Proteinase K is not essential for marine eDNA metabarcoding (original) (raw)
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Effect of Proteinase-K on Genomic DNA Extraction from Gram-positive Strains
Direct extraction of DNA from natural environment and clinical samples has become a useful alternative for the phylogenetic identification and in situ detection of individual microbial cells without cultivation. In this study, three different Gram positive microorganisms (B. cereus, B. subtilis, and S. aureus) were chosen for genomic DNA extraction. High salt SDS (Sodium Dodesyl Sulfate) based extraction method was followed to extract genomic DNA with addition of three different lysis protocols to observe the effect of proteinase-K on total genomic DNA yield, lysis steps were carried with SDS, SDS with 3 l proteinase-K and SDS with 6l proteinase-K. High molecular weight intact DNA bands were observed only for Bacillus subtilis when the extraction procedure was carried out in presence of SDS, SDS with proteinase-K (3l) and SDS with increased amount of proteinase-K (6l). In presence of SDS and increased amount of proteinase-K (6l) the mean value of DNA concentration for Bacillus cereus, Bacillus subtilis, and Staphylococcus aureus were found to be 1.530.15, 1.360.10 and 1.650.10 g/l respectively. However, in absence of proteinase-K, the mean values of DNA concentration were found to be decreased (1.280.10, 1.340.15, 1.230.10 g/l for B. cereus, B. subtilis, and S. aureus respectively) for all these stains. Although in case of B. subtilis the overall effect of proteinase-K was not found to be significant in terms of DNA concentration and DNA band intensity, however, for B. cereus, and S. aureus sharp decrease in total extracted DNA concentration was observed suggesting the increased lysis effect of proteinase-K on the thick peptidoglycan layer of Gram-positive cell wall such as B. cereus, and S. aureus.
The assessment of DNA from marine organisms via a modified salting-out protocol
Cellular & Molecular Biology Letters, 2006
We developed a rapid, practical and non-toxic salting-out method for the extraction of DNA from marine organisms, and tested it on two representative species of Porifera and Cnidaria, both living in association with symbiotic zooxanthellae. We tested the efficiency of the protocol by comparing the output of the method for fresh tissue, frozen tissue and tissue stored in ethanol. It proved to be effective for extracting DNA in the case of the methods of preservation considered here, and for obtaining quantities of DNA comparable to those obtained via the traditional approach. The DNA from both species was of good quality. The DNA obtained was amplified by PCR using specific primers for the large ribosomal subunit, allowing the identification of the presence of both the host and symbiont genomes.
Review Article DNA, RNA, and Protein Extraction: The Past and The Present
Extraction of DNA, RNA, and protein is the basic method used in molecular biology. These biomolecules can be isolated from any biological material for subsequent downstream processes, analytical, or preparative purposes. In the past, the process of extraction and purification of nucleic acids used to be complicated, time-consuming, labor-intensive, and limited in terms of overall throughput. Currently, there are many specialized methods that can be used to extract pure biomolecules, such as solution-based and column-based protocols. Manual method has certainly come a long way over time with various commercial offerings which included complete kits containing most of the components needed to isolate nucleic acid, but most of them require repeated centrifugation steps, followed by removal of supernatants depending on the type of specimen and additional mechanical treatment. Automated systems designed for medium-to-large laboratories have grown in demand over recent years. It is an alternative to labor-intensive manual methods. The technology should allow a high throughput of samples; the yield, purity, reproducibility, and scalability of the biomolecules as well as the speed, accuracy, and reliability of the assay should be maximal, while minimizing the risk of cross-contamination.
Scientia Marina, 2005
Assay protocols for RNA and DNA in crude plankton extracts using the fluorochrome SYBR Green II are developed here. The method is based on the fluorescence in 3 aliquots: the first measures RNA after DNA digestion; the second measures DNA after RNA digestion; and the third measures residual fluorescence after digestion of both DNA and RNA. This residual fluorescence measurement is critical for accurate calculations of the nucleic acids. Optimisation of the assay conditions are described: fluorochrome concentration, buffer composition, fluorescence stability, temperature and duration of nuclease incubation. In the optimised procedure, the assays are performed in 5 mM Tris buffer (containing 0.9 mM CaCl 2 •2H 2 O and 0.9 mM MgCl 2 •6H 2 O, pH 8.0); DNase and RNase incubations are conducted at 37ºC for 20 min; the fluorochrome is added to all assays at a final concentration of 3.5x10-4 and readings are done within the 10-60 min period following the SYBR Green II addition. The study evidenced the importance of the residual fluorescence after nuclease digestion, which is especially taken into account in the calculation of the nucleic acid concentrations. Finally, the variability of the fluorescent response to different RNA and DNA standards is examined; from the performed tests, calculations are based on rRNA from calf liver and DNA from calf thymus standards. The accompanying paper (Berdalet et al., 2005) describes the development of the extraction protocol, as well as the application of both protocols in measuring RNA/DNA ratios in natural plankton samples, and a comparison with ethidium bromide based methods.
