共查询到20条相似文献,搜索用时 31 毫秒
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Mithun Raj Vishnu S. Nath M. Senthil @ Sankar M.L. Jeeva 《Archives Of Phytopathology And Plant Protection》2013,46(6):753-760
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. 相似文献
3.
Takemon Y Yamamoto A Nakashima M Tanida K Kishi M Kato M 《Molecular biology reports》2006,33(1):65-70
We describe here a simple and efficient protocol for genomic DNA isolation from adult males of insects: e.g., Ephemeroptera,
Odonata, Orthoptera and Dictyoptera. To minimize contamination of external DNA source, the sperm vesicles were isolated from
male individuals from which high molecular weight genomic DNA was extracted. According to this protocol, the genomic DNA samples
obtained were high quality (intact), and abundant enough for genotyping analyses and molecular cloning. The protocol reported
here enables us to process a huge number of individuals at a time with escaping from cross-contamination, and thus it is quite
useful for conducting genetic studies at least in some species of insects. The large yield of high molecular weight DNA from
single individual may be advantageous for non PCR-based experiments. As a case study of the protocol, partial coding sequences
of histone H3 and EF-1α genes are determined for some insects with PCR-amplified DNA fragments. 相似文献
4.
改良CTAB法用于多年生植物组织基因组DNA的大量提取* 总被引:49,自引:0,他引:49
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. 相似文献
5.
A rapid and hazardous reagent free protocol for genomic DNA extraction suitable for genetic studies in plants 总被引:2,自引:0,他引:2
Protocols for genomic DNA extraction from plants are generally lengthily, since they require that tissues be ground in liquid
nitrogen, followed by a precipitation step, washing and drying of the DNA pellet, etc. This represents a major challenge especially
when several hundred samples must be screened/analyzed within a working day. There is therefore a need for a rapid and simple
procedure, which will produce DNA quality suitable for various analyses. Here, we describe a time and cost efficient protocol
for genomic DNA isolation from plants suitable for all routine genetic screening/analyses. The protocol is free from hazardous
reagents and therefore safe to be executed by non-specialists. With this protocol more than 100 genomic DNA samples could
manually be extracted within a working day, making it a promising alternative in genetic studies of large-scale genomic screening
projects. 相似文献
6.
We describe a simple and efficient method for genomic DNA extraction from woody fruit crops containing high polysaccharide
levels. This method involves a modified CTAB or SDS procedure employing a purification step to remove polysaccharides by using
water-saturated ether and 1.25 M NaCl. Precipitation with an equal volume of isopropanol caused a DNA pellet to form. After
being washed with 70% ethyl alcohol, the pellet easily dissolved in TE buffer. Using this method, DNA was extracted from samples
of more than 1000Citrus spp., including young leaves, old leaves, frosted old leaves, withered old leaves, and callus lines. The average yield of
DNA ranged from 50–500 μg/g of sample. DNA was suitable for PCR and RFLP analyses and long-term storage. Recently, the procedure
was used to isolate DNA from withered old leaves of more than 20 tropical and subtropical fruit crops. 相似文献
7.
Mohammad S. Uddin Wenli Sun Xinhua He Jaime A. Teixeira da Silva Qi Cheng 《Biologia》2014,69(2):133-138
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. 相似文献
8.
Akshara George M. L. Jeeva Vishnu S. Nath G. L. Sreelatha M. G. Sujina 《Archives Of Phytopathology And Plant Protection》2018,51(5-6):241-251
Genomic DNA extraction protocol with relatively high quantity and purity is prerequisite for the successful molecular identification and characterisation of plant pathogens. Conventional DNA extraction methods are often time-consuming and yield only very poor quantity of genomic DNA for samples with higher mycelial age. In our laboratory, we have aimed at establishing an efficient DNA isolation procedure, exclusively for the oomycete pathogen Phytophthora colocasiae causing serious leaf blight disease in taro. For this a phenol free protocol was adopted, which involves SDS/Proteinase K-based inactivation of protein contaminants, extraction of nucleic acids using chloroform: isoamyl alcohol and later precipitation of genomic DNA using isopropanol and sodium acetate. The purity of the isolated DNA was analysed by A260/280 and A260/230 spectrophotometric readings and confirmed by restriction digestion with restriction enzyme Eco RI. In this study, a comparative assessment was done with CTAB method and the commercial genomic DNA purification kit (Thermo Fisher Scientific, Fermentas, EU). The extracted DNA was found to be suitable for further downstream applications like ITS amplification of the rDNA ITS region and PCR amplification with species-specific primers. 相似文献
9.
Improved high-throughput sunflower and cotton genomic DNA extraction and PCR fidelity 总被引:1,自引:0,他引:1
The extraction of high-quality genomic DNA for PCR amplification from sunflower (Helianthus annuus) and cotton (Gossypium spp.) is challenging because of the presence of polysaccharides, secondary metabolites, and polyphenolics in the tissues.
