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Download Note 8.2 - DNA Sequencing
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UNIT 3: Molecular Genetics Chapter 8: Genetic Technologies pg. 364 - 421 8.2: DNA Sequencing pg. 376 – 385 DNA sequence is amplified by PCR, it is then separated and purified. Gel electrophoresis uses the physical and chemical properties of the DNA sequence to separate it into fragments. The gel electrophoresis uses an agarose gel to act like a sieve to separate nucleic acids and proteins by the rate of their migration through the gel. The gene fragments are usually negatively charged and migrate towards the positive electrode. The smaller fragments will migrate at a faster through the pours of the gel, then their larger fragment counterparts. This causes the separation of fragments. Figure 3: The four steps in the separation of DNA fragments by agarose gel electrophoresis. There are 4 steps used to perform a gel electrophoresis; Step 1: the agarose gel is prepared and placed in a gel box between two electrodes. The gel is prepared with a series of wells, inset, at one end of the gel. A buffer solution is poured over the gel to allow the charge to pass from one electrode to another. Step 2: Load the DNA sample solutions, PCR products, into the wells using a micropipette. The first well is load with DNA marker which acts as measuring scale to compare DNA migrations to. The size of the DNA fragments migration distances are compared to the markers to determine fragment sizes. A loading dye is used to keep the DNA within well in the gel. Also the dye moves a little faster then the DNA fragment. This allows you to see the pace of the electrophoresis. Step 3: The electric current is applied to the gel. The DNA fragments that are negatively charged start to migrate towards the positive electrode. Shorter DNA moves through the gel at a faster rate then the larger fragments. Once the electric current is turned off the fragment distance are compared to the markers. Distance traveled will determine the size of the fragments. Step 4: DNA fragments are hard to see therefore a stain (Ethidium bromide) is applied to the gel staining the DNA fragments, which are easily seen under an ultra violet light. Gel electrophoresis: is a method for separating large molecules, such as DNA, RNA, and proteins. Molecule Marker: is a fragment of a known size that is run as a comparison standard for gel electrophoresis. Ethidium Bromide: is a large molecule that resembles a bas pair, which allows it to insert itself into DNA; used for staining electrophoresis gels.