DNA Isolation Methods PDF

Isolation of Genomic DNA from Oral Mucosal Epithelium, Analysis of the Purity of Isolated DNA Department of Molecular Biology and Genetics Molecular Biology  Purpose of isolation The primary goal of isolation is to obtain, with maxi...

Isolation of Genomic DNA from Oral Mucosal Epithelium, Analysis of the Purity of Isolated DNA Department of Molecular Biology and Genetics Molecular Biology  Purpose of isolation The primary goal of isolation is to obtain, with maximum efficiency:  high molecular weight DNA,  purified from proteins and enzyme inhibitors that can impede subsequent DNA-related work. Applications include:  genetic disease detection,  assessment of predisposition to cancer development,  production of next-generation drugs,  cell therapy,  genetic engineering,  Choosing a DNA Isolation Method The choice of a suitable isolation method depends on:  the type of nucleic acid that we want to obtain (genomic, mitochondrial, plasmid DNA),  its origin (plant, animal, bacterial, viral, etc.),  the type of material from which the isolation is performed (tissues, organs, cell cultures, etc.),  the expected results (purity, quality, isolation time, etc.),  the intended purpose (PCR, cloning, etc.).  Material for isolation For molecular studies on humans, DNA isolated from peripheral blood is most commonly used. Blood should be collected in a tube containing EDTA (ethylene diamine tetraacetic acid), which prevents blood clotting and inhibits the activity of deoxyribonucleases. Collecting blood in heparin-containing tubes can have a significant impact on subsequent stages of work with the analyzed sample (it may partially transfer into the isolate and contribute to PCR reaction inhibition)  Material for isolation In human-related studies, DNA is also isolated from:  epithelial cells,  fibroblast cultures,  amniotic fluid cells (AFC),  chorionic villus sampling (CVS),  hair follicles. Less commonly used materials for diagnostic studies are: blood spots, semen, tissue fragments obtained by fine needle biopsy, and bone marrow  Stages of DNA isolation The isolation of total DNA consists essentially of several consecutive stages:  collection of the biological material sample,  transport,  homogenization of the collected material,  protein lysis (denaturation),  purification of DNA from the remnants of the lysate,  dissolving DNA,  qualitative and quantitative evaluation of the obtained DNA isolate  Transport The transportation of samples requires proper:  careful labeling of the samples,  secure sealing of the tubes or containers,  protection against damage and contamination  protection against changes in transportation conditions Principles of biological material transport  Storage DNA isolation should begin immediately after sample collection. Tissues can be stored at +4°C for several days without significant DNA degradation. If the material that cannot undergo DNA isolation within 48 hours of collection, it should be frozen and stored at -20°C or -80°C. Both the yield of isolation and the quality of DNA decrease with longer storage  Homogenization The method of homogenizing biological material depends on the origin of the material:  soft animal tissues: homogenization,  cell suspensions and biological fluids: sonication (ultrasound) or chemical lysis only,  plant cells: mechanical grinding,  bacterial cells and those from cell cultures: chemical lysis  Cell lysis Lysis is the disruption of cell membranes to release DNA/RNA from cellular structures, denature proteins, and inactivate nucleases. Enzymatic lysis: Proteinase K: a non-specific serine protease that inactivates nucleases, chitinase (fungi), collagenase, trypsin. Chemical lysis: Buffers containing chaotropic salts, e.g., guanidine isothiocyanate, NaI, detergents (CTAB, SDS, Triton X-100). Physical lysis: Heating the sample, freezing, osmotic shock. Mechanical lysis: Grinding with glass beads, aluminum oxides.  DNA Isolation Methods The most popular DNA isolation methods. Solvent-based methods:  DNA isolation using phenol and chloroform extraction (organic method).  DNA isolation with protein precipitation Chromatographic methods:  DNA isolation by binding DNA to a carrier (silica columns).  DNA isolation by magnetic separation  Purification of DNA – organic method Purifying DNA involves centrifugation to separate it from associated compounds using, for example:  a saturated phenol solution buffer,  chloroform with isoamyl alcohol,  chloroform with octanol,  a mixture of phenol with chloroform and isoamyl alcohol (25:24:1).  Scheme of nucleic acid isolation by phenol method incubation centrifugation Lysis mix with 1: DNA ethanol phenol 2: phenol + impurities precipitation  Scheme of nucleic acid isolation by salt method. incubation centrifugation Lysis mix with NaCl protein precipitation ethanol precipitation  DNA isolation by column Method Lysis occurs in the presence of proteinase K, a lysis buffer, and chaotropic salts. High concentrations of chaotropic salts cause DNA to adsorb to ion-exchange membranes by removing the "water shell" from its surface. The removal of impurities is achieved by washing the column with solutions of lower ionic strength. The final step is the elution of DNA from the membrane through the action of a low- salt buffer. DNA is detached from the membrane and enters the solution.  Scheme of nucleic acid isolation by column method Lysis DNA adsorption Washing Elution Isolate  Scheme of nucleic acid isolation by magnetic method Lysis Binding to paramagnetic Washing Elution Isolate beads  Quantification and Quality Assessment One of the crucial steps in working with genetic material is determining the purity and concentration of DNA and RNA preparations. The presence of contaminants like phenol, EDTA, heparin, etc., in DNA and RNA solutions can significantly interfere with many analytical techniques, especially PCR. DNA concentration can be determined in two ways:  spectrophotometric measurement.  agarose gel electrophoresis.  Spectrophotometric measurement DNA and RNA samples for absorbance measurement must be pre-diluted, typically with water or TE buffer. Quantification is performed using absorbance measurement (spectrophotometry). The same buffer used for diluting the samples is employed for spectrophotometer calibration. Determination of DNA and RNA solution concentration relies on measuring light absorption (optical density, OD) at the following wavelengths:  260 nm (maximum absorption for DNA and RNA)  280 nm (maximum absorption for proteins).  Spectrophotometric measurement The OD260/OD280 ratio (A260/A280) describes the purity of a DNA or RNA sample. Contaminant-free double-stranded DNA (dsDNA) has an A260/A280 ratio of approximately 1.8. For pure RNA, the A260/A280 ratio should be around 2.0. A value below 1.8 indicates the presence of proteins in the sample. An A260/A280 value of 1.5 signifies about 50% protein contamination. Purity of preparations