Isolating plant genomic DNA without liquid nitrogen

DNA was isolated from leaves of 10 plant species (Cuminum cyminum, Vigna aconitifolia, Pennisetum typhoides, Tecoma stans, Lycium barbarum, Anogeissus acuminata, Tecomella undulata, Zizyphus mauritiana, Phoenix dactylifera, andEruca sativa) and a fungus (Fusarium oxysporum) using the CTAB method. Three fixing solutions (alcohol, alcohol and chloroform, alcohol and EDTA) were used to produce high molecular weight DNA (>40 kb). DNA quality and quantity was comparable for the 3 fixing solutions and liquid nitrogen grinding. Adding chloroform or EDTA to fixing solutions offered no advantage over absolute alcohol. Isolated DNA was suitable for RAPD analysis, restriction digestion, and cloning. This method does not require liquid nitrogen for fixation, grinding, or storage at −80°C, making it advantagenous over other common protocols.     

Genomic DNA extraction from medicinal plants available in Malaysia using a TriOmic(TM) improved extraction kit

DNA extraction was carried out on 32 medicinal plant samples available in Malaysia using the TriOmic(TM) extraction kit. Amounts of 0.1 g flowers or young leaves were ground with liquid nitrogen, lysed at 65°C in RY1(plus) buffer and followed by RNAse treatment. Then, RY2 buffer was added to the samples and mixed completely by vortexing before removal of cell debris by centrifugation. Supernatants were transferred to fresh microcentrifuge tubes and 0.1 volume RY3 buffer was added to each of the transferred supernatant. The mixtures were applied to spin columns followed by a centrifugation step to remove buffers and other residues. Washing step was carried out twice by applying 70% ethanol to the spin columns. Genomic DNA of the samples was recovered by applying 50 μL TE buffer to the membrane of each spin column, followed by a centrifugation step at room temperature. A modification of the TriOmic(TM) extraction procedure was carried out by adding chloroform:isoamyl alcohol (24:1) steps in the extraction procedure. The genomic DNA extracted from most of the 32 samples showed an increase of total yield when chloroform:isoamyl alcohol (24:1) steps were applied in the TriOmicTM extraction procedure. This preliminary study is very important for molecular studies of medicinal plants available in Malaysia since the DNA extraction can be completed in a shorter period of time (within 1 h) compared to manual extraction, which entails applying phenol, chloroform and ethanol precipitation, and requires 1-2 days to complete.     

A rapid and inexpensive method for isolation of total DNA from Trichoderma spp (Hypocreaceae)

Extraction of high-quality genomic DNA for PCR amplification from filamentous fungi is difficult because of the complex cell wall and the high concentrations of polysaccharides and other secondary metabolites that bind to or co-precipitate with nucleic acids. We developed a modified sodium dodecyl sulfate/phenol protocol, without maceration in liquid nitrogen and without a final ethanol precipitation step. The A(260/280) absorbance ratios of isolated DNA were approximately 1.7-1.9, demonstrating that the DNA fraction is pure and can be used for analysis. Additionally, the A(260/230) values were higher than 1.6, demonstrating negligible contamination by polysaccharides. The DNA isolated by this protocol is of sufficient quality for molecular applications; this technique could be applied to other organisms that have similar substances that hinder DNA extraction. The main advantages of the method are that the mycelium is directly recovered from culture medium and it does not require the use of expensive and specialized equipment.     

高质量毕赤酵母基因组DNA提取方法比较

旨在比较5种毕赤酵母基因组DNA的提取法,以便获得简便高效的提取高质量酵母基因组DNA的优化方法。分别使用蜗牛酶破壁法,超声波破碎法,液氮研磨法,Lyticase破壁法,试剂盒法提取毕赤酵母基因组DNA,然后进行DNA电泳检测以及紫外分光光度计测定DNA浓度和纯度。结果显示,5种方法均能提取出酵母基因组DNA,而酶法所提取的酵母基因组DNA质量最好。由此证实,蜗牛酶法成本低、效果好,是理想的提取高质量酵母基因组DNA的方法,完全满足后续试验要求。     

