Lecture25S10n - BIS101/Engebrecht Lecture25 5/28/10...

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BIS101/Engebrecht Lecture25 5/28/10 Restriction mapping and gel electrophoresis: Once a piece of DNA has been identified, we need to analyze it. Using restriction enzymes, one can obtain a map of a fragment of DNA. Such a map indicates the position of the recognition sequences of different restriction enzymes on a piece of DNA in relation to each other. Thus, in a restriction digest, the resulting fragments will have a defined size in base pairs and defined ends (the ends generated by digestion with the restriction enzyme). The size of the fragments can be determined by gel electrophoresis. This works because the size of a DNA fragment is directly proportional to charge because of the repeating nature of the phosphate backbone of the double helix. These phosphate groups are negatively charged so in a matrix (agarose or polyacrylamide), the DNA is loaded on top of the gel and an electric field is applied such that the DNA moves toward the positive end. The migration distance is directly proportional to the size (= charge). Using marker DNA with known sizes it is possible to calibrate the gel to determine the size of the fragments generated by restriction digest. To visualize the DNA on the gel, you need to stain the gel with a dye that binds to DNA. Usually, we use ethidium bromide. This intercalates into the DNA molecule and is fluorescent by epifluorescence (so that if you shine a UV light on the gel, you will see the DNA glowing). I showed you a picture from your book of what such a gel looks like. We went through Handout29Restriction mapping. CQ1: You are told that there is a single restriction recognition site for each of the enzymes.
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Lecture25S10n - BIS101/Engebrecht Lecture25 5/28/10...

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