An effective method for isolation of DNA from pig faeces and comparison of five different methods

Polymerase chain reaction (PCR) detection of microorganism in faecal specimens is hampered by poor recovery of DNA and by the presence of PCR inhibitors. In this paper, we describe a new modified method for extracting PCR-quality microbial community DNA from pig faecal samples, which combines the pretreatment with polyformaldehyde, and subsequent DNA lysis in the presence of CTAB, salt, PVP, and β-mercaptoethanol, followed by isolation of nucleic acids using chloroform (no phenol) based protocol. The method resulted in a 1.3- to 11-fold increase in DNA yield when compared to four other widely used methods. Genomic DNA extracted from all five methods was assessed by both agarose gel electrophoresis and polymerase chain reaction for amplification of 16S rDNA specific fragments. The results showed that the improved method represented a reproducible, simple, and rapid technique for routine DNA extraction from faecal specimens and was notably better than using the QIAamp^® DNA Stool Mini Kit.     

A comparison of five methods for DNA isolation from liver and rumen flukes to perform ITS-2+ amplification

Five different DNA isolation methods (4 commercial kits and a modification of phenol-chloroform method) were compared for the discrimination of adults of Fasciola hepatica and Dicrocoelium dendriticum (liver flukes), and Calicophoron daubneyi (rumen fluke) collected from sheep in southern Italy. The second internal transcribed spacer (ITS-2) of ribosomal DNA (rDNA) plus flanking 5.8S and 28S sequence (ITS-2+) was amplified by polymerase chain reaction (PCR) from serial diluted DNA templates (6 ng - 60 fg) of each fluke species. Overall, in terms of efficiency in detection limit, the best results were obtained either with phenol-chloroform purification or with QIAamp DNA Mini Kit (Qiagen), but using this latter method, rapid, safe and not expensive, an increased level of sensitivity sufficient to detect small amounts of target-DNA was achieved. In addition, electrophoresis analysis following PCR also showed that ITS-2+ could be useful as a genetic marker for the molecular identification of F. hepatica, D. dendriticum and C. daubneyi in definitive and intermediate hosts. Furthermore, for the first time, the ITS-2 sequence of D. dendriticum was defined.     

An efficient method for DNA isolation from red algae

A simple, inexpensive and efficient method was developed for rapid isolation of totalgenomic DNA from 15 red algal species. It resulted in 0.1 g high quality DNAfrom 1 mg fresh algal material, with an A₂₆₀/A₂₈₀ratio of 1.68–1.90.Using this rapidly isolated DNA, the 18S ribosomal RNA genes (rDNA) and the nuclearribosomal DNA of the internal transcribed spacer (ITS) regions were amplified. Thetested DNA was suitable for restriction endonuclease digestion, genetic markeranalysis and polymerase chain reaction (PCR) amplification, and may be valid forother genetic manipulation.     

An isolation method of DNA from Pneumocystis carinii: a quantitative comparison to known parasitic protozoan DNA

1. A method for isolating DNA from Pneumocystis carinii is described. 2. The DNA content per nucleus is 0.22-0.34 pg. 3. This finding is consistent with other parasitic protozoa DNA content per nuclei.     

A comparison of five methods for extraction of bacterial DNA from human faecal samples

The purity of DNA extracted from faecal samples is a key issue in the sensitivity and usefulness of biological analyses such as PCR for infectious pathogens and non-pathogens. We have compared the relative efficacy of extraction of bacterial DNA (both Gram negative and positive origin) from faeces using four commercial kits (FastDNA kit, Bio 101; Nucleospin C+T kit, Macherey-Nagal; Quantum Prep Aquapure Genomic DNA isolation kit, Bio-Rad; QIAamp DNA stool mini kit, Qiagen) and a non-commercial guanidium isothiocyanate/silica matrix method. Human faecal samples were spiked with additional known concentrations of Lactobacillus acidophilus or Bacteroides uniformis, the DNA was then extracted by each of the five methods, and tested in genus-specific PCRs. The Nucleospin method was the most sensitive procedure for the extraction of DNA from a pure bacterial culture of Gram-positive L. acidophilus (10(4) bacteria/PCR), and QIAamp and the guanidium method were most sensitive for cultures of Gram-negative B. uniformis (10(3) bacteria/PCR). However, for faecal samples, the QIAamp kit was the most effective extraction method and led to the detection of bacterial DNA over the greatest range of spike concentrations for both B. uniformis and L. acidophilus in primary PCR reactions. A difference in extraction efficacy was observed between faecal samples from different individuals. The use of appropriate DNA extraction kits or methods is critical for successful and valid PCR studies on clinical, experimental or environmental samples and we recommend that DNA extraction techniques are carefully selected with particular regard to the specimen type.     

An efficient method for isolation of plasmid DNA from methylotrophic bacteria

A method, suitable for the isolation of closed circular plasmid DNA from methylotrophic bacteria is described. Improvement of cell lysis was achieved by butanol extraction of cells before application of the lytic agent. Using this method, cryptic plasmids of 7.8, 14, 36 and 200 kb were purified from soil-isolated methylotrophs.     

