利用氯化苄提取适于分子生物学分析的真菌DNA

随着生物技术的飞速发展,更加需要简便、快速地提取高质量的DNA。以往报导的提取真菌DNA的方法大都采用液氮研磨或酶解破坏细胞壁和膜的方式,从而导致繁琐、复杂和费时的提取过程。根据氯化苄在弱碱条件下与多糖上的羟基反应形成醚从而使多糖长链断裂的事实,我们发展了一种全新的真菌DNA提取方法,该方法使氯化苄在pH9.0时与细胞壁多糖作用,破坏细胞壁,基因组DNA因而得以从细胞中释放出来。新方法具有简便、快速、价廉的优点,得到的DNA蛋白质污染少、质量较高、产量稳定。对该法提取的DNA作进一步的分子生物学分析,如限制性内切酶酶解、RFLP分析、RAPD扩增,都取得了令人满意的结果。利用氯化苄提取真菌DNA的研究,迄今在国际、国内均尚未见报道。     

利用氯化苄提取适于分子生物学分析的真菌DNA

随着生物技术的飞速发,更加需要简便、快速地提取高质量的ＤＮＡ。以往报导的提取真菌ＤＮＡ的方法大都采用液氮研磨或酶解破坏细胞壁的膜的方式,从而导致繁琐、复杂和费时的提取过程。根据氯化苄在弱碱条件下与多糖上的羟基反应形成醚从而使多糖长链断裂的事实,我们发展了一种全新的真菌ＤＮＡ提取方法,该方法使氯化苄在ｐＨ９．０时与细胞壁多糖作用,破坏细胞壁,基因组ＤＮＡ因而得以从细胞中释放出来。新方法具有简便、快速     

氯化苄法提取染色体DNA

本文介绍了一种简便、快速提取细菌和真菌的基因组DNA的方法一氯化苄法。使用氯化苄法抽提基因组DNA,不仅具有快迅、简便、耗资少的优点;而且得到的基因组DNA纯度高,质量好,可直接用于酶切、杂交和PCR扩增等用途。该法可用于多种细菌和真菌基因组DNA的提取。     

丝状真菌三孢布拉霉DNA的提取研究

用氯化苄提取丝状真菌三孢布拉霉DNA.用100mMTris*HCl;40mMEDTApH9.0;2%SDS作为提取液,使氯化苄在碱性条件下与三孢布拉霉细胞壁中的多糖成分发生反应,从而达到破坏细胞壁结构,释放DNA的作用.实验得到的DNA污染少,质量高.     

一种高效提取真菌总DNA的方法

<正>在真菌的分子生物学研究中,快速高效地提取质量优良的DNA有着重要意义。目前使用的真菌DNA提取方法步骤大体相似,主要差别在于采用何种方法来打破细胞壁这一关键环节。常用的破壁方法主要有冷冻干燥研磨法、玻璃珠机械破壁法、酶解法和氯化苄法等(吴志红2001)。通常冷冻干燥研磨法多用液氮来处理,一般是直接在液氮中研磨     

植物细胞壁多糖合成酶系及真菌降解酶系北大核心CSCD

秸秆类植物细胞壁多糖高效降解转化对我国农业经济的绿色可持续发展具有重要意义,然而植物细胞壁在长期进化过程中形成了复杂结构限制了工业化酶解转化的过程。一方面从植物细胞壁多糖合成酶系的多样性、细胞壁多糖成分的复杂性、超分子结构的异质性等方面综述了形成植物细胞壁抗降解屏障的原因;另一方面从真菌降解植物细胞壁酶系的多样性、不同菌株降解酶组成差异性等分析降解转化植物细胞壁时发挥的不同作用,从而为工业转化合理复配真菌降解酶系,提高秸秆生物质的利用效率提供理论支持。     

植物细胞壁多糖合成酶系及真菌降解酶系

秸秆类植物细胞壁多糖高效降解转化对我国农业经济的绿色可持续发展具有重要意义,然而植物细胞壁在长期进化过程中形成了复杂结构限制了工业化酶解转化的过程。一方面从植物细胞壁多糖合成酶系的多样性、细胞壁多糖成分的复杂性、超分子结构的异质性等方面综述了形成植物细胞壁抗降解屏障的原因;另一方面从真菌降解植物细胞壁酶系的多样性、不同菌株降解酶组成差异性等分析降解转化植物细胞壁时发挥的不同作用,从而为工业转化合理复配真菌降解酶系,提高秸秆生物质的利用效率提供理论支持。     

