一种高效可直接用于PCR分析的土壤总微生物DNA抽提方法

以CTAB-溶菌酶-蛋白酶K-冻融裂解法直接抽提土壤总微生物的基因组DNA,利用G8000沉淀和纯化DNA.结果表明,该方法是一种简便、有效可直接应用于PCR分析的土壤总微生物基因组DNA的抽提方法.采用含聚乙烯吡咯烷酮(PVP)的缓冲液预洗,添加CaCl₂和BSA,可以去除腐殖酸;用PEG8000沉淀DNA,可以提高DNA质量;采用冻融法破碎细胞,CTAB、溶菌酶和蛋白质酶K共同作用以裂解细胞,可以保证获得大片段的DNA,提高DNA产率.用该方法抽提的七子花林下土壤总微生物DNA产率为9.22 μg·g^-1,A260/A280为1.65,可适用于 PCR扩增及扩增rDNA限制酶切分析(ARDRA)技术,适宜的模板DNA浓度为0.67 ng·μl^-1.快速、有效、可直接用于PCR分析的土壤总微生物DNA提取方法的建立,为大规模的土壤微生物分子生态学研究提供了可能.     

An Efficient Method for the Isolation and Purification of Oligoribonucleotides

Abstract

Problems associated with the use of tetrabutylammonium fluoride like incomplete desilylation and removal of the tetrabutylammonium salts during large scale syntheses of oligoribonucleotides (RNA) have been eliminated by the use of triethylamine trihydrofluoride and precipitation of the RNA with 1-butanol. An efficient anion-exchange HPLC method has been developed for the purification of chemically synthesized RNA and the resulting product precipitated directly by the addition of 1-propanol. A new activator, 5-ethylthio-1H-tetrazole significantly enhances the synthesis quality and yield of oligoribonucleotides. RNA synthesized using these improvements has been shown to be biologically active by a comparative ribozyme-substrate assay.     

Purification and characterization of pregastric esterase from calf

Calf pregastric esterase (PGE) was purified from calf gullet tissues. The tissue was excised and lyophilized, and lipid materials were extracted with acetone and n-butanol at -20 degrees C. Proteins were extracted from the delipidated tissue with a buffer containing a chaotropic salt (NaSCN) to solubilize hydrophobically bound protein aggregates. Calf PGE precipitated from the crude extract at pH 5.0. The precipitated, solubilized proteins were subjected to anion-exchange chromatography on DEAE-Sephacel, and the enzymatic activity was eluted using a linear gradient from 0.10 to 0.50 M NaCl at pH 8.0. Fractions with high specific activities were then chromatographed twice using gel filtration on Sephadex G-100. The resultant enzyme was shown to be pure upon discontinuous electrophoresis in 12% polyacrylamide containing 0.1% sodium dodecyl sulfate (SDS-PAGE). From SDS-PAGE gel patterns, a molecular weight of 49,000 was determined. The amino acid composition of the enzyme allowed calculation of an "average hydrophobicity" (Bigelow index) of 1150 cal/residue. This indicates that calf PGE is relatively hydrophobic, being similar to proteins such as alpha-lactalbumin and bovine serum albumin in average hydrophobicity.     

Isolation of alpha-lactalbumin,beta-lactoglobulin,and bovine serum albumin from cow's milk using gel filtration and anion-exchange chromatography including evaluation of their antigenicity

The aim of this study was to introduce a simple, reproducible, and less expensive method for isolation of alpha-lactalbumin, beta-lactoglobulin, and bovine serum albumin from cow's milk while retaining their antigenicity. Whey (lactoserum) was obtained by isolating casein from defatted milk using hydrochloric acid. Globulins were then precipitated from whey by half-saturated ammonium sulfate and beta-lactoglobulin was purified further using Sephadex G-50 gel filtration. The proteins in the supernatant were also fractionated using diethylaminoethyl cellulose chromatography in which beta-lactoglobulin was separated from alpha-lactalbumin and bovine serum albumin. The latter two proteins that co-eluted in anion-exchange chromatography were then gently isolated from each other by Sephadex G-50 gel filtration. Pure beta-lactoglobulin was also obtained by anion-exchange chromatography of the ammonium sulfate-precipitated globulins. Using enzyme-linked immunosorbent assay (ELISA), Western blotting, and ELISA inhibition assay, antigenicity of the purified proteins was evaluated. Our results showed high purity and well-preserved antigenicity of alpha-lactalbumin, beta-lactoglobulin, and bovine serum albumin thus purified.     

