Understand how to use a restriction digestion map to identify a sample DNA.Learn to separate DNA on an agarose gel using electrophoresis.Understand what a DNA restriction enzyme is and how it works.The gels may be stained overnight prior to photographing or recording results. The restriction digestion takes place overnight and can be kept in the freezer until the next class period when it will be be used for gel electrophoresis. This laboratory will take approximately 3 days. The gel is then stained with a methylene blue stain to visualize the DNA bands and may be photographed. The DNA fragments are separated by electrophoresis, a process that involves application of an electric field to cause the DNA fragments to migrate into an agarose gel. After overnight digestion, the reaction is stopped by addition of a loading buffer. In this experiment, we will perform a full restriction digestion. This is termed a partial restriction digestion. This will result in fragments ranging in size from the smallest possible (all sites are cut) to in-between lengths (some of the sites are cut) to the longest (no sites are cut). If the virus DNA is exposed to the restriction enzyme for only a short time, then not every restriction site will be cut by the enzyme. Since the whole sequence of λ is already known we can predict where each restriction enzyme will cut and thus the expected size of the fragments that will be produced. This virus is 48,502 base pairs in length which is very small compared with the human genome of approximately 3 billion base pairs. In this experiment, we will use restriction enzymes to cut up DNA from a small virus called Bacteriophage λ. This would include transferring genes that will result in a change in the nutritional quality of a crop or perhaps allow a plant to grow in a region that is colder than its usual preferred area. This DNA may contain genes that allow the organism to exhibit a new function or process. This ability of DNA to repair itself has been utilized by scientists to introduce foreign DNA into an organism. It doesn’t matter if the fragment that matches the cut end comes from the same organism or from a different one. In the presence of specific DNA repair enzymes, DNA fragments will reanneal or stick themselves to other fragments with cut ends that are complimentary to their own end sequence. Since the recognition site or sequence of base pairs is known for each restriction enzyme, we can use this to form a detailed analysis of the sequence of bases in specific regions of the DNA in which we are interested. The size of these fragments is measured in base pairs or kilobase (1000 bases) pairs. The restriction enzyme will continue to do this along the full length of the DNA molecule which will then break into fragments. Once it is located, the enzyme will attach to the DNA molecule and cut each strand of the double helix. This recognition site or sequence is generally from 4 to 6 base pairs in length. These restriction enzymes are able to scan along a length of DNA looking for a particular sequence of bases that they recognize. Special enzymes termed restriction enzymes have been discovered in many different bacteria and other single-celled organisms.
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