An improved method for the isolation of total RNA from Avicennia germinans leaves

Isolation of high-quality RNA of Avicennia germinans L. tissue is difficult due to high levels of phenols and other substances that interfere when using conventional procedures for the isolation. These substances not only decrease the yield but also the quality of RNA is almost poor. We present here a simple RNA protocol and fast methodology that effectively removes these contaminating substances without affecting the yield. The protocol developed is based on the SDS/phenol method with modifications including beta-mercaptoethanol to prevent oxidation of phenolic complexes, and phenol/chloroform extraction is introduced to remove proteins, genomic DNA, and secondary metabolites, and co-precipitated polysaccharides. Both A260/A230 and A260/A280 absorbance ratios of isolated RNA were around 2 and the yield was about 0.3 mg g(-1) fresh weight. Good-quality total RNA from leaves of Avicennia germinans could be easily isolated within 2 h by this protocol which avoided the limitation of plant materials and could provide total RNA for all kinds of further molecular studies.     

An Improved and Rapid Protocol for the Isolation of Polysaccharide- and Polyphenol-Free Sugarcane DNA

We have optimized a simple and rapid method for isolating milligram quantities of high quality DNA from polysaccharide- and polyphenolic-rich tissue such as sugarcane, lettuce and strawberry. The protocol utilizes fresh tissue without making use of liquid nitrogen or freeze-drying for initial grinding of the tissue and it significantly minimize the use of lab materials. At least one hundred samples can be processed daily by one person. The isolated DNA is essentially free of polysaccharides, polyphenols, RNA and other major contaminants, as judged by: its clear color, its viscosity, A260/A280 ratio, digestibility with restriction enzymes, and suitability for Restriction Fragment Length Polymorphism (RFLP)- and Polymerase Chain Reaction (PCR)-based techniques.     

Rapid isolation of DNA from Dioscorea species suitable for PCR,restriction digestion and pathogen screening

The methods employed for DNA extraction from many plants is difficult because of the metabolites that interfere with DNA isolation procedures. We have developed a reliable and efficient method for isolating genomic DNA free from polysaccharide, polyphenols and protein contaminants from Dioscorea spp. The method involves inactivation of contaminant proteins by using CTAB/Proteinase K and precipitation of polysaccharides in the presence of high concentration of salt. The purity of genomic DNA was confirmed by A260/280 and A260/230 ratios calculated from the spectrophotometric readings and further by restriction analysis of the isolated DNA using restriction enzymes Eco RI. The total genomic DNA extracted by the new protocol was used for polymerase chain reaction amplification, RAPD analysis, restriction digestion and pathogen screening. The new protocol can be successfully used for both small- and large-scale preparation of genomic DNA from different tissues of Dioscorea spp. The quarantine of seed tubers and use of pathogen-free tubers for planting is a prerequisite for integrated disease management strategy. The protocol can be used for the isolation of genomic DNA from other crop plants too.     

An improved method to extract DNA from mango Mangifera indica

High quality genomic DNA is the first step in the development of DNA-based markers for fingerprinting and genetic diversity of crops, including mango (Mangifera indica L.), a woody perennial. Poor quality genomic DNA hinders the successful application of analytical DNA-based tools. Standard protocols for DNA extraction are not suitable for mango since the extracted genomic DNA often contains secondary metabolites that interfere with analytical applications. In this study, we employed an additional step to remove polysaccharides, polyphenols and secondary metabolites from genomic DNA extracted from young or mature leaf tissue; then a modified traditional cetyl trimethyl ammonium bromide (CTAB) method was applied. The use of 0.4 M glucose improved DNA quality and avoided contamination and browning by polyphenolics, relative to the traditional CTAB method. This is an easy and efficient method for genomic DNA extraction from both young and mature leaves of mango. The isolated DNA was free of polysaccharides, polyphenols, RNA and other major contaminants, as judged by its clear colour, its viscosity, A²⁶⁰/A²⁸⁰ ratio and suitability for PCR-based reactions. This modified protocol was also used to extract high quality genomic DNA from other woody perennials, including walnut, guava, lychee, pear, grape and sugarcane.     