Novel method for identifying sequence-specific DNA-binding proteins.

We developed a general method for the enrichment and identification of sequence-specific DNA-binding proteins. A well-characterized protein-DNA interaction is used to isolate from crude cellular extracts or fractions thereof proteins which bind to specific DNA sequences; the method is based solely on this binding property of the proteins. The DNA sequence of interest, cloned adjacent to the lac operator DNA segment is incubated with a lac repressor-beta-galactosidase fusion protein which retains full operator and inducer binding properties. The DNA fragment bound to the lac repressor-beta-galactosidase fusion protein is precipitated by the addition of affinity-purified anti-beta-galactosidase immobilized on beads. This forms an affinity matrix for any proteins which might interact specifically with the DNA sequence cloned adjacent to the lac operator. When incubated with cellular extracts in the presence of excess competitor DNA, any protein(s) which specifically binds to the cloned DNA sequence of interest can be cleanly precipitated. When isopropyl-beta-D-thiogalactopyranoside is added, the lac repressor releases the bound DNA, and thus the protein-DNA complex consisting of the specific restriction fragment and any specific binding protein(s) is released, permitting the identification of the protein by standard biochemical techniques. We demonstrate the utility of this method with the lambda repressor, another well-characterized DNA-binding protein, as a model. In addition, with crude preparations of the yeast mitochondrial RNA polymerase, we identified a 70,000-molecular-weight peptide which binds specifically to the promoter region of the yeast mitochondrial 14S rRNA gene.     

甲肝病毒减毒株(H2)RNA的全长RT-PCR扩增

通过RT-PCR技术扩增了甲肝病毒减毒株(H₂)全长RNA,并对长片段RT-PCR扩增进行了方法学上的探讨.采用抗血清特异沉淀病毒;盐酸胍-酸性酚、氯仿一步法分离纯化病毒RNA,可得到高质量的RNA样品;以此RNA为模板,在无RNA酶的逆转录酶作用下,合成单链cDNA;继续以此cDNA为模板,利用32 mer寡核苷酸引物, 在Taq和Deep Vent DNA多聚酶的作用下进行PCR扩增,得到7.4 kb的扩增产物.     

噬菌体DNA的快速抽提

介绍一种噬菌体DNA的快速抽提方法.用聚乙二醇沉淀噬菌体颗粒,然后经DEAE纤维素纯化处理和酚抽提.与传统的噬菌体DNA纯化方法相比,改进后的方法方便、快速、经济,可获得高纯度的噬菌体DNA.     

A simple method for DNA extraction from marine bacteria that produce extracellular materials

We present a simple method for extracting DNA from the marine bacteria Hahella chejuensis, a Streptomyces sp., and a Cytophaga sp. Previously, DNA purification from these strains was hindered by the presence of extracellular materials. In our extraction method, the marine bacteria are lysed by freezing and grinding in liquid nitrogen, and treated with SDS. The extracted DNA is purified using a phenol/chloroform mixture, and precipitated in isopropanol. The extracted DNA is of high quality and suitable for molecular analyses, such as PCR, restriction enzyme digestion, genomic DNA blot hybridization, and genomic DNA library construction. We used this method to extract genomic DNA from several other marine bacteria. Our method is a reproducible, simple, and rapid technique for routine DNA extractions from marine bacteria. Furthermore, the low cost of this method makes it attractive for large-scale studies.     

A quick and inexpensive method for removing polysaccharides from plant genomic DNA.

