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PubMed2002

Evaluation of rapid DNA extraction methods for the quantitative detection of fungi using real-time PCR analysis.

Three comparatively rapid methods for the extraction of DNA from fungal conidia and yeast cells in environmental (air, water and dust) samples were evaluated for use in real-time PCR (TaqMan) analyses. A simple bead milling method was developed to provide sensitive, accurate and precise quantification of target organisms in air and water (tap and surface) samples. However, quantitative analysis of dust samples required further purification of the extracted DNA by a streamlined silica adsorption procedure.

DNA Primers↗

PubMedDate not supplied

Integrated method for single-cell DNA extraction, PCR amplification, and sequencing of ribosomal DNA from harmful dinoflagellates Cochlodinium polykrikoides and Alexandrium catenella.

A simplified technique was developed for DNA sequence-based diagnosis of harmful dinoflagellate species. This protocol integrates procedures for DNA extraction and polymerase chain reaction (PCR) amplification into a single tube. DNA sequencing reactions were performed directly, using unpurified PCR products as the DNA template for subsequent sequencing reactions. PCR reactions using DNA extracted from single cells of Cocodinium polykrikoides and Alexandrium catenella successfully amplified the target ribosomal DNA regions. DNA sequencing of the unpurified PCR products showed that DNA sequences corresponded to the expected locus of ribosomal DNA regions of both A. catenella and C. polykrikoides (each zero genetic distance and 100% sequence similarity). Using the protocol described in this article, there was little DNA loss during the purification step, and the technique was found to be rapid and inexpensive. This protocol clearly resolves the taxonomic ambiguities of closely related algal species (such as Alexandrium and Cochlodinium), and it constitutes a significant breakthrough for the molecular analysis of nonculturable dinoflagellate species.

Animals↗

PubMed2001

Rapid methods to extract DNA and RNA from Cryptococcus neoformans.

Extraction of nucleic acids from the pathogenic yeast Cryptococcus neoformans is normally hampered by a thick and resistant capsule, accounting for at least 70% of the whole cellular volume. This paper presents procedures based on mechanical cell breakage to extract DNA and RNA from C. neoformans and other capsulated species. The proposed system for DNA extraction involves capsule relaxation by means of a short urea treatment and bead beating. These two steps allow a consistent extraction even from strains resistant to other procedures. Yield and quality of DNA obtained with the proposed method were higher than those obtained with two earlier described methods. This protocol can be extended to every yeast species and particularly to those difficult to handle for the presence of a capsule. RNA purification is accomplished using an original lysing matrix and the FastPrep System (Bio101) after a preliminary bead beating treatment. Yields range around 1 mg RNA from 15 ml overnight culture (10(9) cells), RNA appears undegraded, making it suitable for molecular manipulations.

Centrifugation↗

PubMed2005

A simple automated instrument for DNA extraction in forensic casework.

The Qiagen BioRobot EZ1 is a small, rapid, and reliable automated DNA extraction instrument capable of extracting DNA from up to six samples in as few as 20 min using magnetic bead technology. The San Diego Police Department Crime Laboratory has validated the BioRobot EZ1 for the DNA extraction of evidence and reference samples in forensic casework. The BioRobot EZ1 was evaluated for use on a variety of different evidence sample types including blood, saliva, and semen evidence. The performance of the BioRobot EZ1 with regard to DNA recovery and potential cross-contamination was also assessed. DNA yields obtained with the BioRobot EZ1 were comparable to those from organic extraction. The BioRobot EZ1 was effective at removing PCR inhibitors, which often co-purify with DNA in organic extractions. The incorporation of the BioRobot EZ1 into forensic casework has streamlined the DNA analysis process by reducing the need for labor-intensive phenol-chloroform extractions.

Blood Stains↗

PubMed1997

DNA extraction from paraffin-embedded tissues using a salting-out procedure: a reliable method for PCR amplification of archival material.

Many techniques have been described for the extraction of DNA from paraffin-embedded tissues. Numerous efforts have been directed at simplification of these methods for rapid analysis using PCR. One disadvantage to some of the simpler procedures is inefficient PCR amplification, and for more involved ones using phenol/chloroform extraction, reduction in the yield of DNA. In the present study we report the use of a novel salting-out procedure that was utilized to extract DNA from 259 separate microdissection specimens of formalin-fixed, paraffin-embedded tissue sections. These sections were derived from 97 patients with tumors of the ampulla of Vater resected between 1965 and 1995 at our institution. The mean DNA yield was 22.75 micrograms (median 13.2 +/- 30.25) and the mean 260/280 absorbance ratio was 1.68 (median 1.70 +/- 0.25). All specimens (259/259) were successfully used to amplify K-ras exon 1 by a nested PCR technique. These results indicate that this DNA extraction method produces good yields of quality DNA, even from specimens several decades old.