松墨天牛成虫标本保存及其DNA提取质量比较

【目的】探讨适合DNA提取的天牛成虫标本保存方法。【方法】采用SDS-蛋白酶K消化法对液氮中冷冻保存、无水乙醇-20℃冷冻保存、无水乙醇室温保存和干标本室温保存且保存时间在2年以上的松墨天牛Monochamus alternates Hope成虫标本基因组DNA进行提取,并对不同保存方式提取的DNA样本进行了质量比较和分析。【结果】在上述常见的松墨天牛成虫标本4种保存方式中,以液氮中冷冻保存效果最佳,其次为无水乙醇-20℃冷冻保存,插针干标本室温保藏效果最差。利用昆虫线粒体基因CO I和CO II的通用引物从上述DNA中均能够成功扩增出目的片段,测序结果证实扩增片段符合预期。【结论】液氮和无水乙醇-20℃冷冻保存适合松墨天牛成虫标本长期保存,且不影响后续的PCR扩增和测序。     

三种提取乳酸菌中内源性质粒DNA方法的比较

采用3种不同抽提质粒DNA的方法普查了6株不同乳酸菌的内源性质粒DNA,并对3种不同方法进行比较。在相同条件下,琼脂糖凝胶电泳检测的结果显示:效果最佳者为改进的Anderson-Mckay方法,其次为液氮反复冻融方法,最差者为改进的碱裂解方法。为抽提革兰阳性细菌乳酸菌类的内源性质粒DNA提供了行之有效的方法。     

An Efficient and Reliable Method of DNA Extraction from Meyna spinosa — A Traditional Medicinal Plant from North-East India

A simple, efficient and reliable CTAB method is standardized for genomic DNA isolation from fresh young leaves of a traditional medicinal plant Meyna spinosa. Key steps in the modified procedure include additional chloroform: isoamyl alcohol (24:1, v/v) extraction, addition of 4% PVP in the extraction buffer and an overnight isopropanol precipitation at room temperature. This procedure yields a high amount (46 μg DNA g^?1 fresh leaf tissue) of good quality DNA free from contaminants. The isolated DNA is suitable for digestion with EcoRI and HindIII restriction enzymes and can be used in other DNA manipulation techniques.     

改良CTAB法提取番石榴叶片总DNA

目的:从番石榴叶片中快速提取高质量的总DNA。方法:改良CTAB法。主要改进之处在于不用液氮,而是直接研磨硅胶干燥样品;用高浓度CTAB、低浓度乙醇与NaCl盐析相结合等方法去除多糖。结果:应用改良后的方法可以快速提取番石榴叶片总DNA,有效去除组织中的多糖、蛋白质,抑制提取过程中的组织褐变。提取的DNA可用于限制性内切酶酶切和PCR扩增。结论:传统CTAB法经过改良,可用于快速提取番石榴高质量DNA。     

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.     

Rapid, automated nucleic acid probe assays using silicon microstructures for nucleic acid concentration

A system for rapid point-of-use nucleic acid (NA) analysis based on PCR techniques is described. The extraction and concentration of DNA from test samples has been accomplished utilizing silicon fluidic microchips with high surface-area-to-volume ratios. Short (500 bp) and medium size (48,000 bp) DNA have been captured, washed, and eluted using the silicon dioxide surfaces of these chips. Chaotropic (GuHCl) salt solutions were used as binding agents. Wash and elution agents consisted of ethanol-based solutions and water, respectively. DNA quantities approaching 40 ng/cm2 of binding area were captured from input solutions in the 100-1000 ng/mL concentration range. For dilute samples of interest for pathogen detection, PCR and gel electrophoresis were used to demonstrate extraction efficiencies of about 50 percent, and concentration factors of about 10x using bacteriophage lambda DNA as the target. Rapid, multichannel PCR thermal cycling modules with integrated solid-state detection components have also been demonstrated. These results confirm the viability of utilizing these components as elements of a compact, disposable cartridge system for the detection of NA in applications such as clinical diagnostics, biowarfare agent detection, food quality control, and environmental monitoring.     

New antibiotic no. 792 formed by Actinomyces bottropensis

An actinomycetes strain 792 producing a new antibiotic was isolated under the programme of antitumor antibiotic screening. By its morphological and cultural properties strain 792 was classified as belonging to species Actinomyces bottropensis. Antibiotic 792 was recovered from the culture fluid of the strain by the extraction method in the form of a crystalline orange substances. lambda max 235, 305, 410 nm (E 1% 1cm 705, 105, 168), m. p. 232-255 degrees (dec), molecular weight 340, C 67 per cent, H 4.8 per cent, no nitrogen, sulphur or halogens. The antibiotic was inactivated in alkaline solutions forming a hardly soluble compound crystallizing in the form of red needles, lambda max 256, 485 nm (E 1% 1ct 800, 195), m. p. 202-204 degrees (dec), molecular weight 320, C 69.5 per cent, H 4.7 per cent. Antibiotic 792 had antitumor and antimicrobial activity.     

