6b.+Restriction+Enzyme+Digest

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Introduction
Now that we have bacterial colonies that we are sure include our plasmid, we want to be able to recover the plasmid from those colonies. This will require two steps. First, we will perform a restriction enzyme digest. The selected colonies will be lysed (split) open and the DNA will be separated from the rest of the cellular matter. Then, the DNA will be cut by restriction enzymes. Lastly, the cut DNA pieces will be separated by size using gel electrophoresis.

Restriction Enzymes
Restriction enzymes are used to make recombinant DNA.
 * Gene cloning and genetic engineering were made possible by the discovery of restriction enzymes that cut DNA molecules at specific locations.
 * In nature, bacteria use restriction enzymes to cut foreign DNA, to protect themselves against phages or other bacteria.
 * They work by cutting up the foreign DNA, a process called //restriction.//[[image:http://www-math.mit.edu/%7Elippert/18.417/lectures/02_PartialDigest/Pictures/RestrictionEnzymes.gif width="306" height="336" align="right"]]
 * Most restriction enzymes are very specific, recognizing short DNA nucleotide sequences and cutting at specific points in these sequences.
 * Bacteria protect their own DNA by methylating the sequences recognized by these enzymes.
 * Each restriction enzyme cleaves a specific sequence of bases or restriction site**.**
 * These are often a symmetrical series of four to eight bases on both strands running in opposite directions.
 * If the restriction site on one strand is 3’-CTTAAG-5’, the complementary strand is 5’-GAATTC-3’.
 * Because the target sequence usually occurs (by chance) many times on a long DNA molecule, an enzyme will make many cuts.
 * Copies of a DNA molecule will always yield the same set of restriction fragments.
 * Restriction enzymes cut covalent sugar-phosphate backbones of both strands, often in a staggered way that creates single-stranded sticky ends**.** In the diagram, BamHI, HindIII, and EcoRI produce these sticky ends.
 * Sticky ends, however, do not always occur. When a restriction enzyme cleaves both strands in the same location, blunt ends are produced. AluI and HaeIII are examples of restriction enzymes which produce blunt ends.
 * These extensions can form hydrogen-bonded base pairs with complementary single-stranded stretches (sticky ends) on other DNA molecules cut with the same restriction enzyme.
 * These DNA fusions can be made permanent by DNA ligase**,** which seals the strand by catalyzing the formation of covalent bonds to close up the sugar-phosphate backbone.
 * Restriction enzymes and DNA ligase can be used to make a stable recombinant DNA molecule, with DNA that has been spliced together from two different organisms.


 * In restriction fragment analysis, the DNA fragments produced by restriction enzyme digestion of a DNA molecule are sorted by gel electrophoresis.
 * When the mixture of restriction fragments from a particular DNA molecule undergoes electrophoresis, it yields a band pattern characteristic of the starting molecule and the restriction enzyme used.
 * The relatively small DNA molecules of viruses and plasmids can be identified simply by their restriction fragment patterns.
 * The separated fragments can be recovered undamaged from gels, providing pure samples of individual fragments.
 * We can use restriction fragment analysis to compare two different DNA molecules representing, for example, different alleles of a gene.
 * Because the two alleles differ slightly in DNA sequence, they may differ in one or more restriction sites.
 * If they do differ in restriction sites, each will produce different-sized fragments when digested by the same restriction enzyme.
 * In gel electrophoresis, the restriction fragments from the two alleles will produce different band patterns, allowing us to distinguish the two alleles.

Check out this link for an interactive electrophoresis activity.







Multimedia
DNA fingerprinting using restriction enzymes media type="youtube" key="drC7rR7CIBg" width="425" height="350" align="center"

Practice Quiz
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Practice Free Response Questions
//Answer the following question on a loose sheet of paper (or word process it). When completed, compare it to the rubric below, and determine your score.//

1. A bacterial plasmid is 100 kb in length. The plasmid DNA was digested to completion with two restriction enzymes in three separate treatments: EcoRI, HaeIII, and HaeIII + EcoRI (double digest). The fragments were then separated with electrophoresis, as shown.

a.) Using the circle provided, **construct** a labeled diagram of the restriction map of the plasmid. **Explain** how you developed your map. b.) **Describe** how: c.) **Discuss** how a specific genetically modified organisms might provide a benefit for humans and at the same time pose a threat to a population.
 * recombinant DNA technology could be used to insert a gene of interest into a bacterium
 * recombinant bacteria could be identified
 * expression of the gene of interest could be ensured



scoring rubric