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.     

Rapid and simple methodology for isolation of high quality genomic DNA from coniferous tissues (<Emphasis Type="Italic">Taxus baccata</Emphasis>)

Various investigations have been so far performed for extraction of genomic DNA from plant tissues, in which the extracted intact DNA can be exploited for a diverse range of biological studies. Extraction of high quality DNA from leathery plant tissues (e.g., coniferous organs) appears to be a critical stage. Moreover, for some species such as Taxus trees, bioprocess engineering and biosynthesis of secondary metabolites (e.g., paclitaxel) is a crucial step due to the restrictions associated with extinction of these species. However, extraction of intact genomic DNA from these plants still demands a rapid, easy and efficient protocol. To pursue such aim, in the current work, we report on the development of a simple and highly efficient method for the extraction of DNA from Taxus baccata. Based upon our protocol, interfering phenolic compounds were removed from extraction using polyvinylpyrrolidone and RNA contamination was resolved using LiCl. By employing this method, high quality genomic DNA was successfully extracted from leaves of T. baccata. The quality of extracted DNA was validated by various techniques such as RAPD marker, restriction digestions and pre-AFLP. Upon our findings, we propose this simple method to be considered for extraction of DNA from leathery plant tissues.     

An efficient protocol for genomic DNA extraction from<Emphasis Type="Italic">Citrus</Emphasis> species

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.     

GENOMIC DNA ISOLATION FROM GREEN AND BROWN ALGAE (CAULERPALES AND FUCALES) FOR MICROSATELLITE LIBRARY CONSTRUCTION1

A method for isolating high‐quality DNA is presented for the green algae Caulerpa sp. (C. racemosa, C. prolifera, and C. taxifolia) and the brown alga Sargassum muticum. These are introduced, and invasive species in Europe, except for the native C. prolifera. Previous methods of extraction, using cetyl trimethyl ammonium bromide or various commercial kits, were used to isolate genomic DNA but either no DNA or DNA of very low quality was obtained. Genomic libraries were attempted with Caulerpa sp. on three occasions and either the restriction enzyme, the Taq polymerase, or the T4 ligase was inhibited, probably by the large amount of polysaccharides in these algae. The method presented here consists of the rapid isolation of stable nuclei, followed by DNA extraction. Yields of 6–10 μ g genomic DNA from 1 g fresh blades were obtained. After genomic DNA was isolated from fresh material, the quality was checked by agarose gel. Quantification of DNA concentration was performed using UV spectrophotometric measurement of the A ₂₆₀/ A ₂₈₀ ratio. The DNA was suitable for PCR, cloning, and hybridization. The DNA isolated using this method allowed successful construction of microsatellite libraries for Caulerpa species and S. muticum . The technique is inexpensive and appropriate for the isolation of multiple samples of DNA from a small amount of fresh material.     

Evaluation of the impact of DNA extraction methods on BAC bacterial community composition measured by denaturing gradient gel electrophoresis

Aims: The impact of DNA extraction methods on biological activated carbon (BAC) DNA yield and bacterial community was evaluated. Methods and Results: Three different DNA extraction methods were compared: method a, method b and method c. Method c with ultrasonic pretreatment improved cell lysis efficiency (from 34% to 87%) and DNA yield [from 10·58 μg g^?1 (dry wt) of carbon to 21·42 μg g^?1 (dry wt) of carbon]. denaturing gradient gel electrophoresis profiles obtained by method c recovered the five seeded bacteria (Bacillus subtilis Strain WSO 6, Pseudomonas putida Strain WSO 7, Acinetobacter lwoffii WSO 10, Pseudomonas pertucinogena WSO 11 and Brevibacterium mcbrellneri WSO 13). Conclusions: The results showed method c with ultrasonic pretreatment was the most successful for the analysis of BAC bacterial community because it was effective in the detachment of bacteria and cell lysis, thereby resulting in good yields. Significance and Impact of Study: These results must be taken into consideration when extracting DNA for analysing BAC bacterial community.     