A high-throughput DNA extraction protocol was needed in our laboratory for simple sequence repeats (SSR)-marker screening
and other molecular analyses that do not require organic extraction steps of phenol or chloroform. Here we describe 2 improved
highthroughput protocols for DNA extraction and in-PCR modification that result in successful PCR amplification of sunflower
and cotton. While the sunflower DNA extraction protocol uses reducing agents such as sodium metabisulfite and dithiothreitol
(DTT), the cotton protocol uses polyvinylpyrrolidone (PVP) in PCR reactions and reducing agents in the DNA extraction procedure. 相似文献
10.
Extraction of plant genomic DNA for subsequent genetic studies is often lengthy and difficult. In addition, it requires the
use of toxic reagents that remove secondary plant products, which otherwise interfere with the polymerase chain reaction steps.
Here we describe a simple cost-efficient one-step protocol for PCR-based analysis in Arabidopsis thaliana. This protocol is quick, performed at room temperature without the need for DNA extraction. Potential applications in higher
plants are here discussed. 相似文献
11.
Genomic DNA of high quality and quantity is needed to analyze genetic diversity with AFLP.Carpobrotus plant species, like most succulents, contain high amounts of polysaccharides and polyphenols, making PCR amplification difficult.
Our protocol eliminates contaminants before DNA isolation by using leaf callus as plant material. This simple and inexpensive
technique gives an average DNA yield of 1800 ng/g of callus and high reproducible profiles in AFLP. Our results indicate that
no genetic variability is associated with callus culture conditions. This technique is suitable for studying genomic polymorphism
in succulents and other plants when classic DNA extraction procedures fail. 相似文献
12.
José A. Mercado Iman El Mansouri Silvia Jiménez-Bermúdez Fernando Pliego-Alfaro Miguel A. Quesada 《In vitro cellular & developmental biology. Plant》1999,35(2):152-153
Summary In this paper we describe a simple and efficient DNA extraction protocol for Fragaria species, a highly recalcitrant genus due to the large amount of polyphenols and polymeric carbohydrates present in strawberry
tissues. The protocol yields a high quality DNA that can be amplified by polymerase chain reaction and digested with restriction
endonucleases. 相似文献
13.
High-quality plant DNA extraction for PCR: an easy approach 总被引:1,自引:0,他引:1
I. Ahmed M. Islam W. Arshad A. Mannan W. Ahmad B. Mirza 《Journal of applied genetics》2009,50(2):105-107
Polymerase chain reaction has found wide applications in modern research involving transformations and other genomic studies.
For reproducible PCR results, however, the quantity and quality of template DNA is of considerable importance. A simple and
efficient plant DNA extraction procedure for isolation of high-quality DNA from plant tissues is presented here. It requires
maceration of plant tissue of about 1.0 cm2 (e.g. of a leaf blade) in DNA extraction buffer (100 mM Tris-HCl, 100 mM EDTA, 250 mM NaCl) using 1.5-mL microfuge tubes,
followed by cell lysis with 20% SDS, and DNA extraction with phenol: chloroform: iso-amyl alcohol (25:24:1). Hydrated ether
is then used to remove polysaccharides and other contaminants from the DNA preparation. Average DNA yield is 20–30 μg cm−2 for fresh tissues, and ratio of absorbance at 260 nm to absorbance at 280 nm is 1.5–1.8. The DNA is quite suitable for PCR
using microsatellites, RAPD and specific markers for recombinant selection. Amplifications have been obtained for these markers
by using template DNA extracted from fresh as well as frozen leaf tissues of various plants, including barley, oat, potato
and tomato. DNA stored for more than 2 years has been successfully amplified with microsatellite markers, which shows suitability
of this method after long-term storage of DNA. Besides, the ease of use and cost-effectiveness make the procedure attractive. 相似文献
14.
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. 相似文献
15.
Echevarría-Machado I Sánchez-Cach LA Hernández-Zepeda C Rivera-Madrid R Moreno-Valenzuela OA 《Molecular biotechnology》2005,31(2):129-135
A protocol is described for rapid DNA isolation from Malvaceae plant species and different tissues of Bixaceae that contain
large amounts of polysaccharides, polyphenols, and pigments that interfere with DNA extractions. The method is a modification
of Dellaporta et al. The current protocol is simple, and no phenolchloroform extraction, ethanol, or isopropranol precipitation
is required. The method is based in the incubation of soluble DNA with silica, mix in batch during the extraction. The procedure
can be completed in 2 h and many samples can be processed at the same time. DNA of excellent quality was recovered and used
for polymerase chain reaction (PCR) amplification, restriction enzyme digestion, and Southern blot analysis. The method was
used with healthy Bixa orellana and virus-infected Malvaceae plants. 相似文献
16.