Ampulla of Vater↗

PubMed2005

Comparison of six DNA extraction methods for recovery of fungal DNA as assessed by quantitative PCR.

The detection of fungal pathogens in clinical samples by PCR requires the use of extraction methods that efficiently lyse fungal cells and recover DNA suitable for amplification. We used quantitative PCR assays to measure the recovery of DNA from two important fungal pathogens subjected to six DNA extraction methods. Aspergillus fumigatus conidia or Candida albicans yeast cells were added to bronchoalveolar lavage fluid and subjected to DNA extraction in order to assess the recovery of DNA from a defined number of fungal propagules. In order to simulate hyphal growth in tissue, Aspergillus fumigatus conidia were allowed to form mycelia in tissue culture media and then harvested for DNA extraction. Differences among the DNA yields from the six extraction methods were highly significant (P<0.0001) in each of the three experimental systems. An extraction method based on enzymatic lysis of fungal cell walls (yeast cell lysis plus the use of GNOME kits) produced high levels of fungal DNA with Candida albicans but low levels of fungal DNA with Aspergillus fumigatus conidia or hyphae. Extraction methods employing mechanical agitation with beads produced the highest yields with Aspergillus hyphae. The Master Pure yeast method produced high levels of DNA from C. albicans but only moderate yields from A. fumigatus. A reagent from one extraction method was contaminated with fungal DNA, including DNA from Aspergillus and Candida species. In conclusion, the six extraction methods produce markedly differing yields of fungal DNA and thus can significantly affect the results of fungal PCR assays. No single extraction method was optimal for all organisms.

Aspergillosis↗

PubMed2004

Qualitative and quantitative evaluation of the genomic DNA extracted from GMO and non-GMO foodstuffs with four different extraction methods.

The presence of DNA in foodstuffs derived from or containing genetically modified organisms (GMO) is the basic requirement for labeling of GMO foods in Council Directive 2001/18/CE (Off. J. Eur. Communities 2001, L1 06/2). In this work, four different methods for DNA extraction were evaluated and compared. To rank the different methods, the quality and quantity of DNA extracted from standards, containing known percentages of GMO material and from different food products, were considered. The food products analyzed derived from both soybean and maize and were chosen on the basis of the mechanical, technological, and chemical treatment they had been subjected to during processing. Degree of DNA degradation at various stages of food production was evaluated through the amplification of different DNA fragments belonging to the endogenous genes of both maize and soybean. Genomic DNA was extracted from Roundup Ready soybean and maize MON810 standard flours, according to four different methods, and quantified by real-time Polymerase Chain Reaction (PCR), with the aim of determining the influence of the extraction methods on the DNA quantification through real-time PCR.

DNA↗

PubMed1998

DNA extraction from Ascaris suum muscle tissue.

A new method for the extraction of DNA from Ascaris suum muscle has been developed. It combines a standard SDS-based extraction with a plant DNA extraction procedure. The use of SDS and proteinase K allows the elimination of proteins, while CTAB and polyclar AT eliminate glycogen and polyphenols. The DNA thus obtained can easily be digested by endonucleases and amplified by PCR.

Animals↗

PubMed1987

Methods for DNA extraction from Candida albicans.

Three different methods are described for the extraction of total genomic DNA from the dimorphic fungus Candida albicans. One method, which enables a large number of cultures to be processed simultaneously, involves pulverizing dried cells with glass beads and then allowing the disrupted cells to break apart, autolyse, by incubation in a solution which includes sorbitol and a nonionic detergent. DNA extraction by a second method with a French pressure cell can be utilized on cultures in any phase of growth, but is not practical for processing numerous samples. The third method, which involves induction of spheroplasts, is commonly used for DNA extraction from various yeasts but is not suited for processing many samples simultaneously. The DNA extracted with the three procedures is comparable in quality; in particular, it is of high molecular size (greater than 30 kbp) and reacts readily with DNA-modifying enzymes such as restriction endonucleases.

Candida albicans↗

PubMed2024

High-throughput DNA extraction and cost-effective miniaturized metagenome and amplicon library preparation of soil samples for DNA sequencing.

