section18_ak

section18_ak - MIT Department of Biology 7.014 Introductory...

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MIT Department of Biology 7.014 Introductory Biology, Spring 2005 Recitation Section 18 Answer Key April 13-14, 2005 Recombinant DNA—Libraries and Forensics A. Library construction Recall that earlier in the semester we considered the C ys4 yeast gene that encodes a CBS protein. Recall also that yeast mutant in Cys4 are cysteine auxotrophs, while humans mutant in a homologous gene have a host of medical problems from mental retardation to hear disease and ocular degeneration. Today we will talk about finding the yeast gene responsible. In Section 22 we will turn to the question of using a yeast system to diagnose this particular human disease. Suppose we wanted to find the particular gene responsible for the cysteine auxotrophy phenotype. We decide to use cloning by complementation, and we use one of our cysteine auxotroph cys4 - mutant strains as a host strain. 1. What is a library? A collection of DNA molecules, derived from restriction fragments that have been cloned in vectors, that includes all or part of the genetic material of an organism. 2. What is a library used for? We are talking about using a library to clone by complementation. There are other possible uses, such as cloning by hybridization—finding a vector that contains a fragment of the genome that is complementary to the nucleic acid probe. 3. What is a common principle between complementation by mating that we did in Genetics and identification of a gene using genomic library? In both cases we seek to create an organism that is diploid in the gene/genes of interest to determine whether the presence of the second copy of the gene would complement the deficiency. In complementation by mating, we create a diploid organism. In the course of cloning by complementation using a library we create a partial diploid—an organism diploid for the gene or genes carried by the particular vector that was taken up by the cell. We need a vector and a source of genomic DNA to construct a library. 4. What must the genotype at the Cys4 locus be of the strain of yeast that we use as the source of genomic DNA? What phenotype must it have? Why? The strain we use must be Cys4+. Because we are cloning by complementation, and our host strain
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section18_ak - MIT Department of Biology 7.014 Introductory...

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