A DNA extraction procedure that does not require hazardous materials, such as CTAB, phenols, or liquid nitrogen, was optimized
forAnthurium andreanum, a plant rich in polysaccharides and polyphenolics. Three DNA isolation techniques were tested. The modified Rowhani protocol
(1983), with slight modifications, was found to yield up to milligrams of DNA suitable for RAPD from spathe and leaf tissues.
High-quality DNA was obtained readily from spathe tissues, while a spermine precipitation step was found to be essential when
DNA was extracted from the leaf tissues. 相似文献
17.
A simple method for DNA extraction from marine bacteria that produce extracellular materials 总被引:9,自引:0,他引:9
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. 相似文献
18.
肠道微生物群落结构和多样性与人体疾病密切相关。然而,相关群落结构分析结果可能受到DNA提取质量等实验因素影响。因此,评估不同DNA提取方法对肠道特定种属的提取效果,对于全面、准确获取人体肠道微生物谱,深入探究肠道微生物群落结构具有指导意义。本研究旨在借助实时荧光定量PCR(real-time quantitative polymerase chain reaction,RT qPCR)技术,以DNA提取纯度、浓度,以及对肠道中特定种属微生物基因组DNA的提取丰度为指标,对5种DNA提取方法进行比较分析。结果表明,试剂盒Q的提取效果最佳,特别是对乳杆菌属和双歧杆菌属等革兰氏阳性菌的提取效果较好。N试剂盒的平均DNA提取浓度较Q试剂盒低,但在纯度方面,二者无显著性差异。与其他3种商用试剂盒(M、PSP、TG)相比,N方法对肠道内指定微生物基因组的提取效果仅次于Q试剂盒,位居第二。相比之下,M试剂盒提取所得DNA,质量较高,但浓度偏低,对于肠道内革兰氏阳性菌的提取效果不很理想。TG试剂盒和PSP试剂盒提取所得DNA在浓度、质量以及细菌丰度方面均不及其他验证的试剂盒。综上,Q试剂盒可作为肠道微生态研究相关实验中获取高质量基因组DNA的提取方法。本研究结果为肠道微生态研究相关实验中基因组DNA提取方法的选择提供参考依据。 相似文献
19.
In recent years, animal ethics issues have led researchers to explore nondestructive methods to access materials for genetic studies. Cicada exuviae are among those materials because they are cast skins that individuals left after molt and are easily collected. In this study, we aim to identify the most efficient extraction method to obtain high quantity and quality of DNA from cicada exuviae. We compared relative DNA yield and purity of six extraction protocols, including both manual protocols and available commercial kits, extracting from four different exoskeleton parts. Furthermore, amplification and sequencing of genomic DNA were evaluated in terms of availability of sequencing sequence at the expected genomic size. Both the choice of protocol and exuvia part significantly affected DNA yield and purity. Only samples that were extracted using the PowerSoil DNA Isolation kit generated gel bands of expected size as well as successful sequencing results. The failed attempts to extract DNA using other protocols could be partially explained by a low DNA yield from cicada exuviae and partly by contamination with humic acids that exist in the soil where cicada nymphs reside before emergence, as shown by spectroscopic measurements. Genomic DNA extracted from cicada exuviae could provide valuable information for species identification, allowing the investigation of genetic diversity across consecutive broods, or spatiotemporal variation among various populations. Consequently, we hope to provide a simple method to acquire pure genomic DNA applicable for multiple research purposes. 相似文献
20.
Shumi Yoshida-Yamamoto Sayaka Nishimura Teruko Okuno Miki Rakuman Yukio Takii 《Molecular biotechnology》2010,46(1):41-48
Owing to the increasing importance of genomic information, obtaining genomic DNA easily from biological specimens has become
more and more important. This article proposes an efficient method for obtaining genomic DNA from nail clippings. Nail clippings
can be easily obtained, are thermostable and easy to transport, and have low infectivity. The drawback of their use, however,
has been the difficulty of extracting genomic material from them. We have overcome this obstacle using the protease solution
obtained from Cucumis melo. The keratinolytic activity of the protease solution was 1.78-fold higher than that of proteinase K, which is commonly used
to degrade keratin. With the protease solution, three times more DNA was extracted than when proteinase K was used. In order
to verify the integrity of the extracted DNA, genotype analysis on 170 subjects was performed by both PCR–RFLP and Real Time
PCR. The results of the genotyping showed that the extracted DNA was suitable for genotyping analysis. In conclusion, we have
developed an efficient extraction method for using nail clippings as a genome source and a research tool in molecular epidemiology,
medical diagnostics, and forensic science. 相似文献