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.     

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 robust universal method for extraction of genomic DNA from bacterial species

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.     

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.     

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.     

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.     

Rapid method for DNA isolation from a tough cell wall green alga <Emphasis Type="Italic">Tetraspora</Emphasis> sp. CU2551

Genetic studies are important to understand the complex biological system of various organisms. Some eukaryotic green organisms have tough cell wall which precludes the efficient extraction of the genetic materials. Here, we developed the method for simple and rapid isolation of high quality DNA from a green alga Tetraspora sp. CU2551. The cell homogenization procedures were combined with physical force plus heat treatment to disrupt the cell envelope of Tetraspora sp. CU2551. Without protease treatment, vortexing with glass bead for 30–105 s at 70 °C led to the isolation of a high purity DNA which was suitable for downstream process. The improved method was successfully developed and could be applied for the rapid isolation of DNA from other unicellular and filamentous green microalgal strains.     

A Rapid Method for the Isolation of Genomic DNA From Aspergillus Fumigatus

Abstract

A majority of Aspergillus induced diseases are reported to be caused by Aspergillus fumigatus. In immunocompromized and post transplant cases it can lead to invasive aspergillosis. Due to this the molecular fingerprinting of aspergillus isolates by RFLP analysis and development of DNA diagnostic probes are gaining importance. Different methodologies are being adopted for extraction of the genomic DNA from fungus. The existing procedures for isolation of DNA are time consuming and range from several hours to few days. The most difficult step in the isolation of DNA from aspergillus species is to disrupt the tough chitin rich ceil wall without causing damage to genomic DNA. We report here a rapid method for extraction of genomic DNA based on the cleavage of chitin with chitinase. The subsequent modification steps included are lysis and microwave treatment. The chromosomal DNA obtained by this procedure is 1.5 – 2.0 μg per mg of wet weight of mycelia and is observed to be minimally sheared. It is pure enough for restriction analysis and for use in the PCR to detect the gene coding for 18 kDa allergen which has been identified in our laboratory using western blot analysis with human patient sera.     

Three Modifications of the Dodecyl Sulfate Method of Kay et al. for Isolating DNA from Calf Thymus

Two rapid methods for the isolation of calf thymus DNA are described which are modifications of the method of Kay et al. ¹ for isolating DNA. The principal changes introduced are that degelation of the DNA-nucleo-protein gels is accomplished rapidly by the use of mild shear or by pH adjustment in the presence of isopropanol instead of by the more time-consuming autolytic proteolysis used in the original method.     

Molecular Typing of Cyst‐Forming Nematodes Globodera pallida and G. rostochiensis,Using Real‐Time PCR and Evaluation of Five Methods for Template Preparation

Globodera pallida and G. rostochiensis are two cyst‐forming nematodes known to infest potato crops, causing severe economic losses worldwide. In this study, a real‐time TaqMan PCR assay was developed and optimized for the simultaneous detection of G. pallida and G. rostochiensis. The assay's analytical and diagnostic sensitivity and specificity were evaluated using reference isolates. Four different DNA extraction methods and one rapid crude template‐preparation procedure were compared in terms of extraction purity, efficiency for PCR applications, utility and cost. Extraction methods A and B included two commercially available kits that utilize silica columns and magnetic beads, respectively. Method C was based on DNA isolation using Chelex resin, and method D was a standard chemistry in‐house protocol. Procedure E included the direct use of crude mixture composed of disrupted cysts in Tris–EDTA buffer. The multiplex TaqMan PCR assay successfully discriminated the two nematode species from all reference cyst samples and its recorded diagnostic sensitivity (Dse) and specificity (Dsp) was 100%. On the contrary, in conventional (Co) PCR tests, the overall Dsp and Dse were lower and estimated at 94 and 87% for G. pallida, and 97 and 88% for G. rostochiensis, respectively. Spectrophotometric results showed that DNA extraction methods A, B and C yielded the purest DNA and gave the lowest mean C_t values as well as the most consistent results in Co PCR. Alternative crude preparation method E resulted in statistically similar and C_t values consistent with those obtained with methods A to C when tested by TaqMan PCR. The developed assay, using crude template‐preparation E, allows the simple, accurate and cost‐effective testing of a large number of cyst samples and can be applied in surveys and certification schemes.     

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.     

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.     

Rapid isolation of terminal sequences from cloned plant DNA fragments

The isolation of DNA clone termini is an important step in the development of DNA contigs utilized for a range of applications, including physical mapping, genetic map-based cloning, insertion mutagenesis cloning, and isolation of complete gene sequences. We describe a rapid PCR-based method for the isolation of vector-insert junctions, or insert terminal sequences, of cloned plant DNA fragments. PCR amplification is performed using a vector-specific primer and a nonspecific primer, originally designed for use in animal systems, containing degenerative bases that we have shown can also anneal to plant insert DNA. Using this method we have successfully isolated end-terminal sequences from plant genomic clones harbored in YAC, BAC, and bacteriophage λ vectors. Termini of genomic clones from both tomato andArabidopsis were isolated demonstrating the utility of this technique among a range of plant species.     

A general method for plasmid isolation in lactobacilli

A simple procedure for rapid isolation and detection of plasmid DNA fromLactobacillus species is described. Using an alkaline-detergent lysis method, plasmid DNA was released and characterized from cells treated with either mutanolysin or lysozyme for 1 h at 0°C. Treatment of cells with either enzyme at 37°C for 1h was detrimental to plasmid isolation and charaterization in someLactobacillus species. The procedure was effective with small volumes of cells and allowed rapid characterization of plasmid DNA inLactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus helveticus, andLactobacillus bulgaricus strains.     

