DNA Extraction Principles and Techniques PDF

DNA Extraction Principles and Techniques 7 October 2024 Assist: Lecturer Salma Zaher 1  “A DNA extraction is described as the separation of DNA DNA Extraction from the cell membrane and nuclear membrane using...

DNA Extraction Principles and Techniques 7 October 2024 Assist: Lecturer Salma Zaher 1  “A DNA extraction is described as the separation of DNA DNA Extraction from the cell membrane and nuclear membrane using chemicals, enzymes, or physical disturbances.” Definition “Nucleic acid extraction” or “DNA extraction” is the process of extracting nucleic acid from the rest of the cell organelle 7 October 2024 Assist: Lecturer Salma Zaher 2  Applications Genetic Engineering of Plants DNA extraction is integral to the process of genetic modification of plants. Many agricultural companies use genetic extraction to isolate DNA from organisms with desirable traits, which they then transplant into the plant’s genome. 7 October 2024 Assist: Lecturer Salma Zaher 3  Applications Altering Animals DNA extraction is also the first step in genetic engineering of animals. Genetic engineering of animals is a very broad field that ranges from editing a single gene to transplanting genes from one animal into another. For example, a Taiwanese research lab transplanted jellyfish genes into pigs, causing them to glow in the dark. 7 October 2024 Assist: Lecturer Salma Zaher 4  Applications Pharmaceutical Products DNA extraction is used as the initial step in manufacturing a number of pharmaceuticals. Hepatitis B vaccine and human growth hormone (hGh). In addition to a number of other hormones created using DNA extraction, one of the most widely used is insulin. 7 October 2024 Assist: Lecturer Salma Zaher 5 Applications Medical Diagnosis Diagnosis of certain medical conditions can often be made from DNA extracted from a patient. Conditions that can be diagnosed by genetic testing include cystic fibrosis, sickle-cell anemia, fragile x syndrome. 7 October 2024 Assist: Lecturer Salma Zaher 6 Applications Identity Verification A well-known use for genetic extraction is genetic fingerprinting, a process that matches genetic material from an individual with other genetic material available. One example is paternity testing, to determine someone’s biological father. Another common use for DNA extraction in identity verification is for forensic purposes. Genetic material from an individual can be compared to genetic material at a crime scene. 7 October 2024 Assist: Lecturer Salma Zaher 7  The cytoplasm and cell membrane/cell wall make up the cell. Several organelles, such as mitochondria, ribosomes, nucleus, and endoplasmic reticulum, are found in the cytoplasm. How to obtain The cell wall is absent in animal cells, although it is present in plant cells and (most) bacterial cells. DNA? To isolate DNA-deoxyribonucleic acid, we must first breach the cell wall/cell membrane, as well as the nuclear envelope. Other cellular organelle debris must also be removed. Precipitation and purification of the DNA are the final steps. 7 October 2024 Assist: Lecturer Salma Zaher 8 The Outline of the process Lysis of cell wall/ cell membrane Chemical disruption (Chelating agents, detergants) Enzymatic disruption (Lyases, glycosyl-hydrolases) Mechanical disruption (Ultrasonication) Lysis of nuclear membrane: Chemical lysis Enzymatic lysis Removing cell debris Centrifugation 7 October 2024 Assist: Lecturer Salma Zaher 9 7 October 2024 Assist: Lecturer Salma Zaher 10  Step 1. Breaking cells open to release the DNA The cells in a sample are separated from each other, often by a physical means such as grinding or vortexing, and put into a solution containing salt. A detergent What does DNA is then added. The detergent breaks down the lipids in the cell membrane and nuclei. DNA is released as these membranes are disrupted. extraction involve? Step 2. Separating DNA from proteins and other cellular debris To get a clean sample of DNA, it’s necessary to remove as much of the cellular debris as possible. This can be done by a variety of methods. Often a protease ( protein enzyme) is added to degrade DNA-associated proteins and other cellular proteins. Alternatively, some of the cellular debris can be removed by filtering the sample. 7 October 2024 Assist: Lecturer Salma Zaher 11  Step 3. Precipitating the DNA with an alcohol Finally, ice-cold alcohol (either ethanol or isopropanol) is carefully added to the DNA sample. DNA is soluble in water but insoluble in the presence of salt and alcohol. Step 4. Cleaning the DNA What does DNA extraction The DNA sample can now be further purified (cleaned). It is then resuspended in a slightly alkaline buffer and ready to use. involve? Step 5. Confirming the presence and quality of the DNA For further lab work, It is important to know the concentration and quality of the DNA. 7 October 2024 Assist: Lecturer Salma Zaher 12 DNA Extraction Techniques Phenol chloroform extraction Solid phase reversible immobilization extraction Chelex extraction 7 October 2024 Assist: Lecturer Salma Zaher 13 7 October 2024 Assist: Lecturer Salma Zaher 14  Phenol Chloroform Extraction 7 October 2024 Assist: Lecturer Salma Zaher 15  Solid-Phase Reversible Immobilization Extraction 7 October 2024 Assist: Lecturer Salma Zaher 16 Principle The beads consist of a polystyrene core surrounded by a thin layer of magnetite, which makes it paramagnetic (i.e., the beads will only clump together under a magnetic field). On the surface, the bead is coated by carboxyl molecules, which provide the charge groups for DNA binding. In the presence of polyethylene glycol (PEG) and salt, which work together as “crowding agents,” you can activate the beads to bind to DNA and the binding is reversible. A typical procedure involves the following five simple steps. 7 October 2024 Assist: Lecturer Salma Zaher 17  Steps 1 2 3 4 5 Step 1. Binding Step 2. Separation. Step 3. Wash. Step 4. Elution. Step 5. and mixing. A powerful magnet Rinse with ethanol The buffer separates Transfer. Aliquot This step is crucial pulls the beads (70%)* the DNA from the the DNA to a fresh because the ratio down, which are beads, which are tube for downstream between the PEG bound to the DNA still bound to the sequencing work and the DNA fragment you want magnet determines the size to purify. of the DNA that will bind to the beads. 7 October 2024 Assist: Lecturer Salma Zaher 18  Differential Extraction of Sperms from Mixed Samples The process of differential extraction used to separate male 7 October 2024 sperms from female epithelial cells. 19 Assist: Lecturer Salma Zaher