ionenpaar-Umkehrphasen-Hochdruckflüssigkeitschromatographie angewandt, die beide eine hochauflösende Trennung von nur einigen wenigen Nukleotiden bis hin zu tausenden Basenpaaren ermöglichen.…”

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