Reductions in sequencing costs have enabled widespread use of shotgun metagenomics and amplicon sequencing, which have drastically improved our understanding of the microbial world. However, large sequencing projects are now hampered by the cost of library preparation and low sample throughput, comparatively to the actual sequencing costs. Here, we benchmarked three high-throughput DNA extraction methods: ZymoBIOMICS™ 96 MagBead DNA Kit, MP BiomedicalsTM FastDNATM-96 Soil Microbe DNA Kit, and DNeasy® 96 PowerSoil® Pro QIAcube® HT Kit. The DNA extractions were evaluated based on length, quality, quantity, and the observed microbial community across five diverse soil types. DNA extraction of all soil types was successful for all kits, however DNeasy® 96 PowerSoil® Pro QIAcube® HT Kit excelled across all performance parameters. We further used the nanoliter dispensing system I.DOT One to miniaturize Illumina amplicon and metagenomic library preparation volumes by a factor of 5 and 10, respectively, with no significant impact on the observed microbial communities. With these protocols, DNA extraction, metagenomic, or amplicon library preparation for one 96-well plate are approx. 3, 5, and 6 hours, respectively. Furthermore, the miniaturization of amplicon and metagenome library preparation reduces the chemical and plastic costs from 5.0 to 3.6 and 59 to 7.3 USD pr. sample. This enhanced efficiency and cost-effectiveness will enable researchers to undertake studies with greater sample sizes and diversity, thereby providing a richer, more detailed view of microbial communities and their dynamics.

Metagenome↗

PubMed2006

[Modified guanidine hydrochloride method for DNA extraction from cord blood used in HLA genotyping].

To compare two different methods for extracting genomic DNA from cord blood and to evaluate their applications for HLA genotyping, the genomic DNA from 72 samples was extracted by guanidine hydrochloride (Gu * HCl) and modified guanidine hydrochloride, the DNA yield and purity were evaluated by spectrophotometry and detected by PCR with sequence-specific primers. The result showed that the genomic DNA was successfully isolated from whole blood by both methods. The modified Gu * HCl method used was better than Gu * HCl method as the modified method produces better quality of DNA and less ambiguous bands in PCR. It is concluded that modified Gu * HCl method has the advantages of low-cost, simple operation, high quality output and clear positive bands in HLA-genotyping, the modified method is optimal for extracting DNA from multiple samples of cord blood bank.

DNA↗

PubMed1995

Direct DNA extraction for PCR-mediated assays of soil organisms.

By using the rDNA of a plant wilt pathogen (Verticillium dahliae) as the target sequence, a direct method for the extraction of DNA from soil samples which can be used for PCR-mediated diagnostics without a need for further DNA purification has been developed. The soil organisms are disrupted by grinding in liquid nitrogen with the natural abrasives in soil, and losses due to degradation and adsorption are largely eliminated by the addition of skim milk powder. The DNA from disrupted cells is extracted with sodium dodecyl sulfate-phenol and collected by ethanol precipitation. After suitable dilution, this DNA extract can be assayed directly by PCR amplification technologies. The method is rapid, cost efficient, and when combined with suitable internal controls can be applied to the detection and quantification of specific soil organisms or pathogens on a large-scale basis.

Base Sequence↗

PubMed1979

Preparation of competent single-cell suspensions of Mycoplasma hominis tets and Mycoplasma salivarium tets for genetic transformation to tetracycline resistance by DNA extracted from Mycoplamsa hominis tetr.

DNA extracted from Mycomplasma hominis (Sprott strain), resistant to 100 micrograms of tetracycline/ml transformed M. hominis strain H29 and Mycoplasma salivarium strain S9, which are sensitive to 2.5 and 5.0 micrograms of tetracycline/ml, respectively, to resistance. The transformants were selected on agar medium containing 10 micrograms of tetracycline/ml. Some transformants were resistant also to 20 micrograms of tetracycline/ml, a finding confirming that transformation occurred between homologous and heterologous species and that resistance is stepwise and controlled by several genetic loci. Medium containing 10 micrograms of tetracycline/ml was bacteriostatic. Prototype experiments employing mixtures of strains that were tetr and tets (tetracycline-resistant and tetracycline-sensitive, respectively) demonstrated that tetr mutants and transformants formed typical fried-egg colonies when mixtures containing not more than 10(9) mycoplasmas were spread on tetracycline agar plates. No mutants to tetracycline resistance were detected. Both M. hominis and M. salivarium were competent after treatment with MgCl2 and CaCl2, while Mycoplasma orale type 2 was inactivated. During DNA extraction different quantities of DNA formed insoluble precipitates with protein, thus preventing quantitative experiments.

Calcium Chloride↗

PubMed2004

[Examination of DNA extract from kernels and processed foods using silica-base resin].