DNA isolation protocol for the medicinal plant lemon balm (Melissa officinalis, Lamiaceae)

Lemon balm (Melissa officinalis) is a medicinal plant that is widely used as a sedative or calmant, spasmolytic and antibacterial agent and sleep aid. This has led to a high demand for lemon balm products, resulting in the extinction of this species in some of its natural habitats. Molecular techniques have increasingly been used in plant diversity conservation and isolation of PCR amplifiable genomic DNA is an important pre-requisite. Lemon balm contains high levels of polyphenols and polysaccharides, which pose a major challenge for the isolation of high-quality DNA. We compared different genomic DNA extraction protocols, including traditional phenol-chloroform DNA extraction protocols and two commercial kits for DNA purification for their ability to produce good-quality DNA from fresh leaves of five lemon balm genotypes. Quality and quantity of the DNA samples were determined using 0.8% agarose gel electrophoresis and a spectrophotometer. The DNA purity was further confirmed by PCR amplification using barley retrotransposon LTR base primers. The spectral quality of DNA as measured by the A(260)/A(280) ratio ranged from 1.46 to 2.37. The Fermentase genomic DNA purification kit and the CTAB extraction protocol using PVP and ammonium acetate to overcome the high levels of polyphenols and polysaccharides yielded high-quality DNA with a mean A(260)/A(280) ratio of 1.87. The quantity of DNA and its PCR purity were similar with all the protocols, but considering the time and cost required for extraction of DNA from a large number of samples, the CTAB protocol using PVP and ammonium acetate is suitable for lemon balm.     

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.     

Genomic DNA extraction from<Emphasis Type="Italic">Victoria amazonica</Emphasis>

DNA extraction is difficult in many plants because of metabolites that interfere with DNA isolation procedures and subsequent applications such as DNA restriction, amplification, and cloning. We developed the first reliable and efficient method for isolatingVictoria amazonica genomic DNA that is free from polysaccharides and polyphenols. This protocol uses 1.5 M NaCl, 2% polyvinylpyrrolidone (PVP) (Mr 1000), 5% mercaptoethanol, 0.12% sodium sulfite, and an incubation at 65°C for 4 h. The purity of isolated genomic DNA was confirmed by means of high-performance liquid chromatography (HPLC) profile and spectrophotometric analyses (A_260/230 ratio of 1.836, A_260/280 of 1.842). DNA was obtained in the amount of 387 μg per gram of leaf material, and it proved amenable to restriction digestion.     

A Simple and Efficient DNA Isolation Method for Salvia officinalis

We report an efficient, simple, and cost-effective protocol for the isolation of genomic DNA from an aromatic medicinal plant, common sage (Salvia officinalis L.). Our modification of the standard CTAB protocol includes two polyphenol adsorbents (PVP 10 and activated charcoal), high NaCl concentrations (4?M) for removing polysaccharides, and repeated Sevag treatment to remove proteins and other carbohydrate contaminants. The mean DNA yield obtained with our Protocol 2 was 330.6?μg DNA g^?1 of dry leaf tissue, and the absorbance ratios 260/280 and 260/230?nm averaged 1.909 and 1.894, respectively, revealing lack of contamination. PCR amplifications of one nuclear (26S rDNA) and one chloroplast (rps16-trnK) locus indicated that our DNA isolation protocol may be used in common sage and other aromatic and medicinal plants containing essential oil for molecular biologic and biotechnological studies and for population genetics, phylogeographic, and conservation surveys in which nuclear or chloroplast genomes would be studied in large numbers of individuals.     

改良CTAB法用于多年生植物组织基因组DNA的大量提取*

根据多年生植物组织富含多酚、多糖的具体特性,对现有的DNA提取方法进行了改进。通过增加提取缓冲液中b-巯基乙醇用量,简化氯仿/异戊醇抽提液步骤,改用经-20℃预冷异丙醇沉淀DNA等,对CTAB法加以改进。改进后方法具有以下优点：（1）获得的DNA质量良好,提取过程无明显的DNA降解,基本上排除了多酚物质的干扰;（2）用获得的DNA进行Southern杂交,可得到理想的杂交信号,可满足相关的分子研究要求;（3）操作简便。Abstract It is a difficult problem to isolate high quality DNA from plants containing a high contents of polyphenolics and polysaccharose, such as Actinidia plant. The protocol described in this paper is a modified CTAB (hexadecyltrimethylammonium bromide) method. High quality genomic DNA can be isolated from Actinidia plant using the improved method. The DNA is good enough for Southern blot and other uses in DNA research. The protocol is also efficient for quick and macro-DNA extraction.     

