DNA Isolation - Procedures and Methods

**DNA isolation** *Purposes of DNA isolation* =========================== Isolation of DNA is needed for genetic analysis, which is used for scientific, medical, or الطب الشرعيforensic purposes Aim of DNA isolation ==================== To separate DNA present in the nucleus of the cell from othe...

**DNA isolation** *Purposes of DNA isolation* =========================== Isolation of DNA is needed for genetic analysis, which is used for scientific, medical, or الطب الشرعيforensic purposes Aim of DNA isolation ==================== To separate DNA present in the nucleus of the cell from other cellular components. **DNA Extraction kits**  **Sample selection** ***Sources for DNA isolation are very diverse*** - Basically it can be isolated from any living or dead organism. - Common sources for DNA isolation include whole blood , hair, sperm , bones, nails, tissues, blood stains, saliva , epithelial cells, urine, paper cards used for sample collection, bacteria, animal tissues, or plants. PROCEDURE --------- Isolation of DNA basically consists of four major steps. 1- Preparation of a cell extract. 2. Purification of DNA from cell extract. 3. Concentration of DNA samples. 4. Measurement of purity of DNA concentration **.** ***[1. Preparation of a cell extract]*** - The \"Extraction buffer\" helps in separate the cells and disrupt cell membrane. - Chemicals such as EDTA (Ethylene Diamine Tetra Acetate) which removes Mg2+ ions that are essential for preserving the overall structure of the cell membrane, and SDS (Sodium Dodecyl Sulfate) which aids in disrupting the cell membranes by removing the lipids of the cell membranes are included in the extraction buffer.  The removal of insoluble cell debris: Cell debris and partially digested organelles etc. can be pelleted by centrifugation leaving the cell extract as a reasonably clear supernatant **.** ***[2. Purification of DNA from cell extract.]*** - In addition to DNA the cell extract will contain significant quantities of detergents, proteins, salts and reagents used during cell lysis step and RNA. - A variety of procedures can be used to remove these contaminants, leaving the DNA in a pure form. - The most commonly used procedures are: i. Ethanol precipitation. ii. Phenol--chloroform extraction. iii. Minicolumn purification. 3. Concentration of DNA samples ------------------------------- The most frequently used method of concentration is ethanol precipitation. In the presence of salt and at a temperature of -20°C or less, absolute ethanol will efficiently precipitate polymeric nucleic acids. ### a. Spectrophotometer/ nanometer