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DNA Isolation

DNA isolation is a process of purification of DNA from using a combination of physical and chemical methods. The first isolation of DNA was done in 1869 by Friedrich Miescher. Isolation of DNA basically consists of four major steps. i. Preparation of a cell extract. ii. Purification of DNA from cell extract. iii. Concentration of DNA samples. iv. Measurement of purity of DNA concentration. Reagents used for A. Preparation of cell extract 1. Ethylene Diamine Tetra Acetate (EDTA) removes Mg2+ ions. 2. Sodium Dodecyl Sulfate (SDS) disrupts the cell membranes 3. Centrifugation B. Purification of DNA . 1. Ethanol precipitation. 2. Phenol—chloroform extraction. 3. Mini column purification. Removal of Protein impurities 1.adding proteases. 2.precipitated with sodium or ammonium acetate. 3.extracted proteins with a phenol-chloroform mixture C. Concentration of DNA 1.absolute ethanol D. Measurement of purity of DNA Concentration Absorbance is measured at 260 nm by UV absorbance spectrometer. i.1.0 corresponds to 50 µg of ds DNA per ml. ii.A260 nm and 280 nm ratio equal to 1.8, Pure DNA sample ii.Ratio less than 1.8, contaminated, either with protein or with phenol. Application of DNA Isolation 1.Diagnosis : Mutation that is DNA sequence change Genetic polymorphism 2.Forensic medicine: For paternity testing and Identification of individuals. 3.Medicine: To identify specific source of virulence of organisms in community or hospital 4.Constructing genome libraries 5. for RT-PCR eg Corona Virus is an RNA virus whose RNA is separated from Oro and Nasopharyngeal swab samples and then reverse transcribed to DNA and then amplified by PCR