An improved method for isolating high-quality DNA from<Emphasis Type="Italic">Vitis vinifera</Emphasis> nuclei

We have developed a simple and highly efficient protocol for isolating large quantities (150–400 μg/g leaf tissue) of high-quality DNA from fresh and frozenVitis vinifera leaves. Isolated DNA is essentially free of polysaccharides, polyphenols, and other major contaminants as judged by viscosity, clear color, A_260/280 ratio, digestibility by restriction enzymes for Southern blot analysis, and PCR suitability.     

A simple and efficient protocol for isolation of high quality functional RNA from different tissues of turmeric (Curcuma longa L.)

Many experiments in plant molecular biology require processing of a large number of RNA samples and in some cases large quantities are required for a single application. In turmeric, a major spice and medicinal plant, a protocol for RNA isolation is not available. The major difficulty encountered while using other popular protocols is the low yield and quality of RNA which hampers the downstream applications like qRT-PCR, cDNA synthesis and micro RNA isolation. Commercial kits though available are costly and were found to be unsuccessful in case of rhizomes and root tissues that are rich in polyphenols, polysaccharides and alkaloids. It was thus felt that a quick, handy and cheap protocol of total RNA isolation from different tissues of turmeric was required for day to day working in our lab. The new protocol utilizes SDS based extraction buffer including β-mercaptoethanol and PVP with sequential acid phenol:chloroform extraction to remove polyphenols and proteins, followed by the purification with sodium acetate to eliminate polysaccharides. The protocol is simple and can be completed in less than 3 h. The RNA yield from rhizome was higher by more than fivefold with both A_260/280 and A_260/230 ratio in the range of 1.8–2.0. The protocol worked well with leaf, rhizome, pseudostem and root tissues with RIN >7.0 and the isolated RNA could be successfully used for cDNA synthesis, RT-PCR, qRT-PCR and small RNA isolation including microRNA.     

堆肥中微生物总DNA的高效提取

采用化学裂解和酶解相结合的方法,选择加入PVPP的高盐缓冲液作为细胞裂解的反应体系,并以PEG-8000进行DNA沉淀,从高有机含量的堆肥样品中进行微生物总DNA的提取。结果表明,从4种性质不同的堆肥中均获得了高质量的微生物总DNA,所得的DNA分子片段在23kb左右;每克干重堆肥的总DNA提取量为63.54±12.08μg~106.50±28.36μg,A260/A280大于1.6,A260/A230大于1.8,不用经过纯化可以直接进行PCR扩增和限制性酶切;以该DNA为模板进行微生物区系的DGGE分析,显示了丰富的微生物多样性。该方法减少了通常环境样品DNA提取过程中的纯化步骤,减少了DNA的损失,为从事微生物分子生态学,尤其是那些针对高有机含量以及获取极为不易的环境样品的研究而言是十分有益的。     

一种高效快速的鱼类标本基因组DNA提取方法

以95%酒精保存的黄鳝(M onopterus albus)和斑鳢(Channa maculates)标本为材料,采用先沉降DNA再去除杂质的方法从鱼类标本中提取基因组DNA。基因组DNA的琼脂糖凝胶电泳和紫外分光光度法检测以及PCR扩增结果显示,本方法提取的鱼类基因组DNA的电泳主带清晰明亮;A260/A280值在1.7830-2.0144之间;PCR扩增产物条带清晰明亮,且单一整齐没有拖带,表明本方法可从酒精保存的鱼类标本中提取比较纯净的DNA,能够满足一般分子生物学试验需要。与传统苯酚/氯仿法相比,本方法操作简单快速,避免了苯酚等物质对后续实验的影响,可作为一种常规动物组织DNA提取方法。     

一种适用于PCR反应的酵母菌、无绿藻及丝状真菌DNA提取方法

目的 介绍一种从酵母、无绿藻及丝状真菌中提取DNA以用于PCR反应的方法。方法 所用菌种包括临床分离的未知菌株和保藏菌株共23株：未知酵母菌（5株）、真皮毛孢子菌（1株）、糠秕马拉色菌（1株）、季也蒙念珠菌（5株）、未知丝状真菌（6株）、无绿藻（1株）、烟曲霉（2株）、拟青霉菌（1株）、茎点霉（1株）。用溶细胞酶（lyticase）结合Biospin真菌基因组DNA提取试剂盒提取基因组DNA,A260／A280检测纯度并计算质量浓度,用真菌通用引物ITS1／ITS4扩增真菌核糖体基因（rDNA）内转录间区ITS基因,经PCR扩增检验所提取的DNA质量。结果 成功提取所有23株真菌基因组DNA,其纯度及质量浓度能满足PCR反应的要求。结论 用溶细胞酶结合Biospin真菌基因组DNA提取试剂盒从酵母菌、无绿藻及丝状真菌提取的DNA可用于PCR反应。     

