BASE MUTATION ANALYSIS BY A FERROCENYL NAPHTHALENE DIIMIDE DERIVATIVE

An electrochemical DNA sensing technique was developed by using ferrocenyl naphthalene diimide coupled with a probe DNA-immobilized electrode. This technique enabled detection of target DNA quickly and with high sensitivity. Applicability of this technique for single nucleotide polymorphisms (SNPs) analysis was demonstrated by successful analysis of a DNA mismatch on the cancer repression gene p53 with high precision.     

The use of cetyltrimethylammonium bromide for the rapid isolation from Staphylococcus aureus of relaxable and non-relaxable plasmid DNA suitable for in vitro manipulation

Plasmid DNA was isolated from Staphylococcus aureus by a rapid method that depends on the precipitation of DNA from cleared lysates by cetyltrimethylammonium bromide at low salt concentrations. The method was validated by its ability to provide DNA for restriction analysis of a highly relaxable plasmid species that is not isolated by more traditional techniques. The DNA can be digested with restriction endonucleases and used for transformation without further purification. The method also provides the basis for analysing staphylococcal plasmids that display a high frequency of deletion after transfer. Simple modifications of the technique allow plasmid DNA to be isolated from other bacteria and the rapid purification of DNA samples before in vitro manipulation.     

Mapping structurally perturbed sites in DNA by replication arrest and run-off replication

We describe a technique for rapid fine mapping of sites of torsion-induced perturbations of DNA structure. The technique involves strand scission or chemical base modification at structurally perturbed sites, replication arrest in a double-strand DNA sequencing reaction, and size analysis of replication products by electrophoresis on sequencing gels. Besides being less complicated and faster than site identification by conventional end-labeling methods, the technique assures high sequence specificity through the use of oligomeric sequencing primers. This property should be useful for in vivo mapping of DNA structural perturbations with known sequence within complex genomes.     

Flow cytometric DNA content of fresh tumor specimens using keratin-antibody as second stain for two-parameter analysis.

Studies concerning flow cytometric assessed DNA content reveal problems in interpretating DNA histograms of tumor specimens. The main problems are histograms with a broad coefficient of variation in the G0/G1 fraction; a high G2M fraction and samples with a low percentage of tumor cells. Therefore, in the present study, 382 fresh tumor specimens of carcinomas were analysed routinely, double labeled with, on the one hand, propidium-iodide for assessing DNA content and, on the other, a monoclonal keratin-antibody for marking epithelial and tumor cells. Of the 311 tumor samples, using single parameter analysis 165 (54%) were classified as DNA aneuploid and 146 (46%) as DNA "euploid." By double parameter analysis, 224 (72%) samples were keratin positive and 87 (27%) keratin negative and, of the 224 keratin positive tumors, 175 (78%) were DNA aneuploid and 49 (22%) DNA euploid. The DNA histograms of single and double parameter analysis were compared and it was concluded that in 24 cases (11%) keratin labeling was necessary to recognize DNA aneuploidy. In another 23 (10%) cases, keratin labeling was helpful in assessing DNA aneuploidy. Finally when the results of the 311 samples were combined, 215 (68%) were scored as DNA aneuploid and 99 (32%) DNA euploid. Thus the overall gain in assessing DNA aneuploidy using the double labeling technique is 14%. In conclusion, it is shown that keratin labeling on fresh tumor cell suspensions of epithelial tumors is of additional value in establishing DNA content. Because single parameter DNA assessment is adequate in approximately 60% of the tested samples, the double labeling technique can be performed routinely, or after initial single parameter DNA assessment. Histograms having a broad CV and/or a high G2M are good candidates for the double labeling technique. Using this technique, DNA-content assessment becomes more reliable.     

Canine DNA subjected to whole genome amplification is suitable for a wide range of molecular applications

Molecular and genetic studies of canine disease phenotypes can be limited by the amount of DNA available for analysis. New methods have been developed to amplify the genomic DNA of a species producing large quantities of DNA from small starting amounts. Whole genome amplification (WGA) of DNA is now being used in human studies, although this technique has not been applied extensively in veterinary research. We evaluated WGA of canine DNA for suitability in a range of molecular tests. DNA from 93 canine blood extracted and 18 buccal swab samples was subjected to WGA using the GenomiPhi kit (Amersham). Genomic DNA was compared with WGA product using a range of techniques, including reference strand-mediated conformation analysis, denaturing high-performance liquid chromatography analysis, microsatellite genotyping, direct DNA sequencing, and single nucleotide polymorphism allelic discrimination. All samples amplified well, giving an average yield of 3 mug of DNA from 2.5 ng of starting material. Extremely high levels of experimental reproducibility and concordance were observed between source and WGA DNA samples for all analyses used: greater than 95% for blood extracted DNA and greater than 80% for buccal swab DNA. These studies clearly demonstrate the usefulness of WGA of canine DNA as a means of increasing DNA quantities for canine studies. This technique will have major implications for future veterinary research.     

