A simple and efficient method of DNA isolation from orchid species and hybrids

A simple and reliable method for extracting DNA has been developed for orchid species and hybrids. The high quality of DNA obtained is suitable for amplification via the polymerase chain reaction (PCR) for producing random amplified polymorphic DNA (RAPD) markers. This revised version was published online in July 2006 with corrections to the Cover Date.     

A simple protocol for isolation of fungal DNA

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.     

A simple and efficient method for DNA extraction from grapevine cultivars andVitis species

A quick, simple, and reliable method for the extraction of DNA from grapevine species, hybrids, andAmpelopsis brevipedunculata (Vitaceae) has been developed. This method, based on that of Doyle and Doyle (1990), is a CTBA-based extraction procedure modified by the use of NaCl to remove polysaccharides and PVP to eliminate polyphenols during DNA purification. The method has also been used successfully for extraction of total DNA from other fruit species such as apple (Malus domestica), apricot (Prunus armeniaca), cherry (Prunus avium), peach (Prunus persica), plum (Prunus domestica), and raspberry (Rubus idaeus). DNA yield from this procedure is high (up to 1 mg/g of leaf tissue). DNA is completely digestible with restriction endonucleases and amplifiable in the polymerase chain reaction (PCR), indicating freedom from common contaminating compounds.     

A simple method for isolation of megabase DNA from cotton

A simple method for preparation of high-molecular-weight DNA from cotton was developed. This method includes two major steps, (i) isolating nuclei and (ii) embedding nuclei into agarose microbeads. DNA isolated by this procedure is larger than 5.7 Mb in size, and is suitable for physical mapping by PFGE and YAC/BAC cloning.     

A rapid and efficient method for the isolation of restrictable total DNA from plants of the genusAbelmoschus

A rapid, simple and efficient protocol is given for the extraction of restrictable total DNA from plants of the genusAbelmoschus, for which the main obstacle is the stickiness of the solution, after grinding of green leaves. This problem is resolved using cotyledons of dark-grown seedlings.     

A rapid method for tissue collection and high-throughput isolation of genomic DNA from mature trees

Collection of tissue and subsequent isolation of genomic DNA from mature tree species often proves difficult. DNA extraction from needles, leaves, or buds is recommended in many protocols. Collecting these tissues from mature trees generally requires the use of firearms or climbing if sampling is to be nondestructive. As a result, sample collection is a major expense of many tree-based projects. Tree (and plant) tissues generally contain large amounts of polysaccharides and phenolic compounds that are difficult to separate from DNA. Many methods aim to overcom these problems, with most involving extraction in buffers containing the nonionic detergent cetyltrimethyl-ammonium bromide (CTAB), followed by numerous steps to clean contaminants from the DNA, using organic solvents and differential salt precipitation. These steps are time-consuming, such that isolation of DNA becomes the bottleneck in many molecular studies. This paper presents a new, efficient, cambium collection method for tree species and a DNA extraction protocol based on that of Doyle and Doyle (1987), with follow-up purification using the Wizard nuclei lysis and protein precipitation solutions (Promega). Results show a significant improvement in yield and DNA purity compared with other published methods, with consistently high yields of pure genomic DNA and high sample throughput. The relatively low cost per extraction, no requirement for use of liquid nitrogen, no requirement for freezer storage, and long-term sample stability after collection are important additional benefits.     

Extraction of geminiviral DNA from a highly mucilaginous plant (Abelmoschus esculentus)

A protocol is described for the extraction of geminiviral DNA from bhendi yellow vein mosaic virus-infectedAbelmoschus esculentus (known as bhendi or okra) containing high amounts of mucilage and other phenolic compounds. This method involves extraction with a buffer containing sodium citrate at pH 6 and PEG precipitation of the virus followed by alkali lysis. The extraction buffer eliminates the mucilage and other polyphenols, PEG precipitates the viral particles and DNA and the alkali lysis enriches the replicative forms of the viral DNA. The extracted DNA could be digested with restriction enzymes and cloned without any interference from chromosomal DNA. The quality of the DNA extracted by this method was compared to three other common plant DNA extraction protocols and was found superior. This method was used for PCR amplification and cloning of the 2.7 kbp DNA-A of BYVMV.     

An efficient procedure for the isolation of PCR-competent DNA from Bacillus endospores germinated in soil

DNA extraction techniques for endospore-forming bacteria in soil are often labour-intensive and unreliable. Our objective in this work was to investigate whether good quality DNA could be obtained from spores germinated in soil. To this end, endospores from Bacillus subtilis, B. megaterium and B. thuringiensis were inoculated into soil microcosms and germination was induced by addition of LB medium supplemented with l-alanine, glucose, fructose and KCl. Heat resistance count was reduced to 80% for B. subtilis and more than 90% for B. thuringiensis and B. megaterium after a few minutes. Isolation of DNA from soil with a procedure which did not work on spores was shown to be as efficient for in situ-germinated spores as for inoculated vegetative cells. Furthermore, we developed a simple procedure that allowed us to use the recovered DNA in PCR amplifications. The present methodology is simple and efficient; it avoids the use of special equipment and harsh spore rupturing methods and can be carried out with multiple samples.     

A method for extracting genomic DNA suitable for medium-throughput applications from plant tissues rich in contaminants

Isolating nucleic acids from sources rich in contaminants is particularly cumbersome when treating a large number of samples. Several protocols have been published that address the problem of nucleic acid extraction and purification, but few address sample number. We describe a method for extracting DNA from recalcitrant tree species by using a commercial grinding apparatus. This alleviates the hard work of sample preparation prior to lysis and purification. Our method has been tested extensively on different fruit tree species and in projects that require the simultaneous processing of hundreds of samples. Moreover, it does not require the availability of robotic workstations.     

A simple and efficient protocol for isolation of functional RNA from plant tissues rich in secondary metabolites

A protocol is described for rapid RNA isolation from various plant species and tissues rich in polyphenolics and polysaccharides. The method is based on the Nucleon PhytoPure^™ system without the use of phenol. The procedure can be completed within 1 h and many samples can be processed at the same time. The yield ranged from 240 μg up to 3 mg per gram of tissue with an average purity measured as A_260/280 of 1.85. The RNA was of sufficient quality for use in RT-PCR reactions. Quantitation of single-stranded cDNA was carried out with the RiboGreen^™ reagent and of PCR products with the PicoGreen^™ reagent.     

A simple method of DNA extraction from whole tissues and blood using glass powder for detection of transgenic animals by PCR

A very simple and reliable method to extract DNA directly from mouse tail, rabbit ear and blood is described. Tissue was homogenized in a solution of NaI and the DNA was extracted using glass powder. The extracted DNA was obtained in sufficient quantity and purity to allow direct detection of transgene by PCR.     

Mannose: A Potential Selection System for Genetic Transformation of Annatto

The aim of the present work was to evaluate the feasibility of mannose as a selection system for the future genetic transformation of annatto (Bixa orellana L.). Hypocotyl segments, inverted hypocotyls and immature zygotic embryos were inoculated onto a Murashige and Skoog's medium supplemented with B5 vitamins, 87.6 mM sucrose and mannose in different combinations, 2.8 g dm^?3 Phytagel®, and 4.56 μM zeatin (organogenesis) or 2.26 μM 2,4-dichlorophenoxyacetic acid and 4.52 μM kinetin (somatic embryogenesis). Annatto explants did not regenerate on medium with mannose as the only carbon source when inverted hypocotyls and immature zygotic embryos were used. However, organogenesis (5 % frequency) occurred exclusively in hypocotyl-derived explants nearest to the crown (collar) of the seedlings. No further shoot development was achieved. Therefore the substitution of sucrose by mannose inhibited both organogenesis and embryogenesis, and thus the employment of mannose could constitute an alternative selective agent in protocols for genetic transformation of this species.     

A rapid and efficient method for the extraction of total DNA from the sweet potato and its related species

Secondary metabolites, latex/mucilagenous secretions, polysaccharides, and proteins interfere with the extraction of high-quality, restrictable total cellular DNA from sweet potato [Ipomoea batatas (L.) Lamk.] and related species. A method for the DNA extraction is described which overcomes these problems.     

An efficient method for DNA isolation from red algae

A simple, inexpensive and efficient method was developed for rapid isolation of totalgenomic DNA from 15 red algal species. It resulted in 0.1 g high quality DNAfrom 1 mg fresh algal material, with an A₂₆₀/A₂₈₀ratio of 1.68–1.90.Using this rapidly isolated DNA, the 18S ribosomal RNA genes (rDNA) and the nuclearribosomal DNA of the internal transcribed spacer (ITS) regions were amplified. Thetested DNA was suitable for restriction endonuclease digestion, genetic markeranalysis and polymerase chain reaction (PCR) amplification, and may be valid forother genetic manipulation.     

一种快速微量提取植物叶片DNA的方法

介绍了一种快速提取微量DNA的方法。该方法简单易行,无需任何特殊设备,所需样品量少。提取的DNA纯度高,D260nm/D280nm在1．9-2．1之间,可满足RAPD、SSR、转基因植株的PCR检测等以PCR扩增为基础的实验需要。     

A rapid and high yielding DNA miniprep for cotton (Gossypium spp.)

A rapid DNA minipreparation method was developed for cotton and yields 500–600 μg DNA from 1.0 g fresh leaf tissue. Cotton DNA extracted using this method is completely digested with restriction enzymes, supports PCR and Southern DNA analyses and was used successfully in these applications. An erratum to this article is available at .