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Gel Electrophoresis What is Gel Electrophoresis? Gel electrophoresis separates molecules on the basis of their charge and size. The charged macromolecules migrate across a span of gel because they are placed in an electrical field. The gel acts as an obsicle to slow the passage of molecules according to their size and shape. Restriction digest of DNA One of the basic tools of modern biotechnology is DNA restriction enzyme digestion. This is the process of cutting DNA into smaller fragments using enzymes. This allows scientists to analyze and compare DNA from different sources, and to study the smaller more manageable pieces of DNA. Because enzymes are very specific in their effects on a substrate, they are predictable and repeatable in their effects. Restriction Enzymes The ability to cut DNA predictably is due to the restriction enzymes specificity they cut at only one sequence of nucleotides. They were first identified in and isolated from the bacteria that use them as a natural defense mechanism to cut up the invading DNA of bacteriophages – viruses that infect bacteria. They are named for their function of restricting the growth of viruses through the cutting of DNA with enzymes. How does electrophoresis work? • The gel is made from agar, a substance in seaweed. •DNA has a negative charge on its particles. • Molecules sort based on: •Charge - The greater the charge the more pull. •Size – Bigger pieces are slower, smaller are faster. •Shape - Coiled is slower straight is faster. •The negatively charged particles move toward the positive electrode while the positive charge particles Move toward the negative electrode. Agarose makes them move slower and shorter distances if they are big, and faster and further if they are small pieces of DNA. How does it work? • DNA is cut into smaller fragments with restriction enzymes that cut at only certain sequences of nucleotides. • Loading dye is used to indicate the movement of the fragments of DNA. • The negative DNA molecule is attracted to the positive electrode. •The smallest fragments move the greatest distance. Look Closely at the Gel and note similarities and differences in bands and patterns in the samples A - H A B C D E F G H Analyzing a Gel Which lane contains the most fragments of DNA? Which sample of DNA has the smallest and largest fragments cut by the restriction enzyme? Why do some bands of DNA fragments appear in more than one lane of the gel? How could you use this technique to analyze DNA?