木根麦冬（Ophiopogon xylorrhizus）干叶提取DNA用于RAPD分析

木根麦冬（Ｏｐｈｉｏｐｏｇｏｎｘｙｌｏｒｒｈｉｚｕｓ）是我国珍稀濒危植物,分布仅限于云南西双版纳雨林,在植物系统学和保护生物学研究中具有独特的意义。随机扩增多态ＤＮＡ（ＲＡＰＤ）方法是揭示群体遗传多样性的高效、简便方法,但一般均以新鲜材料提取总ＤＮＡ,对一些分布边远地区物种难以采用此法。本文研究从木根麦冬干叶片中提取总ＤＮＡ,进行ＲＡＰＤ分析。样品取自４个居群、４９个个体。选取生长旺盛的叶片,在野外用硅胶快速干燥保存样品。采用高盐低ｐＨ值法提取总ＤＮＡ,每克鲜重所得的干叶可得８０～１６０μｇ。通过对模板ＤＮＡ的各种处理和ＰＣＲ扩增程序的调整,解决了扩增片段边缘弥散、界线模糊、产率低等问题,获得了理想的扩增带型。这一成果对其它从野外直接采样的干叶提取ＤＮＡ进行ＲＡＰＤ研究具有指导意义     

高效的植物DNA提取方法

利用液氮研磨植物幼芽,以苯酚─氯仿─异戊醇─核糖核酸酶法提取了大豆、菜豆、玉米、高梁等几种农作物的总ＤＮＡ。所提取的ＤＮＡ经岛津ＵＶ－２６５紫外分光光度计检测及０．６％的琼脂糖凝胶电泳,结果证明：该方法所提取的植物总ＤＮＡ纯度高,片段长度整齐,约５０ｋｂ左右,符合外源ＤＮＡ导入作物的要求,并且ＤＮＡ收率很高。     

一种提取果树叶片DNA的简便方法

植物ＤＮＡ的提取是分子遗传学及遗传工程的根本前提。通常ＤＮＡ提取方法应满足以下几个主要条件：（１）所得ＤＮＡ具备理想的纯度,如ＲＦＬＰ技术对ＤＮＡ纯度要求高,因为纯度高的ＤＮＡ便于酶切及膜转移等操作,而对于涉及ＰＣＲ扩增的技术,ＤＮＡ提取则尤为注意样...     

一步法提取植物DNA用于大规模RAPD分析

ＲＡＰＤ技术已广泛地用于植物的系统演化、种群多样性、群体遗传学及杂种种子纯度的鉴定等工作中〔１～３〕。本文基于快速节省的原则对碱液法提取植物ＤＮＡ进行一些改进。１材料与方法ＤＮＡ提取方法取１０～２０ｍｇ幼叶 ,加１００μｌ０ ５ｍｏｌ／ＬＮａＯＨ（或附加０ ５ %～１ ５ %巯基乙醇）研磨后 ,取４０μｌ研磨液加入２００μｌ１００ｍｍｏｌ／ＬＴｒｉｓ ＨＣｌｐＨ７ ６缓冲液（或附加０ ５%不溶性聚乙烯吡咯烷酮 ,ＰＶＰ）中 ,８０００×ｇ离心５ｍｉｎ,上清液即可用于ＰＣＲ反应。ＰＣＲ反应体系含１０ｍｍｏｌ／ＬＴｒｉ…     

离子交换树脂法与酚仿法对质粒DNA提取效果的比较

通过两种不同方法对质粒ＤＮＡ提取效果的比较,证实了阴离子交换树脂法能够获得高纯度的质粒ＤＮＡ,满足分子生物学的试验要求,操作简便、快速,不污染实验室的环境,是一种较理想的制备高纯度质粒ＤＮＡ的提取方法。     

