DNA Extraction (DNA Purification) PDF

Molecular biology Dr. Dhama ALsallami DNA extraction (DNA purification) Introduction DNA extraction from a sample is a process of purifying the DNA. The sample can be tissue, plant or animal cells, blood, viral DNA or any other DNA containing...

Molecular biology Dr. Dhama ALsallami DNA extraction (DNA purification) Introduction DNA extraction from a sample is a process of purifying the DNA. The sample can be tissue, plant or animal cells, blood, viral DNA or any other DNA containing the sample. The idea of extracting the DNA is: Disruption of the cell membrane (and cell wall in case of plant cells) to make the DNA exposed and then separate it from the rest of the cell debris. Note: DNA is negatively (-) charged because of the presence of phosphate groups in nucleotides which is due to the presence of bonds created between the phosphorus and oxygen atoms. Basic Isolation Procedure There are five basic steps of DNA extraction: - 1) disruption of the cellular structure to create a lysate. 2) separation of the soluble DNA from cell debris and insoluble material. 3) binding the DNA of interest to a purification matrix. 4) washing proteins and other contaminants away from the matrix 5) elution of the DNA. 1. Creation of Lysate The goal of lysis is to rapidly and completely disrupt cells in a sample to release nucleic acid into the lysate. There are four general techniques for lysing materials: physical methods, enzymatic methods, and chemical methods. A. Physical Methods Physical methods involve some type of sample grinding or crushing to disrupt the cell walls or tough tissue. A common method of physical disruption is (freezing and grinding samples with a mortar under liquid nitrogen). B. Chemical Methods Chemical methods can be used alone with easy-to-lyse materials, such as tissue culture cells or in combination with other methods. Cellular disruption is accomplished with a variety of 1 Molecular biology Dr. Dhama ALsallami agents that disrupt cell membranes and denatures proteins. Chemicals commonly used include detergents (e.g., SDS) and, guanidine salts OR alkaline solutions. C. Enzymatic Methods Enzymatic methods are always used with more structured starting materials in combination with other methods with tissues, plant materials, bacteria and yeast. The enzymes utilized help to disrupt tissues and tough cell walls. Typical enzymatic treatments can include lysozyme, zymolase and liticase, proteinase K, collagenase and lipase. 2. Clearing of lysate Cellular lysates may need to have cellular debris removed before nucleic acid purification to reduce the unwanted materials (proteins, lipids and saccharides from cellular structures). Usually clearing is accomplished by centrifugation, filtration or bead-based methods. 3. Binding to the Purification Matrix DNA isolation systems based on sample lysis by detergents, and purification by binding to matrices (silica, cellulose and ion exchange). Binding capacity of these chemicals is an indication of how much nucleic acid an isolation chemistry can bind before it reaches the capacity of the system and no longer isolates more of that nucleic acid. 4. Washing Wash buffers generally contain alcohols and can be used to remove proteins, salts and other contaminants from the sample or the upstream binding buffers. Alcohols additionally help associate nucleic acid with the matrix. 5. Elution DNA is soluble in low-ionic-strength solution such as TE buffer or nuclease-free water. The purified, high-quality DNA is then ready to use in wide applications, such as multiplex PCR, vitro transcription/translation systems, and sequencing reactions. 2 Molecular biology Dr. Dhama ALsallami Agarose gel Electrophoresis 1. DILUTE concentrated (50X) buffer with distilled water to create 1X buffer. 2. MIX agarose powder with 1X buffer in a 250 ml flask. 3. DISSOLVE agarose powder by boiling the solution. MICROWAVE the solution on high for 1 minute. Carefully REMOVE the flask from the microwave and MIX by swirling the flask. Continue to HEAT the solution in 15-second bursts until the agarose is completely dissolved (the solution should be clear like water). 3 Molecular biology Dr. Dhama ALsallami 4. COOL agarose to 60° C. 5. While agarose is cooling, SEAL the ends of the gel-casting tray with the rubber end caps. PLACE the well template (comb) in the appropriate notch. 6. POUR the cooled agarose solution into the prepared gel-casting tray. The gel should thoroughly solidify within 20 minutes. 7. REMOVE end caps and comb. Take particular care when removing the comb to prevent damage to the wells. 8. PLACE gel (on the tray) into electrophoresis chamber. Completely COVER the gel with 1X electrophoresis buffer. 9. LOAD the DNA samples into wells. 10. PLACE safety cover. CHECK that the gel is properly oriented. Remember, the DNA samples will migrate toward the positive (red) electrode. 11. CONNECT leads to the power source and PERFORM electrophoresis. 12. After electrophoresis is complete, REMOVE the gel and casting tray from the electrophoresis chamber and proceed to VISUALIZATION using UV light trans illuminator. 4 Molecular biology Dr. Dhama ALsallami for 5 Molecular biology Dr. Dhama ALsallami 6