A quick and inexpensive method has been demonstrated to remove polysaccharide contamination from plant DNA. Isolated plant genomic DNA with polysaccharide contaminants was dissolved in TE (10 mM Tris-HCl, pH 7.4, 1 mM EDTA) with NaCl ranging from 0.5-3.0 M, then precipitated with two volumes of ethanol. Most of the polysaccharides were removed effectively in a single high-salt precipitation at 1.0-2.5 M NaCl. At 3.0 M NaCl, the salt precipitated out of solution. Purified DNA was easily digested by either HindIII or EcoRI and was satisfactory as a template for PCR. The results show that high-salt precipitation effectively removed polysaccharides and their inhibitory effects on restriction enzyme and Taq polymerase activity.     

A high-throughput, low-cost method for the preparation of "sequencing-ready" phage DNA template

We have developed a simple and efficient protocol for the isolation of good-quality recombinant phage DNA useful for all downstream processing, including automated sequencing. The overnight-grown phage particles were effectively precipitated (without any contaminating Escherichia coli DNA and other culture media components) by adjusting the pH of the culture medium to 5.2 with sodium acetate, followed by addition of ethanol to 25%. The phage DNA was selectively precipitated with ethanol in the presence of guanidinium thiocyanate under alkaline pH, resulting in uniform quality and quantity of phage DNA. The quality of the phage DNA preparation was demonstrated by DNA sequencing that provided an average read length of >700 bases (PHRED20 quality). This protocol for plating, picking, growing, and subsequent DNA purification of individual phage clones can be completely automated using any standard robotic platform. This protocol does not require any commercial kits and can be completed within 2 h.     

一种高效的哺乳动物粪便DNA提取通用方法

以粪便为材料提取动物DNA进行动物保护遗传学和分子生态学研究的关键是能否提取到高质量的粪便DNA.然而提取方法通用性不好和产物质量不高等问题阻碍了粪便DNA分析技术的推广.本文介绍的改进型十六烷基三甲基溴化铵提取法可广泛适用于各食性哺乳动物粪便DNA提取,在11种不同食性动物的粪便DNA提取实验中验证了它的可靠性和通用性.本方法成本低廉(3元/样),用实验室常规试剂即可完成粪便DNA提取,其产物纯度高于专用试剂盒QIAamp DNA Stool Kit,在拥有超过专业试剂盒提取效果的同时尽可能的降低了实验成本,有利于粪便DNA技术的推广.     

阴离子交换晶胶层析分离质粒DNA

质粒DNA(pDNA)作为重要的基因治疗药物载体,其广泛应用受纯度和产量的限制。为了获得高纯度的pDNA,首先制备超大孔连续床晶胶基质,接枝二乙氨基乙基葡聚糖得到阴离子交换型晶胶介质;然后以pUC19质粒为例,将目标质粒转化至大肠杆菌,培养收集,碱液裂解和离心;最后用阴离子交换型晶胶介质从离心上清液中一步法层析分离pDNA。通过优化层析过程的pH值和洗脱条件,最终在pH值为6.6时,用0.5 mol/L的NaCl溶液洗脱,得到较高纯度的pDNA。整个分离过程中不使用动物源性酶,也不需常规分离中的高毒试剂,使获得pDNA的过程和产物更加安全。     

Combination of methylated-DNA precipitation and methylation-sensitive restriction enzymes (COMPARE-MS) for the rapid, sensitive and quantitative detection of DNA methylation

Hypermethylation of CpG island (CGI) sequences is a nearly universal somatic genome alteration in cancer. Rapid and sensitive detection of DNA hypermethylation would aid in cancer diagnosis and risk stratification. We present a novel technique, called COMPARE-MS, that can rapidly and quantitatively detect CGI hypermethylation with high sensitivity and specificity in hundreds of samples simultaneously. To quantitate CGI hypermethylation, COMPARE-MS uses real-time PCR of DNA that was first digested by methylation-sensitive restriction enzymes and then precipitated by methyl-binding domain polypeptides immobilized on a magnetic solid matrix. We show that COMPARE-MS could detect five genome equivalents of methylated CGIs in a 1000- to 10000-fold excess of unmethylated DNA. COMPARE-MS was used to rapidly quantitate hypermethylation at multiple CGIs in >155 prostate tissues, including benign and malignant prostate specimens, and prostate cell lines. This analysis showed that GSTP1, MDR1 and PTGS2 CGI hypermethylation as determined by COMPARE-MS could differentiate between malignant and benign prostate with sensitivities >95% and specificities approaching 100%. This novel technology could significantly improve our ability to detect CGI hypermethylation.     