水稻线粒体DNA的提取与分析

为了研究水稻细胞质雄性不育的分子基础,我们比较了各种提取线粒体ＤＮＡ（ｍｔＤＮＡ）的方法,并提出了一些改进措施。以丛广４１Ａ、丛广４１Ｂ和杂种一代广优青为材料,对所提取的材料ｍｔＤ－ＮＡ进行了紫外扫描、ＯＤ值测定、电泳、酶切等分析,结果表明,以新鲜材料进行不连续蔗糖密度梯度超速离心对提取高纯度的线粒体ＤＮＡ效果较好。     

香蕉不同组织中总RNA的有效分离

从植物组织中提取总ＲＮＡ是进行植物分子生物学某些方面研究的必要前提。如进行Ｎｏｒｔｈｅｍ杂交分析,纯化ｍＲＮＡ以用于体外翻译或建立ｃＤＮＡ文库,ＲＴ－ＰＣＲ及差示分析等分子生物学实验,都需要高质量的总ＲＮＡ。因此,从植物组织中提取高质量的总ＲＮＡ是顺利进行上述研究的关键所在。目前常用于植物材料ＲＮＡ提取的方法有脱法［１～４］苯酚法［５,６］和ＣＴＡＢ［７,８］法等。这些方法对于提取某些植物的总ＲＮＡ是有效的,但对另外一些植物来说,却并不一定是好的方法。因为不同植物组织的细胞内外成分复杂多变,在使…     

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     

利用微波炉快速制备酵母质粒和基因组DNA PCR模板

制备基因组ＤＮＡＰＣＲ模板一般都比较费工费时 ,研究人员尝试用微波炉提取真核生物基因组ＤＮＡ ,然后做ＰＣＲ扩增取得了较好的效果[1 ,2 ] 。本文描述了一个利用微波炉快速制备酵母质粒和基因组ＤＮＡＰＣＲ模板的程序 ,具有良好的重复性。1　材料与方法1.1　材料酿酒酵母菌种 1ｃ163 6ｄａｒｇ11,ｕｒａ 3为比利时ＯｒｉａｎｅＳｏｅｔｅｎｓ馈赠。酵母载体ｐＹＸ13 3从美国康乃尔大学曹维国处获得 ,并由此构建了粗糙脉孢霉ａｒｇ 13ｃＤＮＡ的酵母表达载体ｐＹＸＡ5。培养基为ＳＤ或ＹＰＤ固体培养基。1.2　酵母质粒提取[3]取 1.5ｍｌ…     

五种提取马尾松基因组DNA方法的比较

对于含有大量多糖如酚、酯、萜等其它二次代谢产物的松科和杉科等针叶植物,要从其组织中提取高质量的基因组ＤＮＡ一般都比较困难。本文以马尾松（ｐｉｎｕｓ ｍａｓｓｏｎｉａｎａ）针叶为材料,分别采取了简易提取法、高盐沉淀法、ＣＴＡＢ沉淀法、Ｚｉｅｇｅｎｈａｇｅｎ法和ＱＬＡＧＥＮ公司ＤＮｅａｓｙ Ｐｌａｎｔ Ｍｉｎｉ Ｋｉｔｓ５种方法提取基因组ＤＮＡ;并通过琼脂糖凝胶电泳、限制性内切酶自理和ＲＡＰＤ３种方法对     

A Rapid DNA Minipreparation Method Suitable for AFLP and Other PCR Applications

A rapid DNA minipreparation method was developed for rice and other plant species. This method uses an Eppendorf tube and 1-ml pipette tip to grind plant tissues, and requires only one transfer for DNA isolation. In a single day, one person can complete DNA isolation from more than 120 leaf samples. The yields of the DNA samples ranged from 2.3 to 5.2 g from 25–50 mg fresh leaf tissue. DNA samples extracted using this method from rice were completely digested with five restriction enzymes (EcoR I, EcoR V, Hind III, Mse I and Pst I) and were successfully used for AFLP and other PCR applications.     

