A Novel Method for Quantification of Circulating DNA in Serum by Capillary Zone Electrophoresis

In this paper, we first presented a novel method for quantification of circulating DNA in human serum based on capillary zone electrophoresis with laser-induced fluorescence detection (CZE-LIF). The serum was digested by proteinase to release free DNA, and then CZE-LIF system was used for the quantification of total circulating DNA. This method was successfully used to quantify the circulating DNA levels in sera from healthy individuals and certain cancer patients.We found the significantly elevated circulating DNA levels in certain prostate cancer patients. Our results demonstrated that CZE-LIF system has good linearity, excellent sensitivity (0.5 ng/mL DNA),satisfactory reproducibility (RSDs in one day and between days were both less than 5%) and reliability, and is well suitable to the quantification of the circulating DNA in human serum or plasma.     

Effect of glutaraldehyde on the activity of some DNA restriction endonucleases

The effect of the bifunctional crosslinking reagent glutaraldehyde on the activity of the restriction enzymes Bam HI,Hind III, EcoRI, and Tthlll I was investigated. The four enzymes exhibited differential sensitivity to inactivation. Tthlll I was the most sensitive, with activity losses occurring at levels of 0.0025% and above.Hind III was the most stable of the four and remained fully active at concentrations as high as 0.075%. Addition of BSA to incubation mixtures generally had a stabilizing effect. Implications of these results for the design of glutaraldehyde-based methods for the immobilization of restriction endonucleases are discussed.     

DNAzyme amplification of molecular beacon signal

This paper reports an improved catalytic molecular beacon. Addition of the target oligonucleotide activates a DNA enzyme (DNAzyme), which, in turn, activates multiple copies of molecular beacons (MB) and gives rise to a strong fluorescence signal. In a previous design, the activated DNAzyme could oligomerize, especially dimerize, and result in inactivation of the DNAzyme. The current design avoids this problem, upon activated by the target DNA, the DNAzyme will stay constantly active. With the improved method, a detection of 10 pM DNA has been demonstrated, which is 1000 times more sensitive than the method previously reported.     

Conformation of Tax-response elements in the human T-cell leukemia virus type I promoter

Background: HTLV I Tax is believed to activate viral gene expression by binding bZIP proteins (such as CRIB) and increasing their affinities for proviral THE target sites. Each 21 by THE target site contains an imperfect copy of the intrinsically bent CRE target site (the TRE core) surrounded by highly conserved flanking sequences. These flanking sequences are essential for maximal increases in DNA affinity and transactivation, but they are not, apparently, contacted by protein. Here we employ non-denaturing gel electrophoresis to evaluate TRE conformation in the presence and absence of bZIP proteins, and to explore the role of DNA conformation in viral transactivation.Results: Our results show that the TRE-1 flanking sequences modulate the structure and modestly increase the affinity of a CREB bZIP peptide for the TRE-1 core recognition sequence. These flanking sequences are also essential for a maximal increase in stability of the CREB-DNA complex in the presence of Tax.Conclusions: The CRE-like TRE core and the TRE flanking sequences are both essential for formation of stable CRIB-TRE-1 and Tax-CREB-TRE-1 complexes. These two DNA segments may have co-evolved into a unique structure capable of recognizing Tax and a bZIP protein.     

Electrochemical Study of the Interactions of DNA with Redox‐Active Molecules Based on the Immobilization of dsDNA on the Sol‐Gel Derived Nano Porous Hydroxyapatite‐Polyvinyl Alcohol Hybrid Material Coating

A novel type of sol‐gel inorganic‐organic hybrid material coated on glassy carbon electrode used for immobilization of double‐stranded DNA (dsDNA) and study of dsDNA with redox‐active molecules was developed. The hybrid material coating was produced by sol‐gel method with nano hydroxyapatite (HAp)‐polyvinyl alcohol (PVA). The optimum composition of the hybrid material was first examined, and the morphology of the nano HAp‐PVA coatings was investigated with the help of Scanning Electron Microscope (SEM). DsDNA was immobilized in/on the nano HAp‐PVA hybrid coatings by adsorption and the characteristics of the dsDNA/HAp‐PVA/GCE were studied by cyclic voltammetry (CV) using the probes of Co(phen) and Fe(CN) . The results indicate that the dsDNA can be immobilized on the nano porous HAp‐PVA coating effectively and its stability can satisfy the necessity of study on the interactions of dsDNA with redox‐active molecules on the electrode surface. Co(bpy) and Co(phen) were used as the model molecule to study the interactions of dsDNA with redox‐active molecules. Information such as ratio (K_Ox/K_Red) of the binding constant for the oxidized and reduced forms of a bound species, interaction mode, including change in the mode of interaction, and “limiting” ratio K /K at zero ionic strength (μ) can be obtained using dsDNA/HAp‐PVA/GCE with about 2 μg of DNA samples.     

DNA与中性红相互作用电化学和光谱研究

利用电化学和光谱方法研究中性红和DNA的相互作用,根据其循环伏安曲线和紫外-可见光谱,在pH=7.05缓冲体系中中性红与DNA发生了嵌插作用.     

野生大豆DNA导入小麦及RAPD分子验证

利用花粉管通道法将野生大豆总DNA导入小麦，以期获得变异系小麦．对小麦球蛋白的SDS—PAGE，印迹表明，冬小麦T2l6#产生了新的蛋白亚基，其分子量为78kD，而有的变异品系一些蛋白亚基消失了；凯氏定氮法和氨基酸分析表明小麦后代T216#的总蛋白含量增加，其氨基酸组成，尤其是赖氨酸含量明显提高．RAPD分析结果表明，新品系基因组出现多态性，并具有供体特异性DNA带，这些变异现象表明该小麦品系为转基因后代．本实验为选育高蛋白、优质的新小麦品种提供了途径．     

Real time observation of the photocleavage of single DNA molecules

A method for real time observation of photo-cleavage of stratched λDNA at single molecular level by a fluorescent microscope coupled with CCD is developed.DNA molecules stained with YOYO-1 are stretched by the mo-lecular combing technique and fixed on a modified slide.Then the Process of Photocleavage and relaxation of DNA under radiation of blue light is observed.We speculate that the conformation change of stretched DNA and the effect of water are likely to facilitate the effect of YOYO photocleav-age DNA molecules.The photocleavage effect of YOYO for stretched DNA may be useful to study DNA elasticity,cancer research as well as the interaction between DNA and dyes.