Isolation of milligram quantities of nuclear DNA from tomato (Lycopersicon esculentum), A plant containing high levels of polyphenolic compounds

We have developed a protocol for isolating milligram quantities of highly purified DNA from tomato nuclei. The protocol utilizes fresh seedlings or leaves without freezing. Tissues are treated with ethyl ether, thoroughly washed, and placed in a buffer containing the nuclear-stabilizing agent 2-methyl-1,4-pentanediol. Nuclei are liberated from tomato cells by homogenization in a Waring blender. The interaction of nuclear DNA with oxidized polyphenols is inhibited by compounds that adsorb polyphenols or prevent oxidation reactions. Chloroplasts and mitochondria are preferentially eliminated with Triton X-100. Nuclei are concentrated using a Percoll gradient and lysed with SDS. DNA is subsequently purified by RNase and protease digestions and phenol/chloroform extractions. The isolated DNA is essentially free of polyphenols and other major contaminants based upon its lack of coloration, A₂₆₀/A₂₈₀ ratio, digestibility with restriction enzymes, melting profile, and reassociation properties.     

Isolation of genomic DNA from medicinal plants without liquid nitrogen

Genomic DNA was extracted from eight medicinal plants using the present DNA extraction protocols (CTAB extraction method) with some modifications. Leaves were fixed in different fixing solutions containing absolute alcohol (99.99%), chloroform and EDTA, but without liquid nitrogen. DNA quality and quantity obtained were comparable to those isolated with liquid nitrogen, as the lambda260/lambda280 ratio with liquid nitrogen was in range 1.3-1.7 and with other fixing solutions it was 1.1-1.5. Absolute alcohol showed best results as fixing solution. Good quality of DNA was isolated without using liquid nitrogen from different medicinal plant species. DNA isolated by this method was suitable for various molecular biology applications.     

胡椒叶片基因组DNA提取方法比较

目的：研究建立胡椒叶片中提取高质量DNA的方法。方法：采用各种CTAB法和SDS法的改良方法,提取胡椒叶片中的总DNA,并对DNA进行紫外和电泳检测。结果：改良CTAB法4和5提取的DNA经电泳检测,有一条明亮主带,且无拖尾现象,样品槽无荧光出现,说明抽出的DNA纯度较高,一致性好;测定其OD260和OD280值,并计算其比值,OD260/OD280值在1.8-2.0之间,提取率在51.667-60.000μg/g之间,获得的基因组DNA质量高。结论：改良CTAB法4和法5可从胡椒幼叶中提取高质量DNA。     

A simple protocol for isolating genomic DNA from chestnut rose (<Emphasis Type="Italic">Rosa roxburghii</Emphasis> tratt) for RFLP and PCR analyses

Isolation of high-quality DNA from rosaceous species is particularly difficult because of their high levels of polyphenols, polysaccharides, and other compounds. The yields and quality of genomic DNA are considerably affected when the common protocol for DNA isolation is applied to the chestnut rose (Rosa roxburghii Tratt). A simple, rapid, and efficient protocol for the extraction of DNA from the chestnut rose is described. The modified hexadecyltrimethylammonium bromide (CTAB) procedure, which uses phenol-absent extraction to enhance the yield, involves a washing step before extraction for the removal of organic molecules and excessive water; the use of high concentrations of polyvinylpyrrolidone (2% [w/v]), CTAB (3% [w/v]), and β-mercaptoethanol (3% [v/v]) in the high-salt-concentration extraction buffer to remove polyphenols and polysaccharides; and the combined use of potassium acetate and chloroform to remove proteins and polysaccharides. Finally, DNA is precipitated with an equal volume of isopropanol and 0.1 vol of sodium acetate. This protocol results in high yields of DNA. The average yield of DNA ranged from 980–1800 μg/g of fresh weight of leaves. Downstream results indicate that DNA quality is sufficient for restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR) analyses.