人体蠕形螨的DNA提取与随机引物PCR检测

【目的】探索人体毛囊蠕形螨和皮脂蠕形螨DNA的提取方法。【方法】采用液氮反复冻融研磨法破碎螨体细胞, 选用改良小昆虫DNA提取法、碱裂解法和试剂盒提取法, 分别提取冻存时间在5个月内和8~10个月的毛囊蠕形螨和皮脂蠕形螨基因组DNA, 并用随机引物PCR方法进行检测。【结果】蛋白核酸测定仪检测结果显示, 试剂盒法提取的DNA纯度较高、量较多, 明显优于改良小昆虫法和碱裂解法。随机引物扩增结果显示清晰的DNA指纹图谱, 两种人体蠕形螨DNA指纹具有明显差异。蠕形螨冻存时间影响DNA提取的量, 但对DNA提取的纯度和RAPD指纹图谱影响较小。不同DNA提取方法提取的同一种蠕形螨DNA指纹图谱基本相似, 试剂盒法和改良小昆虫法提取的DNA样本条带多而清晰, 碱裂解法提取的样本条带少而模糊。【结论】液氮反复冻融研磨法破碎蠕形螨细胞是有效的, 蠕形螨冻存时间不宜超过6个月, 试剂盒提取法是提取蠕形螨DNA的好方法。RAPD技术可以用于这两种人体蠕形螨DNA分子水平上的检测和分类。     

作物根际联合固氮芽孢杆菌的分离鉴定及生态分布

本文报导一种富集和分离根际联合固氮芽孢杆菌的方法,该方法以果胶为唯一碳源的培养基进行富集,中性红为指示剂的培养基进行分离,根据固氮芽孢杆菌能产气以及在分离培养基平板上菌落为红色两个重要的鉴别性特征即可检出,准确性高,简便快速;应用此法从１５个地点６种作物共８８个根系样品中分离出５１株固氮芽孢杆菌,经鉴定均为多粘芽孢杆菌（Ｂａｃｉｌｕｓｐｏｌｙｍｙｘａ）;还初步讨论了多粘芽孢杆菌在几种作物根际的分布情况。     

Comparison of seven DNA extraction and amplification protocols in historical herbarium specimens of juncaceae

Seven DNA extraction protocols were used to obtain DNA from herbarium specimens ofJuncus andLuzula (Juncaceae) of various ages. DNA of historical samples is difficult to extract, and the extracts are seldom of good quality. The quality of DNA obtained was estimated by using a spectrophotometer to measure the A260/280 absorbance ratio. The total DNA yield was measured by a fluorometer. The results indicate the success of using both mixer mill grinding and a DNeasy Plant Kit. Another extraction protocol (grinding with mortar and pestle, using liquid nitrogen) yielded DNA from many samples. Modified CTAB extraction, with a lengthy precipitation, usually provided good amounts of DNA. Other protocols did not give satisfactory results.     

改良CTAB法提取林木树种基因组DNA的研究

目的:验证改良CTAB法提取不同林木树种基因组DNA的效果,寻求一种对于不同林木树种基因组DNA提取普遍使用的方法。方法:采用改良的CTAB法提取22种木本植物基因组DNA,并对其进行定性、定量分析及酶切分析。结果:采用本法可以去除多糖和其他次生代谢物并获得高质量的DNA。结论:该方法可以作为一种适于在实验室进行的林木树种基因组DNA的提取方法。     