一种简单有效的去除植物DNA中多糖等杂质的方法

目的：寻找一种经济、快速、简便的去除多糖等杂质并获得高质量DNA的、适于不同植物而又便于一般实验室操作的基因组DNA提取方法。方法：使用1mol/LNaCl溶液溶解常规方法提取的DNA,离心后弃沉淀,从而使多糖等杂质去除。结果：提取DNA纯化之后经电泳检测以及OD260nm和OD280nm比值的测定,限制性内切酶反应,PCR扩增的结果表明,基因组DNA条带清晰,亮度均一,DNA片段大小一致,无降解弥散,用改进方法提取的DNA,无论在纯度上还是在完整性上都比用常规方法要好。结论：该方法能较好的去除DNA中色素、多糖等干扰物质。     

一种提取真菌染色体DNA简便方法

本文描述了一种快速简便提取白腐真菌Phanerochaete chrysosporium染色体DNA的方法。用该方法可节约药品,提取的染色体DNA降解小,纯度高,并可直接用于限制性酶切和基因文库的构建。这种方法对研究真菌分子遗传学既适合又经济。     

一种快速高效提取病原真菌DNA作为PCR模板的方法

真菌rDNA-ITS序列分析适合于较高等级水平的生物群体间的系统分析。真菌DNA的提取采用传统的方法,步骤繁琐,需要较长时间。采用Chelex-100法提取真菌DNA,使用PCR扩增rDNA-ITS序列评价提取核酸的质量。结果显示,该方法具有经济、简便、快速、高效的特点,是一种比较理想的提取真菌基因组DNA作为PCR模板的方法。     

A rapid and simple method for DNA extraction from yeasts and fungi isolated from Agave fourcroydes

A simple and easy protocol for extracting high-quality DNA from different yeast and filamentous fungal species is described. This method involves two important steps: first, the disruption of cell walls by mechanical means and freezing; and second, the extraction, isolation, and precipitation of genomic DNA. The absorbance ratios (A₂₆₀/A₂₈₀) obtained ranged from 1.6 to 2.0. The main objective of this procedure is to extract pure DNA from yeast and filamentous fungi, including those with high contents of proteins, polysaccharides, and other complex compounds in their cell walls. The yield and quality of the DNAs obtained were suitable for micro/minisatellite primer-polymerase chain reaction (MSP-PCR) fingerprinting as well as for the sequence of the D1/D2 domain of the 26S rDNA.     

Rapid and efficient protocol for DNA extraction and molecular identification of the basidiomycete Crinipellis perniciosa

DNA isolation from some fungal organisms is difficult because they have cell walls or capsules that are relatively unsusceptible to lysis. Beginning with a yeast Saccharomyces cerevisiae genomic DNA isolation method, we developed a 30-min DNA isolation protocol for filamentous fungi by combining cell wall digestion with cell disruption by glass beads. High-quality DNA was isolated with good yield from the hyphae of Crinipellis perniciosa, which causes witches' broom disease in cacao, from three other filamentous fungi, Lentinus edodes, Agaricus blazei, Trichoderma stromaticum, and from the yeast S. cerevisiae. Genomic DNA was suitable for PCR of specific actin primers of C. perniciosa, allowing it to be differentiated from fungal contaminants, including its natural competitor, T. stromaticum.     

Evaluation of transforming activity of cellular DNAs from different origins by NIH3T3 transfection test.

The NIH3T3 cell transfection test, as first described by Cooper, has been optimized, then used to examine the transforming activity of genomic DNA extracted from eucaryotic cell lines commonly used for preparing vaccines or biopharmaceuticals. Accurate assessment of technical parameters of the test has led to improvement in reproducibility, while the demonstration of dose-effect relationships has allowed the definition of applications and limits for quantitative use. We have performed the direct assessment of transforming activity of cellular DNAs from cell lines widely used in biotechnology. In particular, we have shown that genomic DNA extracted from Vero, CHO or MRC5 cells, as well as from human or murine lymphoid cells, has no detectable transforming activity on NIH3T3 cells. Lastly, it has been demonstrated that acidic pH conditions are sufficient to destroy the major part--if not all--of the transforming activity of positive control DNAs.     

Rapid preparation of PCR-amplifiable fungal DNA by benzyl bromide extraction

For isolation of fungal DNA for PCR amplification, we compared three DNA isolation methods: enzymatic cleavage and the use of benzyl chloride or benzyl bromide. Since benzyl bromide is more reactive, its use enabled us to readily isolate the total nucleic acids as a DNA template source from various fungi, including dematiaceous hyphomycetes, for RAPD analysis.     

