bio214k - Biology 214/414 2009 Packet#10 Suggested problems...

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Biology 214/414 2009 Packet #10 Suggested problems: Chapter 13: 1, 3, 6, 8, 12, 14, 16, 20, 22, 26, 28, 30 Recombinant DNA technology I. Almost all modern techniques for detailed analysis of genes (including DNA sequencing) and modern practical genetic applications involve “recombinant DNA technology ”; basic knowledge of genetics was absolutely indispensable in its development. a. Whether used for basic or applied research recombinant DNA technology requires (and gets its name from) the synthesis of “recombinant DNA ” (also called “gene splicing ”). i. This requires insertion of a gene or segment of DNA of interest into a small, self-replicating chromosome or plasmid called a “vector ”. 1. This is done “in vitro ”, ie, in a test tube. 2. The product is a “recombinant DNA molecule ”. ii. Following this is “cloning ” of the recombinant DNA molecule, that is replicating it many times (by virtue of its containing a self-replicating vector) so that there is plenty of material to work with. This requires placing the recombinant DNA molecule in a suitable host cell, where it can replicate. iii. In common usage, both i. and ii. above, when considered together, are called cloning (even though the strict meaning of “cloning” is the replication step). II. Types of vector include: a. plasmids (used in bacteria) ( Figures 13-4, 5, 6, 7 ) b. bacteriophage vectors (used in bacteria) ( Figures 13-8 ) c. “cosmids ” (combinations of plasmid and bacteriophage vectors; used in bacteria; can hold large inserts) ( Figure 13-9 ) d. “artificial chromosomes ” (used in both bacteria (“BACs ”) and yeast (“YACs ”); can hold inserts of enormous size ( Figures 13- 10, 13 )
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e. “shuttle vectors ” (can replicate in both bacterial and eukaryotic cells, or in two quite different bacterial species) f. “expression vectors ” are designed especially for high level expression of the cloned gene of interest in the host cell ( Figure 13-11 ) g. vectors based on transposable elements (for both prokaryotes and eukaryotes) and eukaryotic viruses (especially for mammalian cells/organisms) h. vectors designed especially for genetic engineering of higher plant cells ( Figure 13-14 ) III. Cloning techniques typically require the use of “restriction enzymes ”. a. These are DNases ( Figures 13-1, 2 ); most are “site specific ”, and many of these cleave at specific 4-6 bp long “palindromes ”. The cleavages, or “cuts”, can be either “staggered” or “blunt”. ( Figures 13-2, 3 ). Note that each enzyme (to a first degree of approximation) has its own specific cleavage site ( Figure 13-2 ). b.
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bio214k - Biology 214/414 2009 Packet#10 Suggested problems...

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