An effective method for isolating DNA from historical specimens of baleen

DNA was isolated from an early twentieth century museum specimen of northern right whale baleen. A system of stringent controls and a novel set of cetacean specific primers eliminated contamination from external sources and ensured the authenticity of the results. Sequence analysis revealed that there were informative nucleotide positions between the museum specimen and extant members of the population and closely related species. The results indicate that museum specimens of baleen can be used to assess historical genetic population structure of the great whales.     

Comparison of different methods for the isolation and purification of total community DNA from soil

The efficiency and reproducibility of DNA extraction from soil was tested for variations in lytic and purification treatments and their effect on yield and purity of DNA. The extraction yield was improved by increasing the concentration of EDTA or monovalent ions in isolation buffers, by the introduction of mechanical lysis treatments, and by the use of ethanol precipitation in place of PEG precipitation. Purity was improved using buffers with decreasing concentration of EDTA or by reducing the ionic strength of the buffer, and by all mechanical treatments. No lytic treatment was efficient on its own, the highest purity was achieved using Crombach buffer and a combination of bead-beating with lysozyme and SDS lysis followed by potassium acetate and PEG precipitation, phenol/chloroform purification, isopropanol precipitation, and spermine-HCl precipitation. Sonication sheared the DNA more than bead-beating. Lysozyme and SDS lysis without any mechanical treatments allowed isolation of larger fragments (40-90 kb). Denaturing gradient gel electrophoresis analysis of DNA isolated using a range of lytic treatments revealed alterations in band patterns which might reflect differences in the efficiency of lytic treatments.     

一种经济高效提取实蝇基因组DNA的方法

单头实蝇高质量基因组DNA的获得为实蝇分子生态学研究奠定了重要基础。本文提出一种经济高效的实蝇基因组DNA提取方法,该方法广泛适用于不同虫态、不同保存条件的实蝇标本,与传统的CTAB法和SDS法相比操作简单、耗时短,得率高。     

An improved method for the rapid isolation of chromosomal DNA from Mycoplasma spp

A method is described for the rapid isolation of chromosomal deoxyribonucleic acid from species of the genus Mycoplasma. The method involves incubation of washed cells at elevated temperature in the presence of an ionic detergent, chelating agents, and proteinase K prior to the removal of residual protein and ribonucleic acid with ribonuclease and chloroform. It results in a good yield of high molecular weight material that is shown to be free of endogenous nuclease and substantially free of protein or ribonucleic acid contamination without the use of phenol. The isolated DNA is shown to be an excellent substrate for restriction endonuclease digestion and ligation with T4 DNA ligase.     

Evaluation of isolation methods for pathogenic Yersinia enterocolitica from pig intestinal content

Aims: The aim of this study was to evaluate the efficiency of four isolation methods for the detection of pathogenic Yersinia enterocolitica from pig intestinal content. Methods and Results: The four methods comprised of 15 isolation steps using selective enrichments (irgasan–ticarcillin–potassium chlorate and modified Rappaport broth) and mildly selective enrichments at 4 or 25°C. Salmonella–Shigella‐desoxycholate–calcium chloride agar, cefsulodin–irgasan–novobiocin agar were used as plating media. The most sensitive method detected 78% (53/68) of the positive samples. Individual isolation steps using cold enrichment as the only enrichment or as a pre‐enrichment step with further selective enrichment showed the highest sensitivities (55–66%). All isolation methods resulted in high numbers of suspected colonies not confirmed as pathogenic Y. enterocolitica. Conclusions: Cold enrichment should be used in the detection of pathogenic Y. enterocolitica from pig intestinal contents. In addition, more than one parallel isolation step is needed. Significance and Impact of the Study: The study shows that depending on the isolation method used for Y. enterocolitica, the detected prevalence of Y. enterocolitica in pig intestinal contents varies greatly. More selective and sensitive isolation methods need to be developed for pathogenic Y. enterocolitica.     

小麦线粒体DNA的高效提取方法

以小麦黄化苗为材料, 通过简单差速离心、DNaseⅠ处理得到无核DNA杂质的线粒体, 用SDS和蛋白酶K裂解线粒体, 经酚/氯仿抽提除去蛋白, 并用RNase A消化而得到单纯线粒体DNA(mtDNA)。对所提取的mtDNA进行紫外吸收光度分析, A₂₆₀/A₂₈₀ 平均为1.92, A260/A230 平均为2.09, 平均每克黄化苗可提取mtDNA 26.85 mg; 并对mtDNA进行琼脂糖凝胶电泳和RAPD扩增, 均得到清晰的电泳图谱。结果表明: 此提取方法得到的mtDNA, 不但产率高、结构完整, 而且能有效去除核DNA、RNA和蛋白质等杂质, 获得高质量的mtDNA用于PCR反应和各种遗传学分析。研究还发现, 通过调整线粒体裂解温度(先50℃裂解1 h, 再37℃裂解1 h), 亦可大幅度提高mtDNA的产率。