High-resolution flow cytometry of nuclear DNA in higher plants

Summary High-resolution flow cytometry of nuclear DNA in higher plants has been performed from chopped plant tissues and plant protoplasts. A preparation and staining procedure with the DNA specific fluorochrome DAPI, successfully employed for precise flow cytometric DNA analysis of animal and human cells has been used in a slightly modified manner for the DNA analysis of plant cell material. High-resolution DNA histograms coefficients of variation about 1–1.5% have been obtained routinely from plant species with different DNA content. Staining of nuclei with DAPI in combination with the protein fluorochrome sulforhodamine 101 allows bi-parametric analysis of nuclear DNA and protein. The described simple and precise method might be very promising for the analysis of DNA in basic and applied cytogenetic investigations of plant cell research.Abbreviations CV coefficient of variation - DAPI 4,6-diamidino-2-phenylindole - SR 101 sulforhodamine 101     

Isolation of Pinus radiata Genomic DNA Suitable for RAPD Analysis

A protocol is presented for Pinus radiata genomic DNA isolation based on an alkyltrimethyl-ammonium bromide (CTAB) method described for other woody species. The method involves mortar grinding of tissue, a modified CTAB extraction employing high salt concentrations and polyvinyl pyrrolidone, a RNase A treatment and successive isoamyl alco- hol-chloroform extractions. The yield was approximately 15 g DNA per 100 mg of initial fresh plant material. The genomic DNA obtained by this method was suitable to be used in simple sequence repeat and random-amplified-polymorphic DNA reactions. This extraction method would allow the molecular analysis of shoots from different clones within P. radiata families.     

An improved method of DNA purification from secondary metabolites rich medicinal plants using certain chaotropic agents

The presented work describes good quality DNA isolation method from mature leaves of some medicinally important plant species, viz. Asparagus racemosus, Withania somnifera, Abrus precatorius, Commiphora wightii and Carissa carandas. These plants hold immense medicinal values due to presence of certain secondary metabolites like polyphenols, terpenes, flavonoids, alkaloids, gums, resins, etc. Although these metabolites are accountable for important medicinal properties and authorize these plants to precedence over others, the same compounds disappoint the researcher while isolating high quality DNA. To overcome this problem, we propose a simple method in which DNA is adroitly bounded to diatomaceous earth in a solution of different chaotropic agent and alienated from intrusive compounds. Presented method affirms that secondary products, along with polysaccharides and proteins, can be perceptibly reduced by using silica matrix along with chaotropic agents. The described method is fast, simple and highly reliable for the isolation of DNA from obstinate plant species.     

Effective Extraction and Assembly Methods for Simultaneously Obtaining Plastid and Mitochondrial Genomes

Background

In conventional approaches to plastid and mitochondrial genome sequencing, the sequencing steps are performed separately; thus, plastid DNA (ptDNA) and mitochondrial DNA (mtDNA) should be prepared independently. However, it is difficult to extract pure ptDNA and mtDNA from plant tissue. Following the development of high-throughput sequencing technology, many researchers have attempted to obtain plastid genomes or mitochondrial genomes using high-throughput sequencing data from total DNA. Unfortunately, the huge datasets generated consume massive computing and storage resources and cost a great deal, and even more importantly, excessive pollution reads affect the accuracy of the assembly. Therefore, it is necessary to develop an effective method that can generate base sequences from plant tissue and that is suitable for all plant species. Here, we describe a highly effective, low-cost method for obtaining plastid and mitochondrial genomes simultaneously.

Results

First, we obtained high-quality DNA employing Partial Concentration Extraction. Second, we evaluated the purity of the DNA sample and determined the sequencing dataset size employing Vector Control Quantitative Analysis. Third, paired-end reads were obtained using a high-throughput sequencing platform. Fourth, we obtained scaffolds employing Two-step Assembly. Finally, we filled in gaps using specific methods and obtained complete plastid and mitochondrial genomes. To ensure the accuracy of plastid and mitochondrial genomes, we validated the assembly using PCR and Sanger sequencing. Using this method,we obtained complete plastid and mitochondrial genomes with lengths of 153,533 nt and 223,412 nt separately.

