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Name: ______________________________________________ Date: _____________ Hour: ______ BIOLOGY B Genetic Recombination Lab.2016 DNA fragments from donor cells must become part of the genetic material of living cells before the genes they contain can be activated. For example, DNA fragments may be combined with bacterial DNA so that they can later be inserted into a bacterial cell. Bacteria often contain small circular DNA molecules known as plasmids in addition to their chromosome. Scientists use restriction enzymes to cut segments of genetic material from plasmids. When the plasmids are cut, unpaired bases are left at the ends. These exposed bases are sometimes called ‘sticky ends’ because new segments of DNA can be attached to them by hydrogen bonding as if they were sticky, like tape. In this activity, you will make and use a model of the technique used to combine pieces of DNA. In so doing you will form a new plasmid that contains a piece of foreign DNA. This new plasmid is called recombinant DNA. If a scientist would like to make many copies (clone) a human gene, he can use this technique. First, he must identify the human gene in which he is interested. This gene is called the ‘gene of interest’! He must then treat his sample of human DNA with the same restriction enzyme used to cut the plasmid. By using the same restriction enzyme, the scientist makes certain that his human gene has the same ‘sticky ends’ as the plasmid. A new segment of DNA can be attached to the plasmid only if its exposed bases are complementary to the exposed bases on the DNA of the plasmid. Once the complementary bases are hydrogen bonded to each other, another enzyme, DNA ligase, adds the covalent bonds holding the sugar-phosphate backbone of the DNA together. Once the plasmid is complete, it can be inserted into a bacterium, and induced to transcribe and translate this new gene, making a human protein in a bacterium! Materials: Scissors Pen or pencil of a contrasting color to write on the plasmids Paper plasmids Paper ‘human DNA’ Paper Restriction enzymes Masking tape Procedure: 1. Using your scissors, carefully cut out all 6 columns of the plasmid DNA. Then, tape the six strips together in order. Strip #2 should be attached to the bottom of strip #1, strip #3 should be attached to the bottom of strip #2, and so on. When you are finished with this part, you should have a ring of plasmid DNA. 2. Cut out the restriction enzyme BAM HI. 3. Cut out the segment of human DNA containing a ‘gene of interest’. 4. Now, carefully move the restriction enzyme along the human DNA, and find the two sites at which BAM HI will cut the human DNA. The base sequence that BAM HI will cut is printed on the restriction enzyme. When you find these two sites, mark them with a pencil the same way they are marked on the restriction enzyme. Then, cut the human DNA along the lines you drew. 5. Next, move the restriction enzyme along the plasmid, looking for the one site at which BAM HI will cut the plasmid DNA. As you did with the human DNA, draw the cutting lines as marked on the restriction enzyme, then cut your plasmid along those lines. 6. Match up the human DNA with the gene of interest by complementary pairing of the nitrogenous bases on the ‘sticky ends’. Tape the human DNA in place. You have now made a recombinant plasmid containing a human ‘gene of interest’! 7. Use the knowledge you have gained in doing this lab, reading your text, and from class notes to answer the analysis questions. Analysis Questions: 1. Organisms that contain DNA from two distinctly different organisms are called _______________________. (1) 2. Scientists have many ways to combine DNA from different organisms. The procedure used in this lab is ___________________________________________. (1) 3. Plasmids are rings of DNA that are found in _____________________________. 4. Plasmids often give ______________________________________ to a bacterium. (1) 5. ___________________________________ are enzymes that cut DNA at specific places. (1) This molecules belong to the class of macromolecules called ________________________ (1) and are made in the process of ______________________________________________________________. (1) 6. When you used your scissors to cut the plasmid and the human DNA at the restriction site, what type of bonds were you breaking? ___________________________________________________________ (2) 7. How are the bonds between sticky ends formed? ___________________________________________________________________________ ______________________________________________________________________ (2) 8. How is recombinant DNA technology used in medical science? ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________ 9. Name three chemicals made through the process of recombinant DNA cloning and give their uses in medical science: (6) 1) ______________________________________________________ 2) ______________________________________________________ 3) ______________________________________________________ Extra Credit (2 points) Does bacterial DNA contain introns? What implications does this have for the technique used in recombinant DNA technology? ___________________________________________________________________________ ___________________________________________________________________________ ___________________________________________________________________________