Rapid isolation of high-molecular-weight DNA from agarose gels

We have developed a simple, reliable, and rapid method for recovering DNA from agarose gels. While many methods for DNA extraction have already been described, few provide quantitative recovery of large DNA molecules. These procedures generally require costly apparatus, extended elution times, or considerable handling of the sample after elution. Our method employs a novel electroelution chamber constructed from acrylic plastic. Gel slices containing DNA are placed in the chamber between platinum electrodes. Voltage is applied and a continuous flow of buffer sweeps the eluted DNA from the chamber into an external receptacle. Elution is complete in 7 min. Concentrated DNA is obtained by butanol extraction and alcohol precipitation in 1 h. Recoveries, quantitated by counting radiolabeled DNA or by densitometry of analytical gels, were 94 to 100% for fragments of 4 to 50 kb. The eluted DNA was undegraded and could be digested with restriction enzymes, ligated, end-labeled, or used to transform cells as efficiently as noneluted DNA. Complete elution of a 100-kb plasmid, a 194-kb concatemer of bacteriophage lambda, and of 440- and 550- chromosomes of Saccharomyces cerevisiae was also achieved using the same process. This method is suitable for routine use in a wide range of cloning applications, including the electrophoretic isolation of large DNA molecules.     

鳞翅目成虫乙醇保存标本基因组DNA的提取及在微卫星研究中的应用

高质量的基因组DNA样品是分子生态学研究的先决条件。本研究目的在于探索从东方粘虫Pseudaletia separata (Walker)成虫自然种群的乙醇保存标本中分离高质量基因组DNA的有效方案。在2 mL微型离心管中进行4种提取方案的实验比较,结果发现采用传统的苯酚抽提方法的2种方案提取腹部中段组织的基因组DNA,样品合格率只有7.69%~40%。但是,如果在苯酚抽提以前加入高浓度盐和十六烷基三甲基溴化铵（CTAB）,就会使DNA样品合格率达到68.42%~95.28%,而且DNA平均产量达到5.59~10.04 mg/g,明显高于前者的2.83~5.78 mg/g （统计检验表明,在不同种群中差异显著或不显著）。研究结果还证明腹部组织比胸部组织更适宜提取DNA。对来自一个自然种群的99头东方粘虫DNA合格样品的统计分析表明,DNA提取总量（μg）与组织样品用量（mg）之间存在弱的正相关关系,平均DNA提取量（mg/g）与组织样品用量（mg）之间存在中度负相关关系。总之,在2 mL微型离心管中,用10~20 mg腹部组织,利用CTAB+苯酚抽提方法可以获得高纯度和高含量的基因组DNA样品。用该方案提取的基因组DNA能够顺利地进行微卫星位点的分离和基因分型。     

油气田土壤DNA提取方法及油气指示菌基因定量结果的比较

采用4种提取方法对油田上方6个土壤样品微生物总基因组DNA进行提取,并比较了其纯度和浓度。利用定量PCR技术定量分析各土壤中甲烷单加氧酶基因（pmoA）和丁烷单加氧酶基因（brnoX）。与DNA试剂盒法相比,玻璃珠击打法、液氮研磨法、反复冻融法得到DNA纯度较低,需纯化才能进行后续的PCR扩增。液氮研磨法得到的DNA完整性、纯度和得率较其他提取方法均较好,尤其对于生物量较少的深层油田土壤DNA提取较为实用,成本较低。甲烷单加氧酶基因（pmoA）和丁烷单加氧酶基因（bmoX）的定量PCR结果表明,液氮研磨法提取DNA样品的定量结果在气区和油区均出现一定的高值。液氮研磨法较其他方法更适合于油田土壤DNA的提取。下一步研究可以把丁烷氧化茵作为油气指示茵研究中的重点检测对象。     

琼脂固体培养基培养蜜环菌菌索总DNA的提取

野外采集的蜜环菌[Armillaria mellea(Vahl.ex Fr.)Quel]在提取DNA前需要分离获得纯化的菌丝体。常规液体培养获得菌丝团的方法感杂率较高,采集固体培养基表面cellophane膜上形成的菌丝则难以获得足量的DNA提取材料。蜜环菌细胞内含有大量多醣类物质,也使得蜜环菌高质量DNA的提取存在一定的困难。本研究通过改进试验,提供一个直接从琼脂固体培养基培养的蜜环菌菌索中提取高质量DNA的方法。其中样品的预先冻融处理方法可以促使蜜环菌菌索与琼脂分离;而在裂解提取缓冲液裂解材料细胞后加入1.25 mol/L KAc溶液,则有利于除去蜜环菌细胞内的多醣类物质以及残留的少量琼脂。通过琼脂糖电泳、紫外分光光度计对DNA浓度及OD值的测定、ISSR引物的PCR扩增以及酶切产物的PCR扩增等方法的检测,结果均表明该方法提取的DNA质量较好,符合进一步进行分子生物学研究的要求。