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1.
A simple method of genomic DNA extraction suitable for analysis of bulk fungal strains 总被引:1,自引:0,他引:1
Aims: A simple and rapid method (designated thermolysis) for extracting genomic DNA from bulk fungal strains was described. Methods and Results: In the thermolysis method, a few mycelia or yeast cells were first rinsed with pure water to remove potential PCR inhibitors and then incubated in a lysis buffer at 85°C to break down cell walls and membranes. This method was used to extract genomic DNA from large numbers of fungal strains (more than 92 species, 35 genera of three phyla) isolated from different sections of natural Ophiocordyceps sinensis specimens. Regions of interest from high as well as single‐copy number genes were successfully amplified from the extracted DNA samples. The DNA samples obtained by this method can be stored at ?20°C for over 1 year. Conclusions: The method was effective, easy and fast and allowed batch DNA extraction from multiple fungal isolates. Significance and Impact of Study: Use of the thermolysis method will allow researchers to obtain DNA from fungi quickly for use in molecular assays. This method requires only minute quantities of starting material and is suitable for diverse fungal species. 相似文献
2.
采采用氧化硅超顺磁性纳米磁珠和自己设计的试剂体系及提取流程,建立了一种基因组DNA的快速提取方法,该方法以氧化硅磁珠为固相吸附载体,盐酸胍、 -巯基乙醇和SDS为主要裂解吸附试剂。以全血或培养细胞为实验材料进行基因组DNA的提取结果显示用本文建立的方法提取100 L小鼠抗凝血,可得2~3 g基因组DNA, OD260/OD280为1.8 ± 0.05,其纯度可满足后续的酶切和PCR生物操作要求。该方法整个提取过程只需12分钟,不需特殊实验条件同时可省略蛋白酶K的消化过程和离心操作,适用于一般实验室的需求,是一种操作简便、快速高效的提取方法。 相似文献
3.
A simple extraction method suitable for PCR-based analysis of plant,fungal, and bacterial DNA 总被引:1,自引:0,他引:1
George S. Mahuku 《Plant Molecular Biology Reporter》2004,22(1):71-81
A simple and easy protocol for extracting high-quality DNA from microorganisms and plants is presented. The method involves
inactivating proteins by using SDS/proteinase K and precipitating polysaccharides in the presence of high salt. Further purification
is based on differential solubility of DNA and high-molecular-weight polysaccharides in aqueous media. The procedure does
not use the toxic and potentially hazardous phenol and chloroform, and as many as 100 samples can be processed per day. Absorbency
ratios (A260/A280) of 1.6–2.0 indicated a minimal presence of contaminating metabolites. The DNA was completely digested with 5 restriction
enzymes:EcoR I,RsaI,TaqI,EcoR V, andHind III. PCR analysis using enterobacterial repetitive intergenic consensus (ERIC) sequence, sequence-characterized amplified
region (SCAR), and random amplified microsatellite (RAMS) primers showed the DNA's compatibility with downstream applications.
This procedure is applicable to a range of pathogens and plants and thus may find wide application in quarantine services
and marker-assisted selection (MAS) breeding. 相似文献
4.
A simple method of DNA extraction for Eimeria species 总被引:4,自引:0,他引:4
A new, simple method is described for extracting DNA from coccidia (Eimeriidae) oocysts. In our hands this method works well for all Eimeria oocysts and, presumably, will work equally well for oocysts of other coccidia genera. This method combines the two steps of breaking oocyst and sporocyst walls, and dissolving the sporozoite membrane in one step. This greatly simplifies the currently used DNA extraction procedures for Eimeria species and overcomes the disadvantages of existing DNA extraction methods based on glass-bead grinding and sporozoite excystation procedures. Because all the procedures are done in a 1.5-ml microfuge tube, which minimizes the loss of DNA in the extraction procedures, this method is especially suitable for samples with small number of oocysts. In addition, this method directly lyses the oocyst and sporocyst walls as well as the sporozoite membrane in a continuous incubation; therefore, it does not require the sporozoites to be alive. The results of PCR experiments indicate that this method generates better quality of DNA than what the existing glass-bead grinding method does for molecular analysis, and is suitable for both large or small number (<10(2) oocysts) of living or dead oocyst samples. 相似文献
5.
