DNA Isolation Lecture Notes PDF
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Uploaded by CompactHeptagon
University of Technology
Mohammed Salah Faisal
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This document provides a detailed set of lecture notes on DNA isolation, covering the principles, methods, and applications such as in diagnosis and forensic medicine. The notes cover instruments, cell extraction, sample preparation, and purification techniques. These notes are suitable for undergraduates studying biochemistry.
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University of Technology Applied Sciences Department Applied Chemistry Branch DNA ISOLATION (1) Biochemistry Lab for 3rd class Prepared by Asst. Lec. Mohammed Salah Faisal INTRODUCTION DNA isolation: it is a process of purification of DNA using a combination of...
University of Technology Applied Sciences Department Applied Chemistry Branch DNA ISOLATION (1) Biochemistry Lab for 3rd class Prepared by Asst. Lec. Mohammed Salah Faisal INTRODUCTION DNA isolation: it is a process of purification of DNA using a combination of physical and chemical methods. The first isolation of DNA was done nm 1869 by Friedrich Miescher. INTRODUCTION Significance of DNA Isolation DNA Isolation is needed for genetic analysis, scientific, medical, or forensic purposes. DNA isolation technique provide pure DNA and devoid of contaminants. INSTRUMENTS UV-spectrophotometer Centrifuge or microfuge. Vortex mixer Micropipette Microtube Tips INSTRUMENTS DNA Isolation main steps 1. Preparation of a cell extract. 2. Purification of DNA from cell extract. 3. Concentration of DNA samples. 4. Measurement of purity of concentrated DNA. SAMPLES Common sources for DNA Isolation include: whole blood , hair, sperm, bones, nails, tissues, blood stains, saliva , buccal (cheek) swabs, epithelial cells. urine, paper cards used for sample collection. bacteria, animal tissues, or plants. 1. PREPARATION OF A CELL EXTRACT. To extract DNA from a tissue/cells of interest, the cells have to be separated and the cell membranes have to be disrupted by Extraction Buffer. 1. PREPARATION OF A CELL EXTRACT. Cell Components Extraction A. Ethylene Diamine Tetra Acetate (EDTA) or NaCl removes Mg2+ ions that preserves structure of the cell membrane. B. Sodium Dodecyl Sulfate (SDS) disrupts the cell membranes by removing the lipids of the cell membranes. C. Centrifugation or crushing leaving the cell extract as a clear supernatant. 2. PURIFICATION OF DNA FROM CELL EXTRACT Impurities Detergents Salts and reagents used for cell lysis Cell debris Proteins RNA 2. PURIFICATION OF DNA FROM CELL EXTRACT The most commonly used procedures are for purify is Ethanol precipitation. Phenol-chloroform extraction. Minicolumn purification (Chromotography). 3. CONCENTRATION OF DNA SAMPLES The most frequently used method of concentration is ethanol precipitation, using freezing ethanol Then: A. Draw With glass rod B. Centrifuge 4. MEASUREMENT OF PURITY OF CONCENTRATED DNA DNA concentrations can be accurately measured by UV-spectrophotometer at 260 nm and 280 nm (A260/A280) ratio = 1.8, Pure DNA sample. (A260/A280) ratio < 1.8, contaminated, either with protein or with phenol. 4. MEASUREMENT OF PURITY OF CONCENTRATED DNA APPLICATIONS 1. Diagnosis: DNA Mutation (sequence change in 1% of population) 2. Forensic medicine: For paternity testing Identification of individuals. APPLICATIONS 3. Medicine: To identify specific source of virulence of organisms in community or hospital 4. Constructing genome libraries:
