Simple and Rapid Procedure Suitable for Quantitative Isolation of Low and High Molecular Weight Extracellular Nucleic Acids

The procedure based on binding of nucleic acids with glass surface in presence of chaotropic salts was adapted for efficient isolation of 100–10000 b.p. DNA fragments and 50–10,000 b. RNA fragments. The method provide 90% and 85% efficacy of isolation of 100 b.p. DNA and 100 b. RNA fragments respectively. High molecular weight nucleic acids are isolated with 98% efficacy. Isolated nucleic acids are free from contaminations, influencing nucleic acids modifying enzymes and fluorochromes. The method is rapid, simple and cost‐effective.     

Release of Nucleic Acids by Eukaryotic Cells in Tissue Culture

Extracellular nucleic acids in cultures of A431 and HeLa cells were investigated. The data obtained demonstrate the presence of high weight DNA and RNA in the extracellular medium. Temporal changes of extracellular nucleic acids levels in growth medium were investigated.     

用DNA重组法表达丙肝病毒部分NS_4-NS_5基因

用ＤＮＡ重组法表达丙肝病毒部分ＮＳ_４－ＮＳ_５基因祁自柏,谷金莲,李河民（中国药品生物制品检定所,北京１０００５０）丙型肝炎病毒（ＨＣＶ）是一种ＲＮＡ病毒,为研究其复制机理,制备检测丙肝抗体的诊断试剂,需大量纯化的各种丙肝病毒蛋白,我们在国内首次用Ｄ...     

Amplification of Nucleic Acids by Polymerase Chain Reaction (PCR) and Other Methods and their Applications

Abstract

The in vitro replication of DNA, principally using the polymerase chain reaction (PCR), permits the amplification of defined sequences of DNA. By exponentially amplifying a target sequence, PCR significantly enhances the probability of detecting target gene sequences in complex mixtures of DNA. It also facilitates the cloning and sequencing of genes. Amplification of DNA by PCR and other newly developed methods has been applied in many areas of biological research, including molecular biology, biotechnology, and medicine, permitting studies that were not possible before. Nucleic acid amplification has added a new and revolutionary dimension to molecular biology. This review examines PCR and other in vitro nucleic acid amplification methodologies—examining the critical parameters and variations and their widespread applications—giving the strengths and limitations of these methodologies.     

双裂解法提取血清HBV DNA技术的建立

为进行ＰＣＲ实验建立了一种两步裂解血清ＨＢＶ提取其ＤＮＡ的方法。该法首先用ＳＤＳ及蛋白酶消化裂解病毒,随后再进行碱裂解,操作较酚提取法大为简化,而ＰＣＲ实验进行的两法的灵敏性比较,说明双裂解法至少不低于酚法。     

Synthesis and Nucleic Acids Binding Properties of Diastereomeric Aminoethylprolyl Peptide Nucleic Acids (aepPNA)

A general synthetic method for Fmoc-protected monomers of all four diastereomeric aminoethyl peptide nucleic acid (aepPNA) has been developed. The key reaction is the coupling of nucleobase-modified proline derivatives and Fmoc-protected aminoacetaldehyde by reductive alkylation. Oligomerization of the aepPNAs up to 10mer was achieved by Fmoc-solid phase peptide synthesis methodology. Preliminary binding studies of these aepPNA oligomers with nucleic acids suggested that the “cis-” homothymine aepPNA decamers with (2′R,4′R) and (2′S,4′S) configurations can bind, albeit with slow kinetics, to their complementary RNA [poly(adenylic acid)] but not to the complementary DNA [poly(deoxyadenylic acid)]. On the other hand, the trans homothymine aepPNA decamers with (2′R,4′S) and (2′S,4′R) configurations failed to form stable hybrid with poly(adenylic acid) and poly(deoxyadenylic acid). No hybrid formation could be observed between a mixed-base (2′R,4′R)-aepPNA decamer with DNA and RNA in both antiparallel and parallel orientations.     

Extremely Rapid Extraction of DNA from Bacteria and Yeasts

A very simple and rapid method for extracting genomic DNA from Gram-negative bacteria, Gram-positive bacteria and yeasts is presented. In this method, bacteria or yeasts are lysed directly by phenol and the supernatant is extracted with chloroform to remove traces of phenol. The supernatant contains DNA that is suitable for molecular analyses, such as PCR, restriction enzyme digestion and genomic library construction. This method is reproducible and simple for the routine DNA extraction from bacteria and yeasts.     

Extracellular Circulating Nucleic Acids in Human Plasma in Health and Disease

The concentration of extracellular DNA and RNA in blood plasma of healthy donors, trauma patients, patients with breast and lung cancer, nonmalignant breast tumors and nonmalignant lung diseases were estimated. Significant amounts of extracellular RNA were found in plasma of trauma patients. The concentration of DNA and RNA in plasma of trauma patients correlates with the extent of posttraumatic organ failure. Extracellular RNA was not found in the plasma of breast cancer patients and patients with nonmalignant breast tumors, whereas a very high concentration of extracellular RNA was found in patients with malignant and nonmalignant diseases of lung.     

Pathways for Virus Assembly around Nucleic Acids

Understanding the pathways by which viral capsid proteins assemble around their genomes could identify key intermediates as potential drug targets. In this work, we use computer simulations to characterize assembly over a wide range of capsid protein–protein interaction strengths and solution ionic strengths. We find that assembly pathways can be categorized into two classes, in which intermediates are either predominantly ordered or disordered. Our results suggest that estimating the protein–protein and the protein–genome binding affinities may be sufficient to predict which pathway occurs. Furthermore, the calculated phase diagrams suggest that knowledge of the dominant assembly pathway and its relationship to control parameters could identify optimal strategies to thwart or redirect assembly to block infection. Finally, analysis of simulation trajectories suggests that the two classes of assembly pathways can be distinguished in single-molecule fluorescence correlation spectroscopy or bulk time-resolved small-angle X-ray scattering experiments.     

Changes of Nucleic Acids and Proteins in the Oocysts of Eimeria tenella during Sporulation

SYNOPSIS. The total content of DNA in Eimeria tenella , estimated at 5.8 × 10⁻¹² gm/oocyst, varies little during sporulation. Its buoyant density is 1.682 gm/cm³, reflecting a G + C content of ∼41%. Thymidine is not incorporated into any TCA insoluble fraction of sporulating oocysts, but radioactivity from [³H]uridine and [³H]deoxyuridine are incorporated into RNA at a linear rate during the first 5 hr of sporulation. The labeled RNA, found mainly in the paranuclear bodies of newly formed sporozoites, contains ∼0.15 nmole [³H]uridine/10⁶ oocysts at the completion of sporulation. One nmole of leucine is incorporated into the hot TCA insoluble fraction of 10⁶ oocysts during the first 7 hr of sporulation after an initial lag. The incorporated amino acid is mainly in the cytoplasm of the sporozoites, and an analysis by SDS-gel electrophoresis reveals most of the radioactivity in a narrow band with a molecular weight of ∼50,000 daltons. Incorporation of uridine and leucine, however, can be totally suppressed by respiratory inhibition. Further analysis of the proteins in the oocysts reveals that the total protein content remains relatively unchanged at 2.64 × 10⁻¹⁶ gm/oocyst during sporulation, but there is a shift of 13–14% of total protein from the soluble cytoplasm to the 15,000 g pellets. By polyacrylamide gel electrophoresis, a major protein band. possibly a glycoprotein, is shown in the soluble cytoplasm of unsporulated oocysts. This band disappears during sporulation.     

活性污泥总DNA提取方法的研究

采用冻融＋玻璃珠、溶菌酶＋SDS和冻融＋玻璃珠＋溶菌酶＋SDS的方法提取活性污泥的微生物总DNA,并对以上三种方法进行比较,从中确定最佳的方法,以实现普通实验条件下成功提取符合PCR扩增要求的DNA。经紫外光度分析及电泳结果表明,冻融＋玻璃珠＋溶菌酶＋SDS方法所得的DNA的OD260/OD280为1．81,电泳结果显示,所提DNA片段分子量大于10kb,适于酶解和PCR扩增要求,为PCR技术应用于活性污泥的研究提供了一种简便、可靠的DNA提取方法。     

马蹄金基因组DNA的提取方法比较

对马蹄金基因组DNA的提取方法———快速提取法、小量提取法和大量提取法进行了对比分析,结果表明,利用快速提取法可在20 min内快速可靠地从马蹄金(Dichondra repensForst.)组织中提取DNA,与小量提取法和大量提取法获得的DNA用于PCR检测结果一致,可为转基因植物的快速检测提供方法。     

