U09_F08 - Unit 9 1 UNIT 9 RECOMBINANT DNA METHODS...

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Unit 9 1 U NIT 9 R ECOMBINANT DNA M ETHODS Recombinant DNA techniques have been invaluable to the study of genes and the determination of the biological function of the proteins which they encode. Recombinant DNA technology has also been used to isolate genes so that their protein products can be produced on a large scale in bacteria. The ability to produce these products has had a profound impact on medicine. For example the cloning of the insulin gene has allowed researchers to produce the protein in bacteria on a large scale, instead of isolating it from mammalian sources. Other proteins of commercial and medical interest are the human growth hormone and interleukins. In this unit we will describe the processes whereby a gene is isolated, identified, and manipulated in the laboratory as well as some aspects of genomics and proteomics. Assignment: Nelson & Cox, pp. 292 - 295, 303 - 338, 1145-1146. 1. Use Fig. 8-33 (p. 293) to discuss the Sanger method for DNA sequencing. a. Draw the structure of ddATP (you may use A as an abbreviation for the structure of the base). Explain how incorporation of a dideoxynucleoside will affect subsequent polymerization by DNA polymerase. b. How many reactions are included in a typical DNA sequencing experiment.
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Unit 9 2 1) Describe the composition of each reaction. 2) Review the definition of "radioactive isotope" given in the glossary of the text book (p. G-13). How is radioactivity incorporated into the sequences? c. Given the sequence and primer, write the sequences that would be produced using the dideoxy analog of dATP. Do the same for the other three dideoxy analogs. 3' AGCTATTCGTGAC 5' 5' TCGA 3' d. The bands seen on the electrophoresis gel differ in length by how many nucleotides? e. Do problem 12 on p. 300. 2. In a more modern and more highly automatable version of the original Sanger method, fluorescent tags can be attached to the dideoxynucleotides to make them detectable by fluorescence measurements (Fig. 8-34, p. 294). Review the definition of "fluorescence" given in the glossary of the text book (p. G-6). Distinguish between radioactivity (p. G-13) and fluorescence. a. Why is it possible with this method to have only a single reaction mixture? b. After separating the fragments, how do you know which fragment stopped at which residue? c. Why does this method allow more sequence information to be derived from a single sequencing reaction?
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Unit 9 3 3. Currently several sequencing methods are being developed which do not separate the synthesized DNA, making the process much less expensive. One that is commercially available now is called "Pyrosequencing" because it takes advantage of the PPi that is released when a dNTP is incorporated into the growing DNA strand. dNTP's are added one at a time and the PPi is detected by converting it to ATP and using the ATP to drive light formation by luciferase. In one version of this method thousands of reactions are run simultaneously on a plate and the genome of a bacterium can be sequenced in a single day by one person.
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