DNA Extraction PDF

DNA Extraction DNA extraction is an extraction process of DNA from various source. The aim: is to separate DNA present in the nucleus of the cell from other cellular components. Purpose: To extract genetic material in relatively purified form for genetic, molecular or forensic anal...

DNA Extraction DNA extraction is an extraction process of DNA from various source. The aim: is to separate DNA present in the nucleus of the cell from other cellular components. Purpose: To extract genetic material in relatively purified form for genetic, molecular or forensic analysis Types of DNA: 1. genomic (chromosomal, nuclear) 2. organellar (satellites), 3. plasmid DNA or extra-chromosomal: →mitochondrial (mDNA), chloroplast (clDNA), 4. cDNA (complementary DNA), 5. phage or viral (ds or ss Process (3 basic steps) 1. Break the cells open to expose DNA 2. Remove membrane lipids by adding detergent 3. Precipitate DNA with an alcohol — usually ethanol isopropanol Since DNA is insoluble in these alcohols, it will aggregate together, giving a pellet upon centrifugation. This step also removes alcohol soluble salt. LYSIS in DNA extraction from plants, this step often refers to the breaking of the cell wall & cellular membranes (most importantly, the plasma & nuclear membranes) CW (made of cellulose) is disrupted by mechanical grinding the leaves) +++Detergent = breaks down the cell membranes force (e.g. Detergents = able to disrupt membranes due to the amphipathic (having both hydrophilic & hydrophobic regions) nature of both cellular membranes & detergent molecules; detergent molecules are able to pull apart the membranes End result of LYSIS: contents of plant cells are distributed in solution. Lysis of the Cell. Use Detergent to solubilize the membrane lipid. PRECIPITATION this a series of steps where DNA is separated from the rest of the cellular components In a research lab, 1st part of precipitation uses phenol/chloroform to remove proteins from DNA ) 17 Phenol denatures proteins & dissolves denatured proteins. Chloroform is also a protein denaturant (THIS STEP CANNOT BE PERFORMED IN CLASSROOM LABS!!!) 2nd part of research lab DNA precipitation is the addition of salts Salts interrupt the H bonds between water & DNA molecules. DNA is then precipitated from the protein in a subsequent step with isopropanol or ethanol In the presence of cations, ethanol induces a structural change in DNA molecules that causes them to aggregate & precipitate out of solution. Washing and Re-suspension Washing: Precipitated DNA is laden with acetate salts. It is “washed” with a 70% ethanol solution to remove salts & other water soluble impurities but not re-suspend the DNA. Re-suspension: Clean DNA is now re-suspended in a buffer to ensure stability & long term storage. ✓Most commonly used buffer for re-suspension is called 1xTE Checking the Quality of DNA ✓The product of DNA extraction will be used in subsequent experiments; poor quality DNA will not perform well in PCR ✓You will want to assess the quality of the DNA extracted using the following simple protocol: Mix 10 µL of DNA with 10 µL of loading buffer Load this mixture into a 1% agarose gel Analyze results (the following slides provide guidance Analyzing DNA Samples in a Research Lab If properly done, genomic extraction should result in bright bands in the very high base pair range of a gel electrophoresis. Analyzing DNA Samples in a Classroom Lab A B C D E Ladder Analysis of samples: Barley (A): This sample is fine Corn (B): This sample is fine Oat (C) : This sample is fine Rice (D) : This sample is fine Wheat (E): This sample has severe degradation, can work for PCR but should re-extract 11 DNA Storage Options Salt solution - EDTA – prevents enzyme activity (only short term) Freezing– expensive, risky Alcohol– explosive, needs regular checking (evaporation), degradation, store at cold temperature (4oC, -20oC) Dried- extract and purify, apply to membranes, store dry (make sure it’s dry) Importance in Agriculture An essential part of agricultural biotechnology is the extraction of DNA, which enables scientists to genetically modify organisms for agricultural purposes and helps plant and animal breeders choose more precisely for desired features. Summary of DNA extraction: 1. Cell lysis, to expose the DNA within. 2. Removing membrane lipids by adding a detergents or surfactants. 3. Removing proteins by adding a protease. 4. Removing RNA by adding an Rnase. 5. Precipitating the DNA with alcohol - usually ice cold ethanol. In these alcohols, DNA strand will aggregate together, giving a pellet upon centrifugation. This step also removes alcohol- soluble salt. Spectrophotometers Vertical electrophoresis Gel Electrophoresis