An improved technique for the efficient construction of gene libraries by partial filling-in of cohesive ends

For the preparation of gene libraries, DNA from lambda EMBL3 phage was digested with SalI and EcoRI, and the cohesive ends partially filled-in by addition of dTTP, dCTP and Klenow fragment of DNA polymerase I (PolIk). Genomic DNA was cleaved partially with Sau3A and subsequently incubated with dATP, dGTP and PolIk. The phage and genomic DNAs were then mixed and ligated. The recombinant DNAs were packaged in vitro. The efficiency of packaging was 10(5)-10(6) of infectious phage lambda particles per microgram of the genomic DNA (as compared to approx. 10(7) per microgram for the wild-type lambda DNA). This procedure is very rapid and requires only microgram quantities of genomic DNA for preparing an entire gene library. The other important advantage is that multiple independent insertions of genomic DNA cannot occur in a single recombinant phage and self-ligation of phage DNA is blocked. It is also applicable for other SalI-containing vectors.     

A procedure for the preparation of bacterial DNA that employs dimethyl sulfoxide to induce the lysis of cells

A protocol for the preparation of DNA from Escherichia coli and Bacillus subtilis without the use of lysozyme as a permeabilizing agent is described. This preliminary step is carried out by treating the cells with dimethyl sulfoxide. A 5-min incubation of the cell pellet in the pure solvent, followed by the treatment with sodium dodecyl sulfate, is sufficient to induce cell lysis. The plasmid DNAs obtained by this method were equivalent in purity and quantity to the material prepared from lysozyme-digested cells and amenable to restriction and ligation. Transformation by plasmid and genomic DNAs prepared from dimethyl sulfoxide-treated cells was demonstrated.     

A simple method of genomic DNA extraction suitable for analysis of bulk fungal strains

Aims: A simple and rapid method (designated thermolysis) for extracting genomic DNA from bulk fungal strains was described. Methods and Results: In the thermolysis method, a few mycelia or yeast cells were first rinsed with pure water to remove potential PCR inhibitors and then incubated in a lysis buffer at 85°C to break down cell walls and membranes. This method was used to extract genomic DNA from large numbers of fungal strains (more than 92 species, 35 genera of three phyla) isolated from different sections of natural Ophiocordyceps sinensis specimens. Regions of interest from high as well as single‐copy number genes were successfully amplified from the extracted DNA samples. The DNA samples obtained by this method can be stored at ?20°C for over 1 year. Conclusions: The method was effective, easy and fast and allowed batch DNA extraction from multiple fungal isolates. Significance and Impact of Study: Use of the thermolysis method will allow researchers to obtain DNA from fungi quickly for use in molecular assays. This method requires only minute quantities of starting material and is suitable for diverse fungal species.     

S-adenosylmethionine: DNA-cytosine 5-methyltransferase from a Novikoff rat hepatoma cell line.

Partial purification of DNA methylase from Novikoff rat hepatoma cells is described. Contamination with other proteins persists although the enzyme preparation has a high specific activity and is purified 980-fold over homogenate activity. Evidence suggests, but does not prove, that there may be more than one species of DNA methylase in these cells. The enzyme has two broad pH optima at pH 7.0 and 7.5 and most readily methylates heterologous denatured DNAs although complex reaction kinetics indicate that native DNAs may eventually be methylated to an equal or greater level. The preparation of undermethylated DNA from Novikoff cells is also described. Undermethylated homologous DNA is an 85-fold greater acceptor of methyl groups than fully methylated Novikoff cell DNA. In contrast to other DNA substrates, the enzyme preparation methylates native undermethylated homologous DNA at a 3.5-fold greater than denatured undermethylated homologous DNA.     

Chromosome-specific DNA repeat probes.

In research as well as in clinical applications, fluorescence in situ hybridization (FISH) has gained increasing popularity as a highly sensitive technique to study cytogenetic changes. Today, hundreds of commercially available DNA probes serve the basic needs of the biomedical research community. Widespread applications, however, are often limited by the lack of appropriately labeled, specific nucleic acid probes. We describe two approaches for an expeditious preparation of chromosome-specific DNAs and the subsequent probe labeling with reporter molecules of choice. The described techniques allow the preparation of highly specific DNA repeat probes suitable for enumeration of chromosomes in interphase cell nuclei or tissue sections. In addition, there is no need for chromosome enrichment by flow cytometry and sorting or molecular cloning. Our PCR-based method uses either bacterial artificial chromosomes or human genomic DNA as templates with alpha-satellite-specific primers. Here we demonstrate the production of fluorochrome-labeled DNA repeat probes specific for human chromosomes 17 and 18 in just a few days without the need for highly specialized equipment and without the limitation to only a few fluorochrome labels.