Investigating the Comparative Biology of the Heterokonts with Nucleic Acids1

During the last decade investigations of heterokont protists utilizing molecular methods have challenged established biosystematic concepts. Most investigations emphasized the chloroplast genome or sequences from nuclear-encoded, ribosomal genes. Refinement of DNA isolation protocols, advent of “universal primers” and the polymerase chain reaction, automated sequencing and increased accessibility of DNA sequence databases have expanded data-gathering efficiency and increased dataset sizes. Because independent datasets have been easier to obtain, the testing of specific phylogenetic hypotheses has been facilitated, altering relationship concepts, primarily at phylum/class levels, and perceptions of cellular evolution. New approaches have emphasized ecological studies and extended studies to genus/species levels and poorly investigated genomes. This paper reviews studies documenting these impacts and identifies some current limitations. Additionally, new DNA sequence data from our laboratory on nuclear-encoded rDNA internal transcribed spacers and the chloroplast-encoded psb A gene suggest that these regions will provide taxonomic resolution for the Synu-rophyceae, at the class/order level and subspecies level, respectively.     

十字花科植物种子低分子RNA提取方法比较

非编码RNA不翻译成蛋白质,它们通过转录、转录后及翻译水平调控靶基因表达,在植物生长发育及逆境胁迫中发挥功能。目前,大量种子萌发期特异表达的非编码RNA (Non-coding RNA)已被发现,高效提取种子低分子RNA是对其进行研究的关键。本研究将介绍一种改良SDS RNA提取方法,并与Trizol、CTAB法、RNA提取试剂盒进行比较。结果表明:这种方法可以高效提取用于Northern blotting、RT-PCR等分子生物学分析的十字花科植物种子低分子RNA。改良SDS RNA提取方法为种子非编码RNA研究、种子萌发生理及分子育种研究提供了帮助。     

Single Fish Egg DNA Extraction for PCR Amplification

Modern stock researches on marine biomass are basically genetic and rely increasingly on PCR-based manipulations of informative DNA markers for detecting the genetic diversity. This study developed a simple and rapid single tube method for DNA extraction from a single fish egg. The 15 min protocol was based on the use of Chelex 100 resin and urea to breakdown membrane and connective tissue of eggs. From various sizes of a single egg of walleye pollack (Theragra chalcogramma), the amounts of total nucleic acids were reproducibly obtained to be 18.25 ± 1.92 μg per egg. Using DNA templates diluted ranging 1/10⁰–1/10⁵, PCR amplification for the mitochondrial cytochrome b (Cytb) gene was successfully performed, and the 1/10² diluted template yielded the best result in PCR amplification for three different DNA marker genes. This method is quite simple and economical, and enables to provide the high throughput often demanded by the stock identification of marine biomass, in which large numbers of specimens of single fish eggs must be analyzed.     

Diversity of total and active free-living vs. particle-attached bacteria in the euphotic zone of the NW Mediterranean Sea

The structure of the total and metabolically active communities of attached and free-living bacteria were analysed in the euphotic zone in the NW Mediterranean Sea with the use of DNA- and RNA-derived capillary electrophoresis single-strand conformation polymorphism fingerprinting. More than half (between 52% and 69%) of the DNA-derived operational taxonomic units (OTUs) were common in both attached and free-living fractions in the euphotic layer, suggesting an exchange or co-occurrence between them. However, analysis targeting 16S rRNA showed that only some of them were found in the dominant active bacterial pool. Especially at the deep chlorophyll maximum, less than half of the attached bacterial populations were found to be active, with regard to the high proportion of OTUs present at the DNA level, but not at the RNA level. These results suggest that even if colonization on and detachment of particles appear to be ubiquitous, most of the particulate organic carbon remineralization appeared to be mediated by a rather low number of dominant active OTUs specialized in exploiting such specific microenvironment.     

一种适于转基因水稻PCR检测的微量DNA快速提取法

对已报道的小麦基因组DNA快速提取方法的部分步骤进行了简化,在水稻上进行了尝试。结果表明,简化法提取的水稻基因组DNA完整性好,PCR扩增效果与试剂盒提取法无明显的差异,结果稳定可靠;而且整个提取过程操作简单、花费时间少,样品用量少,仅需5-10mg,适用于大规模转基因水稻的PCR检测。