A rapid and simple DNA extraction method is needed to detect genetically modified recombinant DNA in soybean kernels and processed foods. However, since various kernels and processed foods differ greatly in form, a uniform DNA extraction method has proved elusive. The silica-base resin DNA extraction method does not use any organic solvent, and the operation is simple and the cost per extraction is low, although the frequency of its use is very low and few domestic reports exist. We therefore studied suitable conditions for a silica-base resin method. We also developed the method to get more pure DNA from soybean kernels. The silica-base resin method was found to be adequate for extracting DNA from various processed foods for PCR amplification with endogenous gene primers. In the case of DNA extraction from soybean kernels, pure DNA could be efficiently extracted after pre-heating the soybean suspension in TNE buffer. The extracted DNA showed higher ratios of absorption at 260 nm/280 nm and 260 nm/230 nm than those for samples obtained with previous methods. Moreover, our observations suggested that the extraction time could be reduced to within 30 min for processed foods such as tofu.

DNA, Recombinant↗

PubMed2005

Detection of ostreid herpesvirus-1 (OsHV-1) by PCR using a rapid and simple method of DNA extraction from oyster larvae.

A DNA extraction procedure was developed for the detection of ostreid herpesvirus-1 (OsHV-1) using the polymerase chain reaction (PCR) in oyster larvae. The DNA extraction procedure developed was tested on 8 larval samples. Abnormal nuclei with characteristic features associated with OsHV-1 infections were only observed in samples in which the viral DNA was detected by PCR. A previously described competitive PCR method was applied to detect inhibition during PCR reactions. The results show that the method can be used on small amounts of oyster larvae (3 mg) for the detection of OsHV-1 DNA by PCR.

Animals↗

PubMed2000

STR-genotyping of archaeological human bone: experimental design to improve reproducibility by optimisation of DNA extraction.

The analysis of degraded DNA with the help of short tandem repeat loci (STRs) is an important source of information both in forensic casework and in the anthropological context. The reproducibility of STR-genotyping of highly degraded or "ancient" DNA can be reduced by the generation of artifacts during PCR amplification. The frequency and amount of these artifacts--allelic dropout and the generation of shadow bands--are related to the quality and quantity of the extracted DNA amplified in a PCR reaction. Therefore, one important strategy to increase the reproducibility of STR-genotyping of samples containing degraded DNA is the optimisation of the DNA extraction.

Archaeology↗

PubMed1990

Quantitative and qualitative analysis of DNA extracted from postmortem muscle tissues.

DNA extracted from 33 postmortem muscle specimens was analyzed using MZ 1.3, a hypervariable minisatellite probe, as well as locus-specific minisatellite probes (g3, MS1 and MS43). After storage at -25 degrees C for 10 months, DNA from all the samples was partially (approximately 21% of total DNA) degraded even when autopsy was performed 1 day postmortem. However, more than 90% of DNA samples up to at least 3 days postmortem were suitable to obtain good restriction fragment length polymorphism (RFLP) patterns. When small strips of specimen were stored for 8 days at room temperature in moist chambers, approximately 42% of total DNA was degraded. Only 30% of these DNA samples still showed good RFLP patterns. However, no obvious relation between qualities of DNA analyzed by detection of RFLP and quantities of total and high-MW DNA became apparent. A case of familial relationship was ascertained by DNA fingerprints. Since DNA of good quality can be recovered from muscle tissues in large quantities, DNA extraction from muscle tissues and detection of RFLP patterns should be very useful for individual identification in autopsy cases.

Accidents, Aviation↗

PubMed2004

Fabrication of amino silane-coated microchip for DNA extraction from whole blood.

A simple microchip device for DNA extraction was constructed based on electrostatic interactions between surface amine groups and DNA. Microchannel was fabricated on silicon wafer by photolithography and coated with 3-aminopropyltriethoxysilane (APTES) or 3-[2-(2-aminoethylamino)-ethylamino]-propyltrimethoxysilane (AEEA) to introduce amine groups on the surface. Determination of the number of surface amine groups and optimization of DNA capture condition were demonstrated to characterize the microchip. Capacities of capturing DNA were approximately 97 ng/cm2 in APTES and 194 ng/cm2 in AEEA modified microchips, respectively. The amount of DNA captured in the microchip increased depending on surface amine density. Furthermore, DNA extraction using amine-coated microchip from whole blood was examined. Quantification of DNA and proteins in washing or eluting fraction indicates that proteins were removed at washing steps and only DNA was effectively eluted by changing alkalinity of buffer from pH 7.5 to 10.6. The amount of DNA extracted from whole blood was approximately 10 ng and its recovery ratio was 27-40%. Performance of PCR for the eluted fraction indicates that DNA extracted from whole blood was well purified using amine-coated microchip.

Adsorption↗