A simple and efficient method for isolation of DNA in high mucilaginous plant tissues

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.     

Isolation and amplification of DNA from rhizomes of turmeric and ginger

Turmeric and ginger are used as spices and in alternative systems of medicine. They are rich in polysaccharides, polyphenols, and alkaloids. A simple and rapid method for isolating good quality DNA with fairly good yields from mature rhizome tissues of turmeric and ginger has been perfected. Isolated DNA was amenable to restriction digestion and PCR amplification.     

Rapid Isolation of DNA from Dry and Fresh Samples of Plants Producing Large Amounts of Secondary Metabolites and Essential Oils

The presence of certain metabolites has been observed to interfere with DNA isolation procedures and downstream reactions such as DNA restriction, amplification and cloning. The chemotypic heterogeneity among species may not permit optimal DNA yields with a single protocol, and thus, even closely related species may require different isolation protocols. Here we describe the essential steps of a rapid DNA isolation protocol that can be used for diverse medicinal and aromatic plants, which produce essential oils and secondary metabolites such as alkaloids, flavanoids, phenols, gummy polysaccharides, terpenes and quinones. The procedure is applicable to dry as well as fresh plant tissues. This protocol, in our experiments, permitted isolation of DNA from tissues of diverse plant species and produced fairly good yields. The isolated DNA proved amenable to PCR amplification and restriction digestion.     

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.     

Extraction of high-molecular-weight DNA from dry root tissue of<Emphasis Type="Italic">Berberis lycium</Emphasis> suitable for RAPD

A simple protocol for DNA isolation from dry roots ofBerberis lycium is described. Four-year-old dry roots are used, and the isolated DNA is suitable for analysis by means of restriction enzyme digestion and random amplification of polymorphic DNA (RAPD). The method involves a modified CTAB procedure using 1% PVP to remove polysaccharides and purification using low-melting-temperature agarose. DNA is amplified by means of PCR using 10-mer random primers from Operon Biotechnologies, Inc. (USA), and DNA samples are digested withTaq I,Hind III andEcoR I and examined on agarose gels. The RAPD reaction is performed according to the 1990 protocol by Williams et al.     

A rapid and efficient method for the extraction of total DNA from mature leaves of the data palm (Phoenix dactylifera L.)

A method is presented for the rapid isolation of high-molecular-weight DNA from mature leaves of date palm (Phoenix dactylifera L.), using a CTAB-based buffer. The method yields up to 800 μg of DNA from 1 g of leaf tissues. The procedure was also suitable for DNA extraction from callus or buds from tissue culture. The DNA obtained through this method was a good substrate for at least seventeen restriction endonucleases. This method was also used to extract DNA from mature leaves of coconut and may be applicable to other species of palms.     

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.     

Isolating conifer DNA: A superior polysaccharide elimination method

Genomic DNA was isolated from frozen needles of maturePinus radiata clones using a modified extraction technique incorporating cetyltrimethylammonium bromide (CTAB) for cell lysis. A high sodium chloride concentration (2 M) was used at 2 stages of the extraction procedure to eradicate polysaccharides, yielding pure genomic DNA suitable for restriction enzyme digestion and PCR amplification. Extractions were scaled down to suit 1.5-mL Eppendorf tubes, allowing easier handling and enhanced sterility.     

Isolation of clean and PCR-amplifiable DNA fromAnthurium andreanum

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.     

Simple and rapid protocol for the isolation of PCR-amplifiable DNA from medicinal plants

Medicinal plant species has a valuable economic importance because of its usage as pharmaceuticals, nutritional, as well as its use in popular medication. For DNA-based techniques, nanogram quantities of the purified DNA are requisite to amplify and yield sufficient amounts of PCR products. SDS-based DNA isolation method was used to extract DNA from 11 species of different aromatic and medicinal plants collected from Saudi Arabia. Three hundred milligrams of fresh shredded plant material was necessary. The DNA purity was further confirmed by agarose gel, restriction endonuclease digestion and microsatellite primed-polymerase chain reaction (MP-PCR). DNA yields ranged from 10-20 μg (in 100-μL elution volumes) from all plant material evaluated. The DNA obtained was free of any contaminating proteins, polysaccharides and colored pigments. The extracted genomic DNA was found suitable for restriction digestion and PCR amplification. Our experimental procedure provides an easy, suitable, non-toxic, cheap, and quick process for the amplification of DNA from medical plant tissue.     

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.     

Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components

A relatively quick, inexpensive and consistent protocol for extraction of DNA from expanded leaf material containing large quantities of polyphenols, tannins and polysaccharides is described. Mature strawberry leaves, which contain high levels of these secondary components, were used as a study group. The method involves a modified CTAB extraction, employing high salt concentrations to remove polysaccharides, the use of polyvinyl pyrrolidone (PVP) to remove polyphenols, an extended RNase treatment and a phenol-chloroform extraction. Average yields range from 20 to 84 μg/g mature leaf tissue for both wild and cultivated octoploid and diploidFragaria species. Results from 60 plants were examined, and were consistently amplifiable in the RAPD reaction with as little as 0.5 ng DNA per 25-μL reaction. Presently, this is the first procedure for the isolation of DNA from mature strawberry leaf tissue that produces consistent results for a variety of different species, both octoploid and diploid, and is both stable and PCR amplifiable before and after extended storage. This procedure may prove useful for other difficult species in the family Rosaceae.     

A Simple and Rapid Procedure for Isolation of Total DNA Suitable for Fingerprint Analysis from shape Amaranthus

A simple, efficient and reliable method is described for isolation of total DNA from young leaves of Amaranthus species. This procedure yields a high amount (600–800 µg DNA/g fresh leaf tissue) of good quality DNA free from contaminating proteins, polysaccharides, and coloured pigments. The DNA is suitable for digestion with several restriction endonucleases, preparation of Southern blots, and PCR amplification. The DNA has been successfully used for generating DNA fingerprint profiles and RAPD banding patterns in two species of Amaranthus. The procedure is suitable for processing of a large number of samples simultaneously.     

An improved procedure for the isolation of nuclear DNA from leaves of wild grapevine dried with silica gel

An efficient and convenient method is presented for the isolation of nuclear DNA from leaves of wildVitis species that have been dried with silica gel. The nuclear DNA obtained with this method is suitable for both PCR amplification and digestion with restriction endonucleases.     

Am improved procedure for the isolation of chloroplast DNA fromChlamydomonas reinhardtii

The isolation of chloroplast DNA fromChlamydomonas reinhardtii requires the efficient separation of this AT-rich genome from the GC-rich nuclear genome by density-gradient centrifugation. We describe a simple and efficient method for separating these DNA fractions by using a sodium iodide gradient in combination with the DNA-binding dye, bisbenzimide. The yield of chloroplast DNA is close to the theoretical maximum and the DNA is suitable for restriction enzyme analysis and cloning. This method is applicable to the isolation of AT-rich plastid genomes from other organisms and may be appropriate as a general method for separating species of DNA that differ in their AT/GC ratios. An erratum to this article is available at .     

Copper (II) acetate improves the quality of pear (Pyrus) DNA during extraction

Isolation of high-quality DNA from Pyrus is particularly difficult because of high endogenous levels of polysaccharides, phenolics, and other organic constituents that interfere with DNA isolation and purification. Presence of phenolic compounds caused browning of tissue and supernatant during the extraction process, despite supplementation with PVP, as suggested by standard methods. By modifying the CTAB extraction procedure of Aldrich and Cullis (1993) by including copper (II) acetate treatment, we obtained high-quality DNA. Copper (II) acetate enabled fixation and removal of tannins. The DNA yield from this procedure is high (up to 1.25 μg of DNA/mg of leaf tissue). This DNA is completely digestible with restriction endonucleases and suitable for generation of molecular markers, such as random-amplified polymorphic DNA and amplified fragment length polymorphism analyses, indicating freedom from common contaminating polyphenolic compounds.     

Genomic DNA isolation and amplification from callus culture in succulent plants,<Emphasis Type="Italic">Carpobrotus</Emphasis> species (Aizoaceae)

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.     

Restriction fragment polymorphisms in the rDNA region among seven species ofAlnus andBetula papyrifera

A study of restriction fragment polymorphisms of ribosomal DNA among seven actinorhizal species (Alnus spp.) and a non-actinorhizal species (Betula papyrifera Marsh.) of the Betulaceae was conducted, using a simple method for the extraction of high molecular weight restrictable nuclear DNA from leaf tissues of perennial angiosperms and nine restriction endonucleases. rDNA restriction fragments were variable within and among the species studied, and the variation noted was used to calculate the similarities and infer phenetic relationships among these members of the Betulaceae. The results confirmed the taxonomy of alder based on morphological characters, showing a clear clustering of the species ofAlnus sampled in each of the two different subgeneraAlnus andAlnobetula. Within each subgenus, the closely related taxa often classified as subspecies by their similar morphology and their ability to interhybridize, were similarly shown by restriction fragment polymorphisms to be more closely related to each other than to any other taxon. The analysis also suggested that some alder species may not be more divergent fromBetula papyrifera than from other alder species.     

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.