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 GENE AMPLIFICATION P OLYM ER A SE CHAI N R EAC T ION 4/21/2024 ASS.LECTURER: SALMA ZAHER 1 Gene Amplification Gene amplification refers to an increase in the number of copies of a gene in a genome. Cancer cells, for example, sometimes produce multiple copies of a gene(s) in response to signals from other cells or the environment. One well-known example of gene amplification and cancer is amplification of the HER2 gene in a subset of breast cancers. HER2 gene amplification results in the production of excess HER2 protein on the surface of the cancer cell. 4/21/2024 ASS.LECTURER: SALMA ZAHER 2 Polymerase chain reaction (PCR) PCR is a technique used to make numerous copies of a specific segment of DNA quickly and accurately. The polymerase chain reaction enables investigators to obtain the large quantities of DNA that are required for various experiments and procedures in molecular biology, forensic analysis, evolutionary biology, and medical diagnostics. 4/21/2024 ASS.LECTURER: SALMA ZAHER 3 4/21/2024 ASS.LECTURER: SALMA ZAHER 4 Principle The PCR technique is based on the enzymatic replication of DNA. In PCR, a short segment of DNA is amplified using primer mediated enzymes. DNA Polymerase synthesizes new strands of DNA complementary to the template DNA. The DNA polymerase can add a nucleotide to the pre-existing 3’-OH group only. Therefore, a primer is required. Thus, more nucleotides are added to the 3’ prime end of the DNA polymerase. 4/21/2024 ASS.LECTURER: SALMA ZAHER 5 Components PCR reaction mixture has to include: 1- DNA template 2- Two PCR primers 3- DNA polymerase 4- Deoxynucleotide triphosphates (dNTPs) 5- Buffer solution 4/21/2024 ASS.LECTURER: SALMA ZAHER 6 4/21/2024 ASS.LECTURER: SALMA ZAHER 7 Components 1. (DNA Template): PCR is a highly sensitive technique and requires only one or two DNA templates for successful amplification. 2. (PCR Primers): The PCR requires the knowledge of DNA sequences that flank the DNA template. Primers are short nucleotide sequences (approximately 15–30 bases) that base pair to a specific portion of the DNA being replicated. 3. (Taq Polymerase): Taq DNA polymerase is the most commonly used enzyme for standard PCR amplification. The function of Taq polymerase is to replicate the target DNA. The DNA polymerases recognize primers as start tags. 4/21/2024 ASS.LECTURER: SALMA ZAHER 8 PCR Primer 4/21/2024 ASS.LECTURER: SALMA ZAHER 9 Taq Polymerase 4/21/2024 ASS.LECTURER: SALMA ZAHER 10 Components 4. (dNTPs): Deoxynucleotide triphosphates (dNTPs) are the building blocks from which the DNA polymerase synthesizes a new DNA strand during successive cycles of PCR amplification. dNTPs consist of four basic nucleotides - dATP, dCTP, dGTP, and dTTP. 5. (Buffer): A buffer of the PCR reaction mixture serves as a chemical environment to maintain an activity and stability of the DNA polymerase. The buffer pH is usually between 8.0 and 9.5 and is often stabilized by Tris-HСl 4/21/2024 ASS.LECTURER: SALMA ZAHER 11 Steps There are three major steps in a PCR, This is done on an automated cycler which are repeated for 25 or 35 cycles. “Thermocycler”, which can heat and cool the tubes with the reaction mixture in a very short time. 4/21/2024 ASS.LECTURER: SALMA ZAHER 12 4/21/2024 ASS.LECTURER: SALMA ZAHER 13  Step 1- Denaturation During the denaturation, the double strand melts open to single stranded DNA. The solution contained in the tube is heated to at least 94°C (201.2°F) using a thermal cycler. The heat breaks the hydrogen bonds of the original DNA sample and separates the DNA into single strands (this is termed denaturation of double-stranded DNA). Normally the denaturation time is 1 min at 94oC: it is possible, for short template sequences, to reduce this to 30 sec or less. Increase in denaturation temperature and decrease in time may also work (as recommend 96oC for 15 sec. 4/21/2024 ASS.LECTURER: SALMA ZAHER 14 4/21/2024 ASS.LECTURER: SALMA ZAHER 15 Step 2 - Annealing The sample mixture is then cooled to between 50 to 60°C (122 to 140°F) allowing the DNA primers and the DNA polymerase enzyme to bind to the individual strands of DNA that were separated by the heat (this is termed annealing of the primers). At this point, the nucleotides (A, T, C, G) from the added mixture solution will pair with the individual separated strands of DNA that resulted from the heating process. 