Recitation Section 19 Answer KeyRecombinant DNA—Libraries and ReviewLibrary construction1 Recitation Section 19 Answer Key May 1-2, 2006 Recombinant DNA—Libraries and Review A. Library construction Recall that earlier in the semester we considered the Cys4 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 heart disease and ocular degeneration. Today we will talk about finding the yeast gene responsible. In a future section 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 library is a set of vectors containing DNA inserts that collectively represent all of the DNA in a genome or all of RNAs expressed in a cell. Each vector has only one piece of DNA or cDNA, but collectively the vectors contain all of the information. The DNA molecules used to create a library are usually derived from restriction digests. 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 if we are mating two haploid yeast strains. If we are using this technique in bacteria, we create a particular partial diploid by transforming one of the mutant strains with a vector containing the characteristic mutation of the other mutant strain. In the course of cloning by complementation using a library we create a collection of partial diploids— organisms 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 is cys4-, the library must contain a Cys4+ allele. Otherwise, no complementation is possible. The phenotype of the strain used is not restrictive. We can use cysteine auxotroph strains, as long as they are mutant in genes other than Cys4. 5. What features should the vector we are using have?2 Origin of replication, restriction site, and a selectable marker (for example, Kan resistance) 6. If we ran a gel of the vector and genomic DNA after the restriction digestion, how many bands would you expect to see in the vector digest lanes? What does your answer depend on? We would expect as many bands in the vector lane as there are restriction sites in our original vector for our restriction enzyme of choice. However, depending on the amount of time a digestion is allowed to proceed, we may see the number of bands corresponding to the number of restriction sites for our enzyme, or more, corresponding to various partial digests. In general, in this course you will only see the gels where DNA has had a chance to be fully digested. 7. How many cut sites for our restriction enzyme do you expect in the yeast genome? How many bands would that produce? Many sites distributed randomly throughout the genome. In fact, so many sites that we should see a smear on the gel because we would not be able to distinguish individual bands. Since a cut site is a particular sequence of six base pairs, we would expect to see on average one such site per 46 bases. Of course, the sequence will not show up at regular intervals, so we can expect to see a smear on the gel. 8. What are the next three steps in library creation? What is the goal of each step? Step Goal Ligation To create closed vectors carrying a fragment of yeast genomic DNA each Transformation To introduce the vectors created in the previous step into a culture of yeast. The strain we choose should allow the use of selectable marker present on the vector. Selection To find the yeast colