Isolation, Mapping, DNA Sequence and RFLPs Studies of Random Single-Copy DNA Segments on Human X Chromosome

Using the total human/mouse DNA as the probe, screening has been carried out three times with in situ plaque hybridization to obtain the single-copy DNA sequence from the human X chromosome genomic library. The effective rate of screening is 1. 45%. DNAs from clones containing single-copy inserts have been analyzed by a panel of hybrid cells with or without human X chromosome. Three segments, designated by DXFD52,73,75, are mapped to the X chromosome. DXFD52 has been precisely localized on Xq12-q13 with in situ chromosomal hybridization. DXFD52 has been partially sequenced. The results indicate that DXFD52 is a new isolated single-copy segment on the X chromosome. Great progress in the RFLPs study with DXFD52 has been achieved in the population of Chongqing, Sichuan Province. The results show that the DXFD52 can be used to detect the RFLP with Hind Ⅲ, Bgl Ⅱ, and Hinf Ⅰ. DXFD52 will be a potential "landmark" for the construction of the complete linkage map of human genome and the analysis of genomic s     

SNPs Feasibility of Nonlabeled Oligonucletides on Using Electrochemical Sensing

We have demonstrated an electrochemical gene chip protocol for the SNPs detection of nonlabeled DNA. Using an array consisting of streptavidin‐modified gold electrodes, probe DNA were attached through the application of a direct electric field. Electrochemical response changes originating from the hybridization of nucleic acids to protein‐bound nucleic acids using soluble mediators in K₃Fe(CN)₆ solution could then be observed. The electrochemical protocol developed showed high sensitivity and good reproducibility in the detection of DNA hybridization. Significant changes in electrochemical signals were also observed when using target DNA with a single base mismatch, indicating the applicability of this method to single nucleotide polymorphisms (SNPs) detection.     

Interaction of the mutagen ethidium bromide with DNA, using a carbon paste electrode and a hanging mercury drop electrode

The interaction of ethidium bromide (2,7-diamino-10-ethyl-9-phenylphenanthridinium bromide; EB) with double stranded (ds) calf thymus DNA and thermally denatured single stranded (ss) DNA was studied in solution and at the electrode surface by means of transfer voltammetry using a carbon paste electrode (CPE) as working electrode in 0.2 M acetate buffer, pH 5.0. As a result of intercalation of this dye between the base pairs of dsDNA, the characteristic peak of dsDNA, due to the oxidation of guanine residues, decreased and after a particular concentration of EB a new peak at +0.81 V appeared, probably due to the formation of a complex between dsDNA and EB. The non-intercalated EB gives another peak, but at an increased concentration of the dye. A similar behaviour was observed during the interaction of the dye with ssDNA.Furthermore, the interaction of EB with ds, ss and supercoiled (sc) DNA was studied at the hanging mercury drop electrode (HMDE) surface by means of alternating current voltammetry in 0.3 M NaCl and 50 mM sodium phosphate buffer (pH 8.5) as supporting electrolyte. dsDNA yields a smaller peak at −1.42 V (peak III) compared to the one yielded by ssDNA, since the latter is a relaxed and more accessible form. By addition of EB into the buffer solution an increase of peak III was observed in the dsDNA form as well as in ssDNA resulting from their interaction with EB. Furthermore, the appearance of peak III in covalently closed circular scDNA after exposure to increasing concentrations of EB is a result of the introduction of ‘free ends’ in DNA affecting its structural integrity.     

ISOLATION,CHARACTERIZATION AND EXPRESSION OF THE COMPLEMENTARY DNA FOR HUMAN TUMOR NECROSIS FACTOR (TNF-α)

A cDNA for human TNF-α (615bp) was isolated by means of polymerase chain reaction (PCR) using first strand cDNA from PMA-induced HL-60 cells as template. The result from sequencing the 615 bp cDNA fragment indicated that it corresponded to the entire sequence of mature human TNF coding region. Direct expression of mature human TNF was achieved using a plasmid pHT-1 constructed by ligation of the cDNA and a synthetic DNA. The IPTG-induced bacterial product (hTNF) showed cytotoxicity to mouse L-929 cells. The TNF activity was further identified by neutralization of a specific monoclonal antibody against human TNF-α. Approximately 80,000 units of activity were detected per ml of culture at A600=2.     

三菲咯啉合铁手性配合物键合DNA的立体选择性

应用光谱法研究了手性金属配合物［Ｆｅ（ｐｈｅｎ）３］＾２＋与小牛胸腺ＤＮＡ的作用，确定了△型异构体对Ｂ型右手螺旋ＤＮＡ有优先键合的立体选择性。     

MgCl2 Enhances Cluster Formation by Nanoscale Toroidal DNA Condensates

Multivalent cations can cause DNA to condense from its extended state in solution into high-density toroid-shaped particles. Developing methods to control the size and size distribution of DNA toroids is an important goal for the development of artificial gene delivery systems. Here we demonstrate that changes in salt conditions, prior to condensation by multivalent cations, can significantly affect DNA condensation. Specifically, millimolar concentrations of MgCl₂ are shown to cause the formation of toroid clusters, whereas NaCl at the same ionic strength does not.     

红四氮唑作为电化学嵌合剂的核酸杂交生物传感器

提出了一种以红四氮唑 (TTC)作为嵌合剂 ,采用石墨修饰电极监测核酸杂交过程和测定特定 DNA片段的灵敏的电化学方法 . TTC具有背景电流低 ,电活性强和对双链 DNA(ds DNA)选择性好等一系列优点 ,可在石墨电极表面形成 ds DNA-TTC层 .在电化学测定中 ,TTC的还原电流与靶 DNA浓度具有良好的线性关系 ,对 DNA的检测限可低达 6× 1 0 - 1 1 mol/L .     

Development of an HPLC method with electrochemical detection of femtomoles of 8-oxo-7,8-dihydroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine in the presence of uric acid

A selective method based on high performance liquid chromatography with electrochemical detection (HPLC-ECD) was developed to enable simultaneous detection of 8-oxo-7,8-dihydroguanine (8-oxoGua) and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo), products of DNA oxidative damage, in the presence of uric acid (UA), a strong interferent in their electrochemical detection. The method developed consists of HPLC isocratic elution with amperometric detection on a glassy carbon electrode, enabling a detection limit for 8-oxoGua and 8-oxodGuo lower than 1 nM in standard mixtures. Detection of low concentrations up to 25 nM of 8-oxoGua and 8-oxodGuo in the presence of UA in a 10⁴-fold higher concentration was achieved after one-step solid phase extraction (SPE). The method was tested with urine samples and it was possible to detect and quantify the presence of 8-oxoGua, and to confirm that UA was eliminated after uricase degradation and SPE. The LOD found in urine samples was about 80 nM, a value higher than in standard mixtures, due to the increase of background current in the urine matrix. The results presented here contribute to the development of a methodological approach to simultaneous determination of 8-oxoGua and 8-oxodGuo in urine samples.     

三元配合物钯（Ⅱ）-2,2''-联吡啶-L-天冬氨酸与DNA作用研究

The ternary complex Pd(Ⅱ)-2,2‘-bipyridine-L-asparagic acid was synthesized and characterized by elemental analysis, IR-spectra and molar conductance. The formula of the complex is Pd(bipy)(L-asp). The interaction of the complex with DNA has been studied by UV-spectra, fluorescence spectra, CD-spectra and gel electrophoresis. The results showed that the interaction of the complex with DNA performed mainly in intercalative mode and the extent of interaction was dependent on the concentration of the complex.