A rapid and cost-effective method of genomic DNA isolation from cyanobacterial culture,mat and soil suitable for genomic fingerprinting and community analysis

This study presents a phenol and lysozyme free protocol for genomic DNA isolation of cyanobacteria from culture, mats and soil. For an efficient and pure DNA isolation from cyanobacteria having tough cell wall, extra steps of glass beading and Sepharose 4B purification were added. The modified method gave a higher yield of DNA than the phenol: chloroform extraction method. Four parameters selected for purity testing of the isolated DNA were: (i) restriction digestion with Hind III, (ii) randomly amplified polymorphic DNA-PCR of axenic culture of cyanobacteria to assess phylogenetic relatedness, (iii) denaturing gradient gel electrophoretic (DGGE) analysis of cyanobacterial mat and soil to ascertain the applicability of the isolated DNA for community analysis, and (iv) sequencing of partial 16S rDNA of Hapalosiphon intricatus BHULCR1, Anabaena doliolum LCR1, Anabaena oryzae LCR2, Aulosira fertilissima LCR4, and Tolypothrix tenuis LCR7 and BLAST analysis to confirm their cyanobacterial identity. Data generated from above analyses lead us to conclude that the modified method in question is rapid, cost effective, health and time conscious and promising for genetic fingerprinting and community analysis of cyanobacteria from diverse habitats.     

An improved method for genomic DNA extraction from cyanobacteria

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.     

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 .     

Effectiveness of the postponed isolation (post-frozen isolation) method for PCR-quality <Emphasis Type="Italic">Sarcoptes</Emphasis> mite gDNA

The aim of the present study was to assess whether individual Sarcoptes mites collected from frozen skin (‘postponed isolation’ method) are suitable sources of PCR-quality genomic DNA, and to test the effectiveness of this method in comparison with the ‘direct isolation’ method, often used through force of habit. Hundreds of single Sarcoptes scabiei samples, resulting from direct (live) or postponed (post-frozen) isolation, were tested using a ~450 bp product (ITS-2) and multi-locus 10× genotyping with microsatellite markers. No statistical difference in yield of soluble DNA was found between the two isolation methods. Nevertheless, 19% of the reactions were classified as failed preparations in the direct isolation method, whereas the rate of unsuccessful reactions was 34% in the postponed isolation method. Consequently, post-frozen isolation is suitable and recommendable for Sarcoptes mite gDNA preparation, particularly when performing a balancing act among safety, practicability and profitability. These results have implications for mite collection for DNA extraction, and hence the needed wider leap of Sarcoptes into the genetic era.     

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.     

Rapid small-scale DNA isolation from filamentous cyanobacteria

Abstract A rapid small-scale DNA isolation procedure is described for the filamentous cyanobacteria, which yields enough chromosomal and plasmid DNA for restriction endonuclease digestions, Souther hybridizations, and electroelution from gels for further manipulation. DNA from seven strains of cyanobacteria were isolated and analyzed on agarose gels.     

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 method for the direct extraction of plasmid DNA from yeast

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.     

用于超声造影的微囊藻气囊提取新方法

气囊是一种由蛋白质外壳包裹气体形成的纳米级细胞结构,常见于蓝藻和嗜盐古菌中.气囊中包含的气体在超声波作用下,能够散射声波并产生谐波信号而增强信噪比,具备成为新型超声造影剂的潜质.但目前提取气囊的方法主要是传统的高渗裂解法,该操作过程烦琐、得率低,不适用于气囊的大规模提取.针对这些技术瓶颈,文中建立了一种快速、高效的微囊...     

Adapting molecular-genetic methods for studying the taxonomic diversity of microbial communities associated with macrophytes

The combination of molecular-genetic techniques used in the study is applied to investigate microorganisms associated with macrophytes. The method of enzymatic lysis with phenol-chloroform extraction is optimal for the total bacterial DNA isolation from both periphyton organisms and enriched cultures. Amplifying the total DNA on conservative primers at the two-step PCR regime is recommended. An analysis of the taxonomic diversity of the microbial community from biofilm on the reed grass and in the enriched cultures propagated on various growth media has been carried out. The results have revealed a high diversity of periphyton microorganisms associated with reed grass, including representatives of such phylogenetic lines as proteobacteria (α, β, γ, and δ subgroups), Bacteroidetes/Chlorobi, Chlamydiae/Verrucomicrobia, and cyanobacteria. The low diversity of sequences in enriched cultures is represented by dominating genotypes of Cellvibrio with a high percentage of homology and uncultivated bacilla.     

A Method for Isolation of Chloroplast DNA and Mitochondrial DNA from Sunflower

We present a method for isolation of chloroplast and mitochondrial DNA from sunflower seedlings. The protocol includes: organelle isolation, deoxyribonuclease treatment, lysis, deproteinisation and a final DNA purification with sodium dodecyl sulphate and potassium acetate. The organelle DNA yield is 5–10 micrograms per gram of tissue and the DNA is fully restrictable. The technique is inexpensive and appropriate for the isolation of multiple samples of organelle DNA from a small amount of tissue.     

Isolation and Properties of Macronuclei from Tetrahymena thermophila1

A simple and efficient method is described for the isolation of macronuclei from Tetrahymena thermophila (7B). The steps involved are deciliation and removal of the mucocysts’ contents by dibucaine treatment, digitonin mediated lysis, differential centrifugations, and finally isopyenic sucrose density gradient centrifugation. Judging from the distribution of marker enzymes and electron microscopy, the macronuclei obtained were free of cytoplasmic and paniculate contamination and were highly active in endogenous RNA-synthesis (1.5 pmol UTP incorporation/ng DNA min at 30°C). The ratio of protein: RNA: DNA was 2.0:0.33:1.0 (weight) and each macronucleus contained an average of 17 pg DNA. The average yield of isolation was 50%.     