A simple method for accurate estimation of apoptotic cells

A simple, sensitive, and reliable "DNA diffusion" assay for the quantification of apoptosis is described. Human lymphocytes and human lymphoblastoid cells, MOLT-4, were exposed to 0, 12.5, 25, 50, or 100 rad of X-rays. After 24 h of incubation, cells were mixed with agarose, microgels were made, and cells were lysed in high salt and detergents. DNA was precipitated in microgels by ethanol. Staining of DNA was done with an intense fluorescent dye, YOYO-1. Apoptotic cells show a halo of granular DNA with a hazy outer boundary. Necrotic cells, resulting from hyperthermia treatment, on the other hand, show an unusually large homogeneous nucleus with a clearly defined boundary. The number of cells with apoptotic and necrotic appearance can be scored and quantified by using a fluorescent microscope. Results were compared with other methods of apoptosis measurement: morphological estimations of apoptosis and DNA ladder pattern formation in regular agarose gel electrophoresis. Validation of the technique was done using some known inducers of apoptosis and necrosis (hyperthermia, hydrogen peroxide, mitoxantrone, novobiocin, and sodium ascorbate).     

A simple method for DNA extraction from sporophyte in the brown alga <Emphasis Type="Italic">Laminaria japonica</Emphasis>

A simple method was developed for extracting DNA from brown algae Laminaria japonica, which possess large amounts of acidic polysaccharides. Firstly, the sporophyte were washed by eliminating polysaccaride buffer to remove the polysaccharides and then ground in liquid nitrogen. Secondly, the powders were treated with lysing buffer. Thirdly, KAc was used to eliminate the remaining acidic polysaccharides. The extracted DNA was purified using a chloroform-isoamyl alcohol (24:1 v/v), and precipitated in cold isopropanol. The yield was from 18.7 to 37.5 μg g⁻¹ (wet weight) and the purity of total DNA was determined spectrophotometrically as the ratio of A₂₆₀/A₂₈₀, which was about 1.7–1.9. The extracted DNA was of high quality and suitable for molecular analyses, such as PCR, restriction enzyme digestion. This method is a reproducible, simple, and rapid technique for routine DNA extraction from sporophyte in Laminaria japonica. Furthermore, the low cost of this method makes it attractive for large-scale studies.     

Clinical aspects of sperm DNA fragmentation detection and male infertility

Over the past 25 years, various methods have been developed to measure sperm DNA strand breaks in situ. Currently, there are four major tests of sperm DNA fragmentation, including the Comet, Tunel, sperm chromatin structure assay (SCSA) and the acridine orange test (AOT). The Comet assay is a light microscope technique where the sperm cells are mixed with melted agarose and then placed on a glass slide. The cells are lysed and then subjected to horizontal electrophoresis. The Tunel assay, another light microscope technique, transfers labeled nucleotide to the 3'OH group of a broken DNA strand with the use of terminal deoxynucleotidyl transferase. The fluorescence intensity of each scored sperm is determined as a "yes" or "no" for sperm on a light microscope slide or by channels of fluorescent intensity in a flow cytometer. The light microscope-based AOT, uses the metachromatic properties of acridine orange to stain sperm cells. The SCSA treats sperm with low pH to denature DNA at the sites of DNA strand breaks, followed by acridine orange (AO) staining of green for native DNA and red for denatured DNA as measured by flow cytometry (FCM) as well as % sperm with high DNA stainability (HDS: immature sperm with intact DNA related to decreased fertilization rates). The SCSA method has defined a 27-30% DNA fragmentation index (DFI) as the point in which a man is placed into a statistical category of taking a longer time to in vivo pregnancy, intra uterine insemination (IUI) and more routine in vitro fertilization (IVF) cycles or no pregnancy. Current data suggest that intracytoplasmic sperm injection (ICSI) may help overcome the diminished pregnancy prognosis with high DFI over the other ART or natural methods.     