4/21/2024 ASS.LECTURER: SALMA ZAHER 16 4/21/2024 ASS.LECTURER: SALMA ZAHER 17 Step 3 - Extension Once joined together, they form a new complementary strand of DNA (termed extension of the DNA). Thus, a new duplicate double-stranded DNA molecule has been formed from each of the single strands of the original sample molecule. The temperature cycles from 95°C to 50 to 60°C. The cycle is then repeated about 35 to 40 times using the thermal cycler which automatically repeats the heating and cooling cycles of the process. Resulting DNA sequence is doubled each time the heating/cooling cycle is conducted by the cycler. Thus, what started out as a single short segment of DNA from one sample can be amplified to form millions of copies after 35 doubling cycles 4/21/2024 ASS.LECTURER: SALMA ZAHER 18 4/21/2024 ASS.LECTURER: SALMA ZAHER 19 4/21/2024 ASS.LECTURER: SALMA ZAHER 20 Step 4: Validating the Reaction Once your PCR reaction has run, there are two ways of determining success or failure. The first is to simply take some of the final reaction and run it out on an agarose gel with an appropriate molecular weight marker to make sure that the reaction was successful and if the amplified product is the expected size relative to the maker. 4/21/2024 ASS.LECTURER: SALMA ZAHER 21 Applications of PCR PCR is used in many research labs, and it also has practical applications in forensics, genetic testing, and diagnostics. For instance, PCR is used to amplify genes associated with genetic disorders from the DNA of patients (or from fetal DNA, in the case of prenatal testing). PCR can also be used to test for a bacterium or DNA virus in a patient's body: if the pathogen is present, it may be possible to amplify regions of its DNA from a blood or tissue sample. 4/21/2024 ASS.LECTURER: SALMA ZAHER 22 4/21/2024 ASS.LECTURER: SALMA ZAHER 23 Advantages of PCR over other diagnostic tests Owing to its high sensitivity. Specificity and speed. PCR provides a method for obtaining large quantities of specific DNA sequences from small amount of DNA, including degraded DNA samples. The specificity of the PCR reaction is imposed by the use of priming oligonucleotides and theoretically focuses the amplification activities on only a small region of the target DNA 4/21/2024 ASS.LECTURER: SALMA ZAHER 24  Gel Electrophoresis 11/24/2024 Ass.Lecturer: Salma Zaher 1 What is Gel Electrophoresis ? Gel electrophoresis is a procedure used to separate biological molecules by size. The separation of these molecules is achieved by placing them in a gel with small pores and creating an electric field across the gel. The molecules will move faster or slower based on their size and electric charge. 11/24/2024 ASS.LECTURER: SALMA ZAHER 2  What is Gel Electrophoreses Used For? The purpose of gel electrophoresis is to visualize, identify and distinguish molecules that have been processed by a previous method such as PCR, enzymatic digestion or an experimental condition. Often, mixtures of nucleic acids or proteins that are collected from a previous experiment/method are run through gel electrophoresis to determine the identity or differentiate between molecules. 11/24/2024 ASS.LECTURER: SALMA ZAHER 3  There are basically two types of materials are used to make gels: 11/24/2024 ASS.LECTURER: SALMA ZAHER 4 1. AGAROSE Agarose is natural colloid which is isolated from the seaweed. It is linear polysaccharide. This gel has generally larger pore size, which makes them suitable to separate larger molecules having molecular mass more than 200 kDa. electrophoresis It is most commonly used for the electrophoresis of both protein and nucleic acids. Agarose is used in concentration between 1% and 3%. 11/24/2024 ASS.LECTURER: SALMA ZAHER 5 11/24/2024 ASS.LECTURER: SALMA ZAHER 6 2. POLYACRYLAMIDE GEL Polyacrylamide gel is consisting of chains of acrylamide monomers. In this gel, pore size and resolving power is totally depends upon the concentration of acrylamide and bisacrylamide. The concentration of the gel normally varies from 5% to 25%. This gel is used in electrophoresis for the separation of proteins ranging from molecular weight 200,000 11/24/2024 ASS.LECTURER: SALMA ZAHER 7 11/24/2024 ASS.LECTURER: SALMA ZAHER 8 APPARATUS OF GEL ELECTROPHORESIS Vertical gel apparatus: It is commonly used in SDS PAGE for the separation of proteins. Horizontal gel apparatus: It is used for immune electrophoresis, DNA electrophoresis of DNA and RNA in the agarose gel. 11/24/2024 ASS.LECTURER: SALMA ZAHER 9  Examples of Gel Electrophoresis 1. Agarose gel electrophoresis 2. SDS-PAGE 3. Pulse field gel electrophoresis (PFGE) 4. 2D gel electrophoresis 11/24/2024 ASS.LECTURER: SALMA ZAHER 10  1- Agarose Gel Electrophoresis 11/24/2024 ASS.LECTURER: SALMA ZAHER 11 Requirement/ instrumentation: 1) An electrophoretic unit 2) A power supply 3) Gel casting trays 4) Combs 5) Agarose gel or media 6) Transilluminator: (An ultraviolet light box), which is used to visualize bands in gels. 11/24/2024 ASS.LECTURER: SALMA ZAHER 12 Electrophoresis Buffer Composition and ionic strength of electrophoresis buffer is most important factor for the separation of nucleic acids (DNA or RNA). Most routinely used buffers are: TAE- (Tris-acetate-EDTA), it has lower buffering capacity and generally used to separate larger nucleic acid fragments (>12kb). TBE- (Tris-borate-EDTA), it has high buffering capacity and higher ionic strength and generally used for the separation of low molecular weight compound (