Sina Atashpaz Sajjad Khani Abolfazl Barzegari Jaleh Barar Sepideh Zununi Vahed Reza Azarbaijani Yadollah Omidi 《Microbiology》2010,79(4):538-542
The intactness of DNA is the keystone of genome-based clinical investigations, where rapid molecular detection of life-threatening
bacteria is largely dependent on the isolation of high-quality DNA. Various protocols have been so far developed for genomic
DNA isolation from bacteria, most of which have been claimed to be reproducible with relatively good yields of high-quality
DNA. Nonetheless, they are not fully applicable to various types of bacteria, their processing cost is relatively high, and
some toxic reagents are used. The routine protocols for DNA extraction appear to be sensitive to species diversity, and may
fail to produce high-quality DNA from different species. Such protocols remain time-consuming and tedious, thus to resolve
some of these impediments, we report development of a very simple, rapid, and high-throughput protocol for extracting of high-quality
DNA from different bacterial species. Based upon our protocol, interfering phenolic compounds were removed from extraction
using polyvinylpyrrolidone (PVP) and RNA contamination was precipitated using LiCl. The UV spectrophotometry and gel electrophoresis
analysis resulted in high A
260/A
280 ratio (>1.8) with high intactness of DNA. Subsequent evaluations were performed using some quality-dependent techniques (e.g.,
RAPD marker and restriction digestions). The isolated DNA from 9 different bacterial species confirmed the accuracy of this
protocol which requires no enzymatic processing and accordingly its low-cost making it an appropriate method for large-scale
DNA isolation from various bacterial species. 相似文献
6.
Muhammad Sohail 《Molecular biotechnology》1998,10(2):191-193
Molecular epidemiologic and other studies may require preparation of genomic DNA from large numbers of bacteria in sufficiently
pure form for restriction endonuclease digestion, cloning, RAPD-PCR, Southern hybridization, and so on.Staphylococcus and other Gram-positive bacteria have a rigid cell wall and can be difficult to lyse. Here, a simple and rapid method for
the preparation of genomic DNA from multiple samples is reported. This method produces clean DNA for use in most molecular
biology methods in <90 min. 相似文献
7.
Singh Shailendra P. Rastogi Rajesh P. Häder Donat-P. Sinha Rajeshwar P. 《World journal of microbiology & biotechnology》2011,27(5):1225-1230
We present an improved method for genomic DNA extraction from cyanobacteria by updating the earlier method from our group
(Sinha et al. 2001) that does not require lysozyme treatment or sonication to lyse the cells. This method use lysis buffer to lyse the cells
and also skips the initial treatments to remove the exopolysaccharides or to break the clumps. To test the efficacy of the
method DNA was extracted from the freshwater cyanobacteria Anabaena variabilis PCC 7937, Anabaena sp. PCC 7120, Synechocystis sp. PCC 6803, Synechococcus sp. PCC 6301 and Rivularia sp. HKAR-4 (Accession number: FJ939128). The spectrophotometric and gel electrophoresis analysis revealed high yield and
high quality of genomic DNA extracted by this method. Furthermore, the RAPD resulted in the amplification of unidentified
genomic regions of various lengths; however, rDNA amplification gave only one band of 1.5 kb in all studied cyanobacteria.
Thymine dimer detection study revealed that thymine dimers are induced only by UV-B radiation in A. variabilis PCC 7937 and there is no effect of PAR and UV-A on its genome. Collectively, all these findings put forward the applicability
of this method in different studies and purposes. 相似文献
8.
Genomic DNA was isolated from as little as 2 mg dry biomass of Magnaporthe grisea by microwave treatment within 30 s. The quantity of DNA was good enough for PCR analysis and Dot blot hybridization. This technique can be used for various studies, such as DNA fingerprinting to study the population structure of the phytopathogen in different regions, and for a quick screening of M. grisea transformants. 相似文献
9.
A simple and fast protocol is described for the purification of genomic DNA from 0.3 ml of whole human blood. The recovery of DNA is quantitative and reproducible; the quality is such that it can be used for all relevant molecular biology techniques. 相似文献
10.
A method for the large scale isolation of high transformation efficiency fungal genomic DNA 总被引:5,自引:0,他引:5
Donglu Zhang Yifan Yang Lisa A. Castlebury Carl E. Cerniglia 《FEMS microbiology letters》1996,145(2):261-265
Abstract A procedure for isolation of genomic DNA from the zygomycete Cunninghamella elegans and other filamentous fungi and yeasts is reported. This procedure involves disruption of cells by grinding using dry ice, removal of polysaccharides using cetyltrimethylammonium bromide and by phenol extractions, and precipitation of DNA with isopropanol at room temperature. The isolation method produced large scale (approximate 1 mg DNA/5 g wet cells) and highly purified high molecular mass DNA. Sau 3AI partially digested DNA showed high transformation efficiency (106 / 100ng DNA) when ligated to ZAP-express λ vector. 相似文献
11.
A simple and reliable method was developed for extracting genomic DNA from preserved mantle tissues of Pacific oyster Crassostrea gigas for reproducible PCR amplification. The method was based on destruction of the tissue using Proteinase K, Chelex 100 resin, detergents, and urea, followed by preferential capturing of genomic DNA with silica particles. Approximately 5 mg of mantle tissue provided a sufficient quality and quantity of DNA for several hundreds of PCR reactions amplifying the hypervariable mitochondrial DNA intergenic spacer, which is a useful genetic marker for population structure analysis of Pacific oyster. The method can be applied for DNA preparation from not only fresh and frozen but also ethanol-preserved mantle tissues, so this rapid and economical method can serve for investigating a large number of bivalve specimens collected in the field and next transported in ethanol at ambient temperature. 相似文献
12.