Extraction from natural planktonic microorganisms of DNA suitable for molecular biological studies
Applied and environmental microbiology, 1988
We developed a simple technique for the high-yield extraction of purified DNA from mixed populations of natural planktonic marine microbes (primarily bacteria). This is a necessary step for several molecular biological approaches to the study of microbial communities in nature. The microorganisms from near-shore marine and brackish water samples, ranging in volume from 8 to 40 liters, were collected on 0.22-mum-pore-size fluorocarbon-based filters, after prefiltration through glass fiber filters, to remove most of the eucaryotes. DNA was extracted directly from the filters in 1% sodium dodecyl sulfate that was heated to 95 to 100 degrees C for 1.5 to 2 min. This procedure lysed essentially all the bacteria and did not significantly denature the DNA. The DNA was purified by phenol extraction, and precautions were taken to minimize shearing. Agarose gel electrophoresis showed that most of the final preparation had a large molecular size (>23 kilobase pairs). The DNA was sufficientl...
Extraction of DNA and RNA from Microorganism
2020
The enzyme lysozyme is used for the rupturing of cell wall of bacteria or microorganism. The high salt concentration prevents the conversion of dsDNA to ssDNA. EDTA and sodium citrate present helps to chelate metal ions which activate DNase and RNase enzymes. The protein denaturation is done by sodium chlorate and finally extraction is done either by ethanol or isopropanol method as discussed earlier in section. Material and Reagents: Any bacteria (for the first timers, use Escherichia coli). Solution A: Saline EDTA pH 8.0 in distilled water. Sodium chloride (0.15 mol/liter) ¼ 125 ml. EDTA (0.1 mol/liter) ¼125 ml. Enzyme lysozyme (N-acetylmuramide glycanhydrolase) ¼ 100 mg. Sodium lauryl sulfate (10% w/v) ¼ 50 ml. Sodium chlorate (6 mol/liter) ¼ 100 ml. Chloroform: Isoamyl alcohol (24: 1) ¼ 500 ml. Absolute ethanol. Sodium acetate (3 mol/liter) ¼ 25 ml. Isopropanol ¼ 100 ml. Sodium hydroxide (6 mol/liter) ¼ 50 ml.
A large number of different protocols for the efficient isolation of highly purified DNA from eukaryotic and prokaryotic cells is extant. (1-4) These procedures usually include treatment with proteinase K in the presence of SDS, which efficiently lyses the cells and nuclei and liberates the DNA tightly bound in chromatin. (s) Proteins are then extracted with phenol and chloroform, and the nucleic acids are precipitated with ethanol. This procedure is tedious and time-consuming, and significant amounts of DNA may be lost, especially when working with small specimens (e.g., joint biopsies). Therefore, this approach is not appropriate for diagnostic tests. Direct amplification of digested samples without phenol/chloroform extraction and precipitation is not possible because SDS is inhibitory to Taq polymerase at concentrations as low as 0.01%. (6) Alternative simple DNA extraction procedures have been used but have often resulted in incomplete lysis of the cells. These procedures typically have included detergents (e.g., Triton X-100), chaotropes (e.g., guanidium isothiocyanate or sodium iodide), proteases (e.g., proteinase K), substances that lyse erythrocytes and leukocytes (e.g., saponin), or heat denaturation. Often nonionic detergents such as Tween 20 or Laureth 12 in combination with proteinase K are used, followed by heat inactivation of the enzyme prior to PCR amplification. (7-1°)
Frontiers in Microbiology, 2015
A method for the extraction of nucleic acids from a wide range of environmental samples was developed. This method consists of several modules, which can be individually modified to maximize yields in extractions of DNA and RNA or separations of DNA pools. Modules were designed based on elaborate tests, in which permutations of all nucleic acid extraction steps were compared. The final modular protocol is suitable for extractions from igneous rock, air, water, and sediments. Sediments range from high-biomass, organic rich coastal samples to samples from the most oligotrophic region of the world's oceans and the deepest borehole ever studied by scientific ocean drilling. Extraction yields of DNA and RNA are higher than with widely used commercial kits, indicating an advantage to optimizing extraction procedures to match specific sample characteristics. The ability to separate soluble extracellular DNA pools without cell lysis from intracellular and particle-complexed DNA pools may enable new insights into the cycling and preservation of DNA in environmental samples in the future. A general protocol is outlined, along with recommendations for optimizing this general protocol for specific sample types and research goals.