Conclusion

A simple method for extracting, evaluating, sequencing and assembling plastid and mitochondrial genomes was developed. This method has many advantages: it is timesaving, inexpensive and reproducible and produces high-quality sequence. Furthermore, this method can produce plastid and mitochondrial genomes simultaneously and be used for other plant species. Due to its simplicity and extensive applicability, this method will support research on plant cytoplasmic genomes.     

DNA extraction and PCR amplification method suitable for fresh, herbarium-stored, lichenized, and other fungi

This paper presents a DNA extraction method suitable for fresh, herbarium-stored, lichenized and other fungal specimens. The method is fast and reliable, comparatively inexpensive and is suitable for obtaining PCR amplification quality DNA from large numbers of samples in a short time. The method has been tested with over 300 samples ofAscochyta, Phyllosticta, Ramalina, Parmelia andPhysconia. Amplifiable fungal DNA was extracted from pure cultures, leaf lesions, whole thalli and dissected only-fungal sections of lichenized fungi. In addition, the method has proved suitable for use with herbarium specimens of both lichenized and non-lichenized fungi, stored as dried pure cultures or dried infected plant material.     

An efficient method for preparing high quality DNA from plant DNA libraries in lambda phage

An efficient method has been developed to improve preparation of phage particles by ammonium sulfate precipitation and to yield high quality DNA. The method, that has been used to screen plant DNA libraries constructed in vectors, is inexpensive, does not require purification of phage particles, and can be used from either plate stocks or liquid lysates. Up to 1100 g DNA was produced from 5 ml lysate obtained from agar plates.     

Recycling Isolation of Plant DNA,A Novel Method

DNA is one of the most basic and essential genetic materials in the field of molecular biology.To date,isolation of sufficient and good-quality DNA is still a challenge for many plant species,though various DNA extraction methods have been published.In the present paper,a recycling DNA extraction method was proposed.The key step of this method was that a single plant tissue sample was recycled for DNA extraction for up to four times,and correspondingly four DNA precipitations(termed as the 1st,2nd,3rd and 4th DNA sample, respectively) were conducted.This recycling step was integrated into the conventional CTAB DNA extraction method to establish a recycling CTAB method.This modified CTAB method was tested in eight plant species,wheat,sorghum,barley,corn,rice,Brachypodium distachyon,Miscanthus sinensis and tung tree.The results showed that high-yield and good-quality DNA samples could be obtained by using this new method in all the eight plant species.The DNA samples were good templates for PCR amplification of both ISSR and SSR markers.The recycling method can be used in multiple plant species and can be integrated with multiple conventional DNA isolation methods,and thus is an effective and universal DNA isolation method.     

一种改良的植物基因组DNA通用提取方法

高质量的基因组DNA是分子生物学研究的基础,而从富含糖类和次生代谢物且异质性强的植物材料中分离DNA相对困难。本方法在CTAB法和商业DNA提取试剂盒的基础上,在裂解细胞之前,对植物材料进行预处理．去除干扰DNA提取的代谢物,并在后续步骤中进行了一些优化。该方法适于多种不同的植物种类,所提取的基因组DNA质量较好,能满足下一步基因操作的要求,是一种通用的植物基因组DNA提取方法。     

Clustering of identical oligomers in coding and noncoding DNA sequences.

We develop a quantitative method for analyzing repetitions of identical short oligomers in coding and noncoding DNA sequences. We analyze sequences presently available in the GenBank separately for primate, mammal, vertebrate, rodent, invertebrate and plant taxonomic partitions. We find that some oligomers "cluster" more than they would if randomly distributed, while other oligomers "repel" each other. To quantify this degree of clustering, we define clustering measures. We find that (i) clustering significantly differs in coding and noncoding DNA; (ii) in most cases, monomers, dimers and tetramers cluster in noncoding DNA but appear to repel each other in coding DNA. (iii) The degree of clustering for different sources (primates, invertebrates, and plants) is more conserved among these sources in the case of coding DNA than in the case of noncoding DNA. (iv) In contrast to other oligomers, we find that trimers always prefer to cluster. (v) Clustering of each particular oligomer is conserved within the same organism.