A method for characterization and fractionation of deoxyribonucleic acid by continuous turbidimetry of cetyltrimethylammonium-DNA precipitates

Cetyltrimethylammonium-DNA from solutions containing 1 m NaCl may be precipitated by continuously lowering the NaCl concentration. By monitoring the increasing turbidity of the reaction mixture at 600 nm as a function of dilution, a curve of apparent absorbance versus volume of diluting solution (or decreasing salt concentration) is obtained. Evaluation of these curves allows (a) an estimation of DNA concentration even in the presence of components absorbing at 260 nm; (b) analysis of the molecular weight distribution of DNA samples; (c) separation of native DNA, RNA and proteins on an analytical or preparative level; (d) optimization of yield and purity by simple procedures even with considerable excess of RNA; (e) characterization at the analytical level and partial, eventually complete separation of doublestranded from single-stranded DNA.The controlled fractionation of protein, DNA, and RNA is the basis of a preparation procedure for DNA from practically all biological objects, yielding products of high quality.     

Isolation of DNA from yeasts

Methods are described that allow DNA to be prepared from widely different yeasts (Candida utilis, Saccharomyces cerevisiae, and Schizosaccharomyces pombe). The methods are reliably reproducible, and the DNA obtained is of appropriate quality for the construction of gene libraries (upper limit of size range consistently 50-150 kbp). In method A, yeast cells are converted into spheroplasts by treatment with a highly purified mixture of enzymes from Trichoderma harzianum, the spheroplasts are lysed in a lauroylsarcosinate/EDTA buffer, and the lysate is incubated with proteinase K and then directly centrifuged through a cesium trifluoroacetate gradient. DNA is recovered from the appropriate fractions by ethanol precipitation, and the redissolved precipitate is incubated with ribonuclease. For the rest of the isolation, two protocols are given, one avoiding and one including phenol/chloroform extraction. In this way, DNA up to about 150 kbp in size can be obtained. In method B, spheroplasts are not made. Yeast cells are broken by grinding under liquid nitrogen and are then worked up in a manner similar to method A, protocol 2. Subsequent steps depend on the purpose for which the DNA is required. Traditional methods of sucrose or salt density gradient centrifugation or agarose gel electrophoresis are applicable for size selection. A sodium iodide/silica matrix technique allows fast and effective DNA recovery from agarose gels.     

A bacteriophage lambda DNA purification procedure suitable for the analysis of DNA from either large or multiple small lysates

A method for the efficient preparation of high quality bacteriophage lambda DNA from cleared lysates is described. Advantages of the method include high DNA yields (typically around 0.8 micrograms of DNA/1 ml of cleared lysate), speed of processing (approximately 2 h from lysate to DNA), economy, and the absence of any requirement for phenol or chloroform extractions. The technique involves the concentration of phage particles by standard polyethylene glycol precipitation followed by enzymatic treatment to remove contaminating RNA and DNA. Phage particles are then lysed with sodium dodecyl sulfate (SDS) at elevated pH and temperature. Contaminating protein/SDS complexes are rendered insoluble by the addition of potassium acetate and removed by centrifugation. The quality of the resultant DNA is comparable to that prepared by cesium chloride banding for all standard molecular biological purposes providing that spermidine is included in all restriction endonucleases digestions.     

A novel method for collection and preservation of faeces for genetic studies

Quantitative and qualitative measurements of DNA were used to compare faecal sample storage in ethanol and silica with a novel method (two‐step) in which samples are soaked in ethanol and then desiccated with silica. Silica‐preserved samples had the lowest DNA concentrations. The two‐step method yielded significantly more DNA in high quality samples (average DNA concentrations > 100 pg/µL with all storage methods). However, for lower quality samples, the ethanol and two‐step methods performed similarly. The amounts and rates of sample degradation were not strongly affected by storage method and neither was the percentage of target DNA (< 1%) obtained from the samples.