Isolation and analysis of the total genomic DNA from the date palm (P. dactylifera L.) and related species

The objective of this study was to adapt a plant DNA preparation procedure for the isolation of biologically active DNA and DNA with a high molecular weight from the date palm and other related palms. Mature leaf tissue extractions of the date palm, Phoenix dactylifera L., the coconut tree, Cocos nucifera, and the Mexican Fan Palm, Washingtonia robusta, were characterized for total genomic DNA yield, purity, integrity, as well as restriction digestion and ligation capabilities. It is demonstrated here that the DNA isolation procedure, modified for use with various palm leaf tissues, met the criteria for simplicity and low costs, and yielded DNA of high molecular weight (～50 Kbp) and of sufficient purity suitable for molecular studies.     

DNA isolation from soil samples for cloning in different hosts

Many protocols to extract DNA directly from soil samples have been developed in recent years. We employed two extraction methods which differed in the method of lysis and compared these methods with respect to yield, purity and degree of shearing. The main focus was on the specific isolation of DNA from different microorganisms, especially DNA from actinomycetes, as these cells are very difficult to lyse, in contrast to non-actinomycetes. Thus, we used both methods to isolate DNA from Pseudomonas, Arthrobacter and Rhodococcus and from soil spiked with the respective microorganisms. Both methods rendered high DNA yields with a low degree of shearing, but differed in the type of cells that were lysed. By one protocol (utilizing enzymatic lysis) only DNA from the Gram-negative Pseudomonas strain could be obtained whereas, by the other protocol (utilizing mechanical lysis), all microorganisms that were used could be lysed and DNA extracted from them. Using a combination of both protocols, DNA from those organisms could be obtained selectively. Furthermore, one of the protocols was modified, resulting in higher DNA yield and purity.     

A prokaryotic sucrose synthase gene (susA) isolated from a filamentous nitrogen-fixing cyanobacterium encodes a protein similar to those of plants

Sucrose synthase (SS), a key enzyme in plant carbohydrate metabolism, has recently been isolated from Anabaena sp. strain PCC 7119, and biochemically characterized; two forms (SS-I and SS-II) were detected (Porchia et al. 1999, Planta 210: 34–40). The present study describes the first isolation and characterization of a prokaryotic SS gene, susA, encoding SS-II from that strain of Anabaena. A 7 kbp DNA fragment containing an open reading frame (EMBL accession number AJ010639) with about 30–40% amino acid identity with plant SSs was isolated from an Anabaena subgenomic library. The putative SS gene was demonstrated to encode an SS protein by expression in Escherichia coli. The biochemical properties of the recombinant enzyme were identical to those of the enzyme purified from the cyanobacterial cells. The deduced amino acid sequence of the Anabaena SS diverged from every plant SS reported. The occurrence of SS in cyanobacteria of different taxonomic groups was investigated. The enzyme occurs in several filamentous nitrogen-fixing cyanobacteria but not in two species of unicellular, non-diazotrophic cyanobacteria. Received: 5 January 2000 / Accepted: 7 March 2000     

Isolation of nuclei from rice tissue grown in suspension culture

Summary A simple method is described for the isolation of intact nucleic from rice tissue (Oryza sativa L.) grown in suspension culture. The procedure involves incubation of the tissue for 4 h with cellulase and pectinase prior to disruption of the cells. The yield of nucleic is approximately 40% (DNA basis) and the preparations are capable of synthesizing RNA in vitro. The method may be valuable to biochemically oriented research requiring plant nuclei.     

DNA isolation from fresh,dry plant samples with highly acidic tissue extracts

Current DNA isolation methods are limited in their ability to obtain quality and/or quantity DNA from plants, such asEmblica officinalis, Terminalia belerica, andTerminalia chebula, which have low pH and high amounts of secondary metabolites in tissue extracts. Our modified DNA isolation method yields good-quality, high-molecular-weight DNA that is free of contaminants and colored pigments and is suitable for PCR amplification. This method is also useful for isolating DNA from dry powders.     

Plasmid content and homology of 16 strains of filamentous,nonheterocystous cyanobacteria

We have developed several strain-specific, rapid, small-scale plasmid isolation procedures in order to characterize the plasmid profiles of 16 filamentous, nonheterocstous cyanobacteria. At least one distinct plasmid was found in eight strains, with seven of these containing two or more different plasmids. Eight strains were found to be without plasmid DNA. Both the large, 12.9 kb, and the small, 1.6 kb, plasmids fromPlectonema boryanum 581 were isolated, purified, and cloned. Southern blots of plasmid DNAs from the eight strains were probed with these cloned DNAs and also with ultra-pure plasmid DNA fromPhormidium liridum 426. Four strains ofP. boryanum (485, 581, 594, 1542) andP. luridum 426 have identical plasmid profiles, and plasmid homology is extensive.     

A rapid and easy method for the DNA extraction from <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>

DNA isolation from C. neoformans is difficult due to a thick and resistant capsule. We have optimized a new and rapid DNA isolation method for Cryptococcus using a short urea treatment followed by a rapid method using a chelex resin suspension. This procedure is simpler than previously reported methods.