Rapid isolation of any known genes from whole cells of yeast by PCR

A short protocol was developed that allows the rapid isolation of any knownSaccharomyces cerevisiae gene. Two known genes,APN1 andIMP2, were isolated directly from whole cells of yeast using polymerase chain reaction, without the need for purified template genomic DNA.     

Nuclei isolation protocols for flow cytometry allowing nuclear DNA content estimation in problematic microalgal groups

Microalgae are fundamentally important organisms for global ecosystem functioning with high potential in biotechnology and its applications. The knowledge of their nuclear DNA content has become a prerequisite for many areas of microalgal research. Due to common presence of various pigments, secondary metabolites and complex cell walls, the nuclear DNA content estimation using flow cytometry (FCM) is, however, often laborious or even impossible with the currently used protocols. In this study the performance of six nuclei isolation protocols was compared on various problematic microalgae using FCM. The nuclei isolation methods involved osmotic bursting of cells, razor blade chopping of fresh biomass and two newly introduced protocols, razor blade chopping of desiccated biomass and bead beating. These techniques also involved the use of two different nuclei isolation solutions, Otto I + II solutions, and LB01 buffer. Performance of the particular protocols differed greatly, depending on the used nuclei isolation solution and microalgal group. The most successful method was a newly adopted chopping of desiccated biomass in LB01 buffer. This method seems more appropriate for nuclei isolation in filamentous microalgae; on the other hand, bead beating appears to be more suitable for nuclei isolation in solitarily living algae. Using the optimal protocol for a given species, their nuclear DNA content was estimated, resulting in first DNA content estimates for four investigated taxa (Chlamydomonas noctigama, Gonyostomum semen, Microglena sp. and Stigeoclonium sp.). The estimated DNA content spanned from 0.15 to 32.52 pg.

     

Rapid detection of Phytophthora nicotianae by simple DNA extraction and real‐time loop‐mediated isothermal amplification assay

Phytophthora nicotianae is a phytopathogenic oomycete with a wide host range and worldwide distribution. Rapid detection and diagnosis at the early stages of disease development are important for the effective control of P. nicotianae. In this study, we designed a simple and rapid loop‐mediated isothermal amplification (LAMP)‐based detection method for P. nicotianae. We tested three DNA extraction methods and selected the Kaneka Easy DNA Extraction Kit version 2, which is rapid and robust for LAMP‐based detection. The designed primers were tested using mycelial DNA from 35 species (81 isolates) of Phytophthora, 12 species (12 isolates) of Pythium, one isolate of Phytopythium and one isolate each from seven other soil‐borne pathogens. All of the 42 P. nicotianae isolates were detected by these primers, and no other isolates gave positive results. Three isolates were tested for the sensitivity of the reaction, and the lowest amounts of template DNA that could be detected were 10 fg for two isolates and 1 fg for the third. The target was detected within 25 min in all tested samples, including DNA extracted from both inoculated and naturally infected plants. In contrast, PCR assays with P. nicotianae‐specific primers failed or showed weakened detection in several samples. Thus, we found that the rapid DNA extraction and LAMP assay methods developed in this study can be used to detect P. nicotianae with high sensitivity, specificity and stability.     

Detection of<Emphasis Type="Italic"> Bacillus cereus</Emphasis> group bacteria from cardboard and paper with real-time PCR

The aim of this study was to develop a PCR-based rapid method to detect Bacillus cereus group cells from paper and cardboard. Primers targeting the 16S rDNA and real-time PCR with SYBR green I detection were used in order to be able to also quantify the target. Both autoclaved cardboard samples spiked with B. cereus vegetative cells or spores and naturally contaminated paper and cardboard samples were studied. Results were compared with culturing verified by commercial (API) tests. Several different methods were tested for DNA isolation from the paper and cardboard samples. Two commercial kits intended for soils, the UltraClean soil DNA kit and the FastDNA spin kit for soil, gave the most reproducible results. In spiked samples, the average yield was 50% of added vegetative cells, but spore yield was only about 10%. PCR results from adding vegetative cells correlated with added colony-forming unit (cfu) values (r=0.93, P <0.001) in the range 100–10,000 cfu g^–1. Three out of nine studied paper and cardboard samples contained B. cereus group bacteria, based both on culturing and real-time PCR. The numbers were 10²–10³ bacteria g^–1; and PCR gave somewhat higher results than culturing. Thus, real-time PCR can be used as a rapid semi-quantitative method to screen paper and cardboard samples for contamination with B. cereus group bacteria.     

Simple and Rapid Method for PCR Characterization of Large <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Strain Collections

Polymerase chain reaction (PCR)-based identification of Bacillus thuringiensis toxin genes has become a routine step in most B. thuringiensis isolation and characterization initiatives. In the present study we propose a simplified method for extensive PCR analysis of B. thuringiensis cry and cyt genes of particular interest in large-scale screening programs. Fifty B. thuringiensis strains were screened for the presence of genes of the cry1 subfamily. Identical results were obtained when our method was used in comparison to other methodology based on a standard alkaline lysis preparation of plasmid DNA. Additional tests evidenced the fitness of our method in a particular multiplex–PCR analysis. The main advantages of the proposed methodology are that it is simpler and quicker than commonly used protocols (as DNA template preparation is substituted by direct addition of small amounts of B. thuringiensis liquid cell suspension to the reaction mixture) and that it allows simultaneous handling (bacterial growth and PCR) of up to 96 strains per round.