Extraction of DNA and RNA from Microorganism (original) (raw)

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

ISOLATION OF GENOMIC DNA FROM BACTERIAL CELLS

2021

The isolation of DNA from bacteria is a relatively simple process. The organism to be used should be grown in a favorable medium at an optimal temperature, and should be harvested in late log to early stationary phase for maximum yield. The genomic DNA isolation needs to separate total DNA from plasmid DNA, RNA, protein, lipid, etc. Initially the cell membranes must be disrupted in order to release the DNA in the extraction buffer. SDS (sodium dodecyl sulphate) is used to disrupt the cell membrane. Once cell is disrupted, the endogenous nucleases tend to cause extensive hydrolysis. Nucleases apparently present on human fingertips are notorious for causing spurious degradation of nucleic acids during purification. DNA can be protected from endogenous nucleases by chelating Mg2 ++ ions using EDTA. Mg2 ++ ion is considered as a necessary cofactor for action of most of the nucleases.

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.

ETHANOL EXTRACTION METHOD FOR DNA ISOLATION FROM

Genomic DNA isolation from the tough cell wall of the organism. Published methods for DNA extraction from M. smegmatis reproducible protocol for obtaining good quality DNA. The method is a part of a procedure used for extraction of the cell This method yields significant quantities of DNA as a by Copyright©2016, Karuna Gokarn et al. This is an open access article distributed under the Creative Commons Att use, distribution, and reproduction in any medium, provided the original work is properly cited.

Simple enzymic method for isolation of DNA from diverse bacteria

Journal of Microbiological Methods, 1989

An enzymic method for the extraction of high molecular weight DNA from a wide range of diverse bacteria, including mycobacteria, is described. The method is simple, rapid, requires few manipulations and is suitable for extracting quantitative yields of DNA from as few as-3 x 107 bacterial cells. The DNA produced is of good quality and is suitable for molecular biological manipulations.

Improved method for simultaneous isolation of proteins and nucleic acids

Analytical Biochemistry, 2011

Guanidinium thiocyanate-phenol-chloroform extraction (GTPC extraction) is widely used in molecular biology for isolating DNA, RNA, and proteins. Protein isolation by commercially available GTPC solutions is time consuming and the resulting pellets are only incompletely soluble. In this study ethanol-bromochloropropane-water was used for precipitation of proteins from the phenol-ethanol phase after GTPC extraction of RNA and DNA. The precipitated proteins can be readily dissolved in 4% SDS for subsequent analysis. This technique allows a fast (30 min) and efficient (with a protein recovery of up to 95%) extraction of proteins for the study of transcriptional and posttranscriptional events from the same sample.