Section 1 update: Simplified protocols for the preparation of genomic DNA from bacterial cultures
Molecular Microbial Ecology Manual, 2008
in the handling of the preparations, which are necessary for obtaining genomic DNA of high molecular weight. Thus, in general, the most desirable means of disrupting bacterial cells for obtaining genomic DNA is through enzymatic digestion and detergent lysis. Such a strategy is enhanced by prior treatment of cells with a metal chelating agent, such as ethylenediamine-tetraacetic acid (EDTA). If the cell wall of the organism is susceptible to such treatments, relatively high molecular-weight genomic DNA can be obtained which is applicable for a number of analytical techniques. Further, the lysis should be carried out in a buffered (pH 8-9) medium containing EDTA. The alkaline pH reduces electrostatic interactions between DNA and basic proteins, assists in denaturing other cellular proteins and inhibits nuclease activities. EDTA binds divalent cations, particularly Mg 2+ and Mn 2+ , reducing the stabilities of the walls and membranes and also inhibits nucleases which have a requirement for metal cations. Cell disruption by enzymatic treatments Lysozyme, isolated commercially from chicken egg white, is a member of the broad class of muramidases which catalyse the hydrolysis of the β-1,4-glycosidic linkage between the N-acetylmuramic acid-N-acetylglucosamine repeating unit, comprising a major part of the peptidoglycan layer of the cell walls of most bacteria [18]. Lysozyme is especially effective in disrupting bacterial cells when used in combination with EDTA [15]. Lysozyme and related enyzmes are useful for disrupting the cells of a broad range of bacterial species, although many species are not particularly susceptible to muramidase treatment due, presumably, to layers of protein or capsular slime, which protect the peptidoglycan. Additionally, as their cell walls do not contain peptidoglycan, all described species of Archae are resistant to lysozyme activity. Proteinase K, a serine protease produced by the fungus Tritirachium album, cleaves adjacent to the carboxyl groups of aliphatic and aromatic amino acids involved in peptide bonding [4], including those comprising the peptide crosslinking interbridges of the peptidoglycan layers of the cell walls of bacteria. The applicability of Proteinase K for disrupting bacterial cell walls is enhanced by its insensitivity to specific chelating agents, allowing it to be utilised in combination with EDTA and lysozyme. However, the peptide interbridges of the cell walls of different species, formed by different combinations of component amino acids, with inherently different susceptibilities to cleavage, may be more or less resistant to Proteinase K lysis. While lysozyme and proteinase K are, probably, the enzymes most commonly used for the disruption of bacterial cells, additional bacterial cell-disrupting enzymes also have been reported with broad or narrow specificities. Other muramidases, mutanolysin and lysostaphin react, analogous to lysozyme, at the peptide linkages in the cell walls, although the species which are susceptible to these enzymes differ from those which are affected by lysozyme [2, 20, 26]. Subtilisins are extracellular proteases, produced by Bacillus spp., exhibiting a broad specificity in hydrolysing most peptide and ester bonds [24]. They are not inactivated MMEM-1.01/4 MMEM-1.01/5 MMEM-1.01/8 Figure 3. The recovery of DNA as a function of the amount of DNA in suspension. The recovery of DNA was observed to be dependent on the concentrations of the suspensions. The values indicated represent the means, calculated from the observed recoveries from suspension, after varying centrifugation times. The ranges of observed recoveries are indicated, with the lowest and highest recoveries, for each DNA concentration, corresponding to the shortest and longest centrifugation times (5-30 minutes). The graph was prepared from data taken from Zeugin and Hartley, 1985 [27]. Procedures The specific methods described here are simplified, rapid, protocols observed to be effective for isolating genomic DNA, from a wide range of bacteria, of a quality applicable for PCR. Protocol I-CTAB protocol for the extraction of bacterial genomic DNA This protocol is derived from the "miniprep" method described by Wilson [25]. Broth cultures (2-5 ml) grown to mid-log growth phase are harvested in 2.0 ml Eppendorf tubes by centrifugation in
DNA extraction from formalin-fixed tissue: new light from the deep sea
Scientia Marina, 2010
The present study is a follow-up on previously published protocols on the extraction of DNA from formalin-preserved samples. It is also discussed why, when using formalin-preserved specimens for molecular analyses, only a fraction of the amplifications are successful. DNA samples were separately extracted from ethanol and formalin-fixed tissue using methods based on Tetramethylsilane (TMS)-Chelex. It was found that amplification success does not depend on the dehydration step, or the removal of soluble PCR inhibitors as previously stated, and that DNA is not simply trapped in an indigestible matrix of cross-linked protein. Instead, amplification success was found to depend on the amount of unmodified DNA present in the extracted sample. Moreover, in order to optimise the effort when analysing formalinfixed samples, a staining method is presented which facilitates the targeting of samples with a high content of unmodified DNA.