Following air sampling fungal DNA needs to be extracted and purified to a state suitable for laboratory use. Our laboratory has developed a simple method of extraction and purification of fungal DNA appropriate for enzymatic manipulation and Polymerase Chain Reaction (PCR) applications. The methodology described is both rapid and cost effective for use with multiple fungal organisms. 相似文献
13.
A method for direct soil extraction and PCR amplification of endomycorrhizal fungal DNA 总被引:2,自引:0,他引:2
DNA from endomycorrhizal fungi was extracted directly from a weathered soil (alfisol) mixed with sand. Mycorrhizae were established
in a greenhouse culture of Glomus clarum with Sudan grass (Sorghum vulgare var. sudanense) host plants. The extraction procedure included enzymatic digestion of cell walls, sodium dodecyl sulfate lysis of cells,
polyvinylpolypyrrolidone absorption of organic compounds, and ethanol precipitation of the DNA. DNA in the extracts was amplified
by the polymerase chain reaction using primers from the nuclear 17S rRNA sequence that were general to fungi or were specific
to endomycorrhizae.
Accepted: 17 July 1996 相似文献
14.
Summary A rapid and simple method for the small scale isolation of shuttle plasmid DNA from Saccharomyces cerevisiae is described. It uses glass beads to break cells and reagents which are also used in bacterial mini-preps to yield plasmid DNA without chromosomal contamination in sufficient quantities to enable direct visualisation on agarose gels. 相似文献
15.
16.
Abstract We present a high-throughput cost-effective method to extract DNA suitable for polymerase chain reaction (PCR) from insect tissue. The method uses standard 200 μL-deep 96-well plates in which samples are ground, digested and subsequently purified. The test extraction using four different insect species and controlling for potential contamination showed that the method yields good-quantity and quality DNA. PCR with mitochondrial and nuclear primers was reliable. The proposed extraction protocol combines the speed of commercial 96-well plate methods with the economies associated with readily available and cheap laboratory chemicals, consumables and equipment. Therefore, this method is particularly suitable for low-budget research projects and for laboratories with only basic equipment present. 相似文献
17.
目的 为快速地提取到质量较好的黑翅土白蚁基因组DNA进行白蚁种群多样性的研究,对基因组DNA提取方法进行了比较与改进.方法 先初步采取CTAB法与蛋白酶K法对黑翅土白蚁基因组DNA的提取方法进行比较,再利用正交设计法对蛋白酶K法中裂解液、蛋白酶、RNA酶及作用时间4个因素进行优化.结果 蛋白酶K法获得的基因组DNA的质量与产量稍优于CTAB法;较佳的提取步骤组合为:裂解液150 μL,蛋白酶K 6μL,作用时间1h,RNA酶可不添加.结论 采用优化后的方法获得的基因组DNA为模板进行PCR扩增,得到了清晰、稳定的扩增谱带,完全可用于相关后续实验. 相似文献
18.
19.
A modified mini-prep method for economical and rapid extraction of genomic DNA in plants 总被引:1,自引:0,他引:1
Molecular markers for map-based cloning, marker-assisted selection in crop breeding, and genetic studies require DNA isolation
from a large number of plants in a short span of time. Here we describe a modified DNA extraction method that is economical
in terms of cost, time and labour. The method allows DNA extraction from as little as 0.2–0.3 g of leaves that are homogenized
in zipper plastic bags, followed by DNA isolation in 1.5-mL Eppendorf tubes. By using the modified method, a DNA yield of
700–800 μg/300 mg leaf tissue was obtained from cotton and wheat samples. The quality of the DNA was quite suitable for PCR-based
markers. 相似文献
20.
A simple and reliable method for SSU rRNA gene DNA extraction, amplification, and cloning from single AM fungal spores 总被引:1,自引:0,他引:1
We describe a method that allows quick and easy PCR amplification and cloning of nearly complete SSU rRNA genes from arbuscular
mycorrhizal fungi. The procedure tested on spores from 37 different glomalean isolates was based on magnetic separation with
Dynabeads, followed by nested PCR with two primer pairs. All trials led to visible amplification products of the expected
size. Thereafter, the PCR fragments could be quickly and efficiently cloned by means of a topoisomerase-activated vector (pCR2.1-TOPO).
The technique is rapid, uncomplicated and comparatively inexpensive. The use of single spores for DNA extraction has some
advantages over multispore-preparations, e.g. it is less susceptible to contamination with other organisms present in the
cultures. The method can be used for the quick and reliable preparation of a large number of samples and is highly reproducible.
It could also be used for genes other than the SSU rRNA gene.
Accepted: 25 October 2000 相似文献