一种用于DNA甲基化分析的改进型亚硫酸氢盐转化法

DNA甲基化分析是认识生理、病理条件下基因表达变化的重要途径.亚硫酸氢盐转化是DNA甲基化分析的瓶颈.本文旨在改进琼脂糖 亚硫酸氢盐DNA处理方案(agarose bisulfite method),建立一种简便稳定、适合常规甲基化分析的亚硫酸氢盐转化法.把DNA包入普通琼脂糖,以饱和亚硫酸氢盐在较高的温度下快速处理,然后用离心柱型琼脂糖凝胶DNA回收试剂盒,集DNA凝胶回收、脱盐、脱磺基和纯化于一体,完成整个转化过程.Bisulfite-PCR、克隆测序和酶切法分析转化率、转化特异性和转化物的质量.用该方案处理的HeLa细胞DNA,多个片段的转化率均大于98%,甲基化片段96.2%的CpG保持不变,可以扩增605 bp的较大片段,灵敏度介于普通法和琼脂糖亚硫酸氢盐法之间,而重复性较二者都好.改良后的方案简化了操作流程,快速稳定,易学易用,可实现高效特异转化,适合于一般实验者对常规检材进行DNA甲基化分析.     

Applications of microelectroporation for studies of chick embryogenesis

A technique by which genes can be introduced into the cells and tissues of developing embryos has great potential for studying the roles of genes during vertebrate embryogenesis. The 'microelectroporation' technique, in which DNA is introduced into cells within a restricted area of developing chick embryos with high reproducibility, was developed by the authors. In this review, the advantages and applications of this microelectroporation technique for developmental studies and functional analysis of genes in chick embryos is discussed.     

用RAPD技术检测野生鲫鱼的四个金鱼代表品种的基因组DNA多态性

利用随机扩增多态DNA技术检测了两个不同地区的野生鲫鱼(Carassius a uratus L.)和4个金鱼(Carassius auratus Var.)代表品种的基因组DNA的多态性。用26个随机引物对各品种实验鱼的基因组DNA进行扩增,平均每个品种观察到约134个标记,单个引物获得的标记在1-16个之间。实验结果经统计学分析表明,金鱼和鲫鱼的随机扩增多态DNA共亨度高,进一步证实了金鱼由野生鲫鱼演化而来,聚类结果表明,草金鱼形成后,首先演化为文种鱼,然后由文种再形成龙种和蛋种两个品系。 Abstract:Genomic DNA polymorphisms in the wild crucian from rwo different areas and in four representative varieties of goldfish were detected by using random amplified polymorphic DNA(RAPD)technique.The genomic DNA of each variety of fish was amplified with 26 primers.On average,about 134 RAPD markers were observed by each variety.The markers obtained by a single primer varied from 1 to 16.The statistical analysis of the experimental results indicated that random amplified polymorphic DNA of the goldfish and wild crucian have a high proportion of random amplified polymorphic DNA fragment shared.This further verified that goldfish is evolved from wild crucian.The cluster analysis suggested that after emerging,grass goldfish is evolved to wen goldfish,then through wen goldfish evolved to dragoneye goldfish and oval goldfish.     

Use of DNA fingerprinting to determine parentage in muskrats (Ondatra zibethicus).

The detection of high levels of genetic variability by DNA fingerprinting probes has allowed researchers to accurately assess relatedness. Multiple-mating strategies are characteristic of the mating systems of small mammals. As such, techniques that provide an accurate indication of how individuals are related genetically is of great importance to assess the mating system of a species. In this study, we applied the DNA fingerprinting technique to captive and wild muskrats (Ondatra zibethicus) to determine its usefulness for parentage analysis in wild populations. We found that DNA digested with the restriction enzyme Hae III and probed with Jeffrey's minisatellite 33. 15 identified a large amount of polymorphism in both groups of muskrats. The DNA fingerprinting technique correctly assessed parentage within the captive group. In the wild population, paternity was assigned between two adult males based on diagnostic fragments and similarity of banding patterns. The likelihood that paternity could be misassigned to a full sibling was high in this free-ranging population. However, because natal dispersal in muskrats is male biased, it is unlikely that two brothers would associate with the same female.     

Single-molecule dilution and multiple displacement amplification for molecular haplotyping

Separate haploid analysis is frequently required for heterozygous genotyping to resolve phase ambiguity or confirm allelic sequence. We demonstrate a technique of single-molecule dilution followed by multiple strand displacement amplification to haplotype polymorphic alleles. Dilution of DNA to haploid equivalency, or a single molecule, is a simple method for separating di-allelic DNA. Strand displacement amplification is a robust method for non-specific DNA expansion that employs random hexamers and phage polymerase Phi29 for double-stranded DNA displacement and primer extension, resulting in high processivity and exceptional product length. Single-molecule dilution was followed by strand displacement amplification to expand separated alleles to microgram quantities of DNA for more efficient haplotype analysis of heterozygous genes.     

制备炭疽芽胞杆菌检测基因芯片的初步研究

建立制备炭疽芽胞杆菌检测基因芯片的技术,并探讨研制检测炭疽芽胞杆菌基因芯片的方法。酶切炭疽芽胞杆菌的毒素质粒和荚膜质粒,通过建立质粒DNA文库的方法获取探针,并打印在经过氨基化修饰的玻片上,制成用于炭疽芽胞杆菌检测的基因芯片。收集了290个阳性克隆探针,制备了检测炭疽芽胞杆菌的基因芯片。提取炭疽芽胞杆菌质粒DNA与基因芯片杂交,经ScanArray Lite芯片阅读仪扫描得到初步的杂交荧光图像。通过分析探针的杂交信号初步筛选出273个基因片段作为芯片下一步研究的探针。     

Human cell DNA replication is mediated by a discrete multiprotein complex

A discrete high molecular weight multiprotein complex containing DNA polymerase alpha has been identified by a native Western blotting technique. An enrichment of this complex was seen at each step in its purification. Further purification of this complex by ion-exchange chromatography indicates that the peak of DNA polymerase alpha activity co-purifies with the peak of in vitro SV40 DNA replication activity eluting from the column. The complex has a sedimentation coefficient of 18S in sucrose density gradients. We have designated this complex as the DNA synthesome. We further purified the DNA synthesome by electroeluting this complex from a native polyacrylamide gel. The eluted complex retains in vitro DNA synthetic activity, and by Western blot analysis, contains DNA polymerase delta, proliferating cell nuclear antigen, and replication protein A. Enzymatic analysis of the electroeluted DNA synthesome indicates that the synthesome contains topoisomerase I and II activities, and SDS-PAGE analysis of the electroeluted DNA synthesome revealed the presence of at least 25 major polypeptides with molecular weights ranging from 20 to 240 kDa. Taken together, our evidence suggests that the DNA synthesome may represent the minimal DNA replication unit of the human cell.     

New insights into the cell cycle profile of Paracoccidioides brasiliensis

The present work focuses on the analysis of cell cycle progression of Paracoccidioides brasiliensis yeast cells under different environmental conditions. We optimized a flow cytometric technique for cell cycle profile analysis based on high resolution measurements of nuclear DNA. Exponentially growing cells in poor-defined or rich-complex nutritional environments showed an increased percentage of daughter cells in accordance with the fungus' multiple budding and high growth rate. During the stationary growth-phase cell cycle progression in rich-complex medium was characterized by an accumulation of cells with higher DNA content or pseudohyphae-like structures, whereas in poor-defined medium arrested cells mainly displayed two DNA contents. Furthermore, the fungicide benomyl induced an arrest of the cell cycle with accumulation of cells presenting high and varying DNA contents, consistent with this fungus' unique pattern of cellular division. Altogether, our findings seem to indicate that P. brasiliensis may possess alternative control mechanisms during cell growth to manage multiple budding and its multinucleate nature.     

Preparation of megabase-sized tomato DNA and separation of large restriction fragments by field inversion gel electrophoresis (FIGE)

The Schwartz and Cantor technique for releasing and fractionating megabase-sized DNA from agarose-embedded cells is beginning to bridge the gap in resoluation between classical genetics and current molecular DNA techniques, particularly in mammalian systems. As yet no conditions have been described for preparing plant DNA that is of sufficient length to allow similar long-range restriction mapping experiments in plant systems. In this report, we describe the application of the Schwartz and Cantor technique for preparing high molecular weight DNA from embedded tomato leaf protoplasts, as well as conditions for generating and fractionating large restriction fragments, by field inversion gel electrophoresis (FIGE). The bulk of DNA released from lysed protoplasts was at least 2 Mb in size and amenable to restriction digestion as shown by hybridizing Southern blots with, among others, a probe for the Adh-2 gene of tomato. Restriction fragments as large as 700 kb were detected. Chloroplast DNA is isolated intact, amenable to restriction analysis and, in its native form, not mobile in FIGE.     

Taenia hydatigena: isolation of mitochondrial DNA, molecular cloning, and physical mitochondrial genome mapping

Mitochondrial DNA was isolated from Taenia hydatigena, T. crassiceps, and Echinococcus granulosus using a cetyltrimethylammonium bromide precipitation technique. The technique is simple, rapid, reproducible, and does not require extensive high speed ultracentrifugation. The advantage of using mitochondrial DNA from taeniid cestodes for comparative restriction analysis was demonstrated. Mitochondrial DNA of T. hydatigena was isolated as covalently closed circular molecules. These were linearized by single digestion with BamHI and the molecular weight was estimated from the linear form of 17.6 kb. The mitochondrial DNA of T. hydatigena is therefore similar in size and structure to that of many other animal species. The entire mitochondrial genome was cloned into pBR322 in Escherichia coli and a restriction map of the recombinant molecule was constructed. The potential of using the cloned mitochondrial genome as a probe in speciation studies as well as for providing functional information on the role of the cestode mitochondrion is discussed.     

水稻中期染色体和DNA纤维的高效制备技术

水稻中期染色体和DNA纤维制备是水稻分子细胞遗传学研究中的关键技术。目前,这两个技术还有很多不足,该研究建立了高效制备水稻中期染色体和DNA纤维的方法。该方法制备的染色体,分裂相多、杂质少、背景清晰、染色体分散且形态好,水稻根尖分生组织细胞的分裂指数高达25％。植物细胞的细胞壁是制备DNA纤维的最大障碍,所以必须先提取细胞核,然后裂解细胞核释放出DNA纤维。在这个研究中,还建立了一个用刀切法分离细胞核,进而用SDS裂解核膜,用载玻片拖出DNA来制备水稻DNA纤维的方法。该方法制备的DNA纤维多呈平行的细线,背景清晰,伸展的程度均匀,适合于原位杂交。     

Combination of ARDRA and RAPD genotyping techniques in identification of Acinetobacter spp. genomic species

A total of 10 non-repetitive multi-drug-resist-ant Acinetobacter strains were collected. With reference to A. calcoaceticus (ATCC23055), A. baumannii (ATCC19606), A. lwoffii (ATCC17986), and A. junii (NCTC5866), DNA fingerprint technique, amplified ribo-somal DNA restriction analysis (ARDRA), and random amplified polymorphism DNA (RAPD) were carried out to identify the genomic species of Acinetobacter spp. The distances between them were calculated by the unweighted pair group method with arithmetic (UPGMA). Genotypes ofAcinetobacter spp. were effectively classified and an A. junii together with nine A. baumannii isolates was genomically identified. The combination of ARDRA and RAPD DNA-fingerprint technique shows high com-plementarity, and could be a useful tool in Acinetobacter genomic species identification.     

Switching effect in prey--predator system

Statistical analysis of DNA base sequences generated from nearest neighbor frequencies by a Monte Carlo technique yields distributions of pyrimidine tracts in good agreement with experimental results. Better agreement with experiment is obtained with nearest-neighbor-frequency based calculations than with calculations based on base composition which assume random base arrangements. The nearest-neighbor-frequency method can also be applied to the analysis of high resolution thermal denaturation profiles, the sequence specific interaction of drugs and proteins, and the distribution of photoproducts produced in DNA by ultraviolet radiation.     

Natural occurrence of left-handed (Z) regions in PM2 DNA

Bacteriophage PM2 DNA, a ccc genome of high apparent superhelical density, contains left-handed (Z) regions as detected by competitive radioimmunoassay, agarose gel electrophoresis of DNA: antibody complexes and immunoelectron microscopy. The latter technique, in conjunction with partial blockage of restriction endonuclease sites by bound antibody, was used to map the left-handed regions along the DNA molecule. A cluster of four to five antibody molecules (approximately 25% of bound antibody) was located within map units 0.05-0.18 of the single Hpa II restriction site. Sequence analysis of part of this region showed the presence of several areas of high alternating purine-pyrimidine content. A strong correlation is observed between alternating pyrimidine-purine tracts of significant length and antibody binding sites.     

Use of molecular DNA probes in the diagnosis of mucoviscidosis-- analysis of restriction fragment length polymorphisms (RFLP) in 22 high-risk families]

The results of DNA analysis with the aid of specific molecular probes are discussed. DNA analysis involved 22 families of a high risk of cystic fibrosis. A significance of the obtained results in genetic counselling is also discussed. DNA analysis enabled detection or exclusion of cystic fibrosis gene carrier state in patient's relatives. DNA analysis proved fully informative in case of 17 families being a base to offer these families prenatal diagnosis of the disease in the I trimester of pregnancy, if such a family plans conception, and to accept this diagnostic technique.