Transformation of Intact Aspergillus niger by Electroporation

A new rapid transformation system for Aspergillus niger that uses electroporation to render intact germinating conidia permeable to DNA is described. The transformant colonies appeared earlier than transformants obtained by the protoplast-forming method. Without pretreatment of the conidia the transformation frequencies were 1.2 colonies per μg of integrative vector and 100 colonies per μg of plasmid DNA. When the conidia were treated with a dilute solution of fungal cell wall lytic enzyme, the frequency of transformation was increased by approx. 2-fold when using two vectors. Southern blot analysis of genomic DNA and restriction endonuclease-digested DNA from a random sample of transformants showed homologous and nonhomologous integration of the integrative vector into the genome, as is also observed with the protoplast-forming method. In transformation with the plasmid vector, the transformant DNA was shown to be mostly maintained in free form with minimal integration into the chromosome when transformed by either intact electroporation or the conventional method.     

A Simple Method for the Efficient Isolation of Genomic DNA from Lactobacilli Isolated from Traditional Indian Fermented Milk (<Emphasis Type="Italic">dahi</Emphasis>)

A simple, inexpensive and effective genomic DNA isolation procedure for Lactobacillus isolates from traditional Indian fermented milk (dahi) is described. A total of 269 Lactobacillus isolates from fermented milk collected from four places in North and west India were tested for lysis by an initial weakening of the Gram positive cell wall with Ampicillin followed by Lysozyme treatment. The average genomic DNA yield was ~50 μg/ml log phase culture. Quality and repeatability of the method was found to be adequate for subsequent molecular applications. The quality of the genomic DNA isolated by this method was verified by restriction digestion and polymerase chain reaction (PCR). No inhibition was observed in subsequent PCR amplification and restriction digestion. The presented method is rapid, cheap and useful for routine DNA isolation from gram positive bacteria such as Lactobacillus.     

Variable transmission rates of a B-chromosome in <Emphasis Type="Italic">Myrmeleotettix maculatus</Emphasis> (Thunb.) (<Emphasis Type="Italic">Acrididae: Orthoptera</Emphasis>)

Amounts of Feulgen staining in individual spermatid and primary spermatocyte nuclei ofTricholioproctia impatiens were measured by the two wavelength method of cytospectrophotometry and compared with Feulgen-DNA values found for bull sperm, taken as a presumed reference standard of 3.24×10^–12 g DNA per nucleus. The amount of DNA estimated for the haploid male genome ofTricholioproctia was 0.39×10^–12 g DNA. This value was used to determine the DNA content and degree of polyteny of Malpighian tubule nuclei sampled from the larval stages of development.     

Rapid extraction of genomic DNA from leaves and bracts of dove tree (Davidia involucrata)

Large amounts of polyphenolics in dove tree leaves make it difficult to obtain high-quality genomic DNA during extraction. A rapid DNA minipreparation method was developed for dove tree (Davidia involucrata) and yields 40–50 μg genomic DNA from 0.1 g fresh matured and young leaves and bracts. The yield and quality of the resulting DNA is satisfactory, and the protocol can be scaled up according to sample size. The obtained DNA is suitable for PCR and the restriction enzyme digestion needed for Southern blotting.     

Isolation of DNA from single microsurgically excised bands of polytene chromosomes of Chironomus

A method is described for excising by a glass knife single bands of isolated polytene chromosomes of the salivary glands of Chironomus tentans larvae. DNA strands were isolated from cut-out bands and their contour lengths were determined on electron micrographs. The mean contour length of DNA strands isolated from the double band I-8A was about twice that of the single band I-11B, namely 63 versus 34 m. The described method may be applicable for molecular studies on single bands (e.g., by DNA cloning).     

Rapid extraction of high-quality genomic DNA from Porphyra yezoensis (Bangiales, Rhodophyta)

We developed a simple, rapid and stable method for extraction of high molecular weight DNA from the marine red alga Porphyra yezoensis Ueda using both guanidium treatment and QIAGEN? kit (Funakoshi, Tokyo, Japan). The method does not require expensive equipment and complex steps. The DNA yield averaged 1.5 μg 100 mg^?1 of Porphyra tissue and the A260/A280 and A230/A260 ratios of the DNA were approximately 1.8 and 0.4, respectively. It was of sufficient quality to be used for not only polymerase chain reactions but also other DNA manipulation techniques such as restriction digestion and construction of genomic libraries.     

Protocol for DNA extraction from potato tubers

A method to extract high-quality DNA from potato tubers was developed and tested on 3 wild potato species (Solanum raphanifolium, S. megistracrolobum, S. bukasovii) and on the tetraploid B3 bred population, (population number 393228, derived fromS. tuberosum subsp.tuberosum). The average yield of extracted DNA varied from 10–30 μg of DNA per gram of processed tissue. The DNA was pure and suitable for ligation-mediated polymerase chain reaction (LM-PCR) amplification, producing clear, distinctive, and reproducible banding patterns in polyacrylamide gels.     

An effective method to extract DNA from environmental samples for polymerase chain reaction amplification and DNA fingerprint analysis

A rapid direct-extraction method was used to obtain DNA from environmental soil samples. Heat, enzymes, and guanidine isothiocyanate were utilized to lyse cells. The DNA was purified by agarose gel electrophoresis, amplified with 16S rRNA-based primers by use of the polymerase chain reaction, and then digested with the restriction endonucleasePalI. The extraction method was used to obtain DNA from a variety of plants, bacteria, and fungi includingGossypium hirsucum (cotton),Pseudomonas, Bacillus, Streptomyces, andColletotrichum. Up to 100 g DNA/g (wet weight) of soil and 400 g DNA/g of plant material were recovered. Restriction endonuclease analysis patterns of amplified rDNA from pure microbial cultures and plant species contained three to five different DNA fragments. Amplified rDNA of mixed population DNA extracts from soil samples, digested with the restriction endonucleasePalI, contained 12–20 DNA fragments, appearing as sample fingerprints. Results from eight environmental soil samples that were analyzed suggest that the amplified rDNA fingerprints can be used to help characterize the genetic and biological diversity of the microbial populations in these samples.     

Isolation of High-Quality RNA from <Emphasis Type="Italic">Reaumuria soongorica</Emphasis>, a Desert Plant Rich in Secondary Metabolites

RNA isolation is a prerequisite for the study of the molecular mechanisms of stress tolerance in the desert plant Reaumuria soongorica, an extreme xeric semi-shrub. However, R. soongorica that contains high levels of secondary metabolites that co-precipitate with RNA, making RNA isolation difficult. Here the authors propose a new protocol suitable for isolating high-quality RNA from the leaves of R. soongorica. Based on a CTAB method described by Liu et al., the protocol has been improved as follows: the samples were ground with PVPP to effectively inhibit the oxidation of phenolics, contaminating DNA was removed with DNase I, and NaAc was used along with ethanol for precipitation to enhance the RNA yield and shorten the precipitation time. Gel electrophoresis and spectrophotometric analysis indicated that this isolation method provides RNA with no DNA contamination. Moreover, the yield (183.79 ± 40.36 μg/g) and quality were superior to those using the method of Liu et al., which yields RNA with significant DNA contamination at 126.30 ± 29.43 μg/g. Gene amplification showed that the RNA obtained using this protocol is suitable for use in downstream molecular procedures. This method was found to work equally well for isolating RNA from other desert plants. Thus, it is likely to be widely applicable.     

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 DNA extraction from sporophyte in the brown alga <Emphasis Type="Italic">Laminaria japonica</Emphasis>

A simple method was developed for extracting DNA from brown algae Laminaria japonica, which possess large amounts of acidic polysaccharides. Firstly, the sporophyte were washed by eliminating polysaccaride buffer to remove the polysaccharides and then ground in liquid nitrogen. Secondly, the powders were treated with lysing buffer. Thirdly, KAc was used to eliminate the remaining acidic polysaccharides. The extracted DNA was purified using a chloroform-isoamyl alcohol (24:1 v/v), and precipitated in cold isopropanol. The yield was from 18.7 to 37.5 μg g⁻¹ (wet weight) and the purity of total DNA was determined spectrophotometrically as the ratio of A₂₆₀/A₂₈₀, which was about 1.7–1.9. The extracted DNA was of high quality and suitable for molecular analyses, such as PCR, restriction enzyme digestion. This method is a reproducible, simple, and rapid technique for routine DNA extraction from sporophyte in Laminaria japonica. Furthermore, the low cost of this method makes it attractive for large-scale studies.     

Rapid and efficient extraction of genomic DNA from differentphytopathogenic fungi using DNAzol reagent

A modified procedure using the commercial DNAzol reagent was successfully applied to extract genomic DNA from 25 fungal species. The DNA yield varied from 306 to 1,927 g g^-1 dry mycelia and the A₂₆₀/A₂₈₀ ratio from 1.59 to 1.93. Compared with the method of J.L. Cenis (Nucleic Acids Res. 1992, 20: 2380) this procedure generated a higher DNA yield from 17 species and a higher A₂₆₀/A₂₈₀ ratio from 23 species. But for four species, Cenis (1992) method was more suitable. No inhibitor of polymerase chain reaction was evident for the DNA extracted by the modified procedure, whereas some inhibitors remained in DNA of eight species extracted by the previous method.Revisions requested 8 September 2004; Revisions received 1 November 2004     

DNA extraction conditions fromPorphyra perforata using LiCl

A rapid and economical method of DNA extraction from a red seaweedPorphyra perforata J. Agardh has been developed by the use of lithium chloride. This paper describes the optimization of extraction conditions. Heat treatment of tissues in a solution (0.8 M LiCl, 0.6% Sarkosyl, 10 mM EDTA, 0.2% PVPP, 5% ß-mercaptoethanol, pH 9.0) at 55 °C for 10 min extracts DNA that is of sufficient quality to be used as a template for PCR amplification. Total DNA yield was approximately 30 to 50g g^–1 t of partially dried tissue. Total RNA yield was approximately 400g g^–1 of partially dried tissue. Carbohydrate was contained as approximately 40 to 90 mM (expressed as glucose equivalents) from 1 g tissue, and protein contamination calculated as the O.D. 260/280 ratio was in the range of 1.4 to 1.7. The DNA was characterized by high molecular weight larger than 50 kb.     

Rapid method for detecting Desulfitobacterium frappieri strain PCP-1 in soil by the polymerase chain reaction

A rapid method was developed for detecting in soil Desulfitobacterium frappieri strain PCP-1, an anaerobic gram-positive bacterium, isolated from a methanogenic consortium degrading pentachlorophenol. The method involved the establishment of a protocol for extracting total DNA from soil with the least contamination, and the use of the polymerase chain reaction (PCR) to detect strain PCP-1 with primers targeted with PCP-1 16S rRNA. To optimize the DNA extraction conditions, a glass mill homogenizer and a low-salt buffer containing polyvinylpolypyrrolidone were used on a black soil rich in organic matter. Recovered DNA was further purified with phenol/chloroform extractions, ammonium acetate precipitation and a G200 Sephadex gel-filtration column. DNA was extracted from soil supplemented with different concentrations of PCP-1 cells. Detection of PCP-1 was by PCR. The limit of detection was 800 added PCP-1 cells/g dry soil. This level of detection was achieved when the T4 gene-32 protein and 1 μg soil DNA were added to the PCR mixture followed by a nested PCR. This method is quick, sensitive, and can process several samples at the same time. Received: 22 October 1996 / Received revision: 24 January 1997 / Accepted: 10 February 1997     

Direct sequencing of tobacco chloroplast genome by the polymerase chain reaction

We have developed a polymerase chain reaction (PCR) method for sequencing of tobacco chloroplast genome. In a mixture containing chloroplast DNA, 5-end-labeled oligonucleotide primer, Taq DNA polymerase and reaction buffer, we were able to sequence a segment of chloroplast 16S rRNA gene. The results showed that the 750 bp of DNA sequenced were identical to the sequence reported, indicating that direct sequencing method that we have developed is useful for the sequencing of chloroplast genome. To analyze the chloroplast genome more rapidly in those in vitro grown plantlets, we also developed a simple method which is applicable for the amplifications and sequencing of chloroplast 16S rRNA fragment from either 0.15 g of tobacco leaf or stem tissue. The readable sequences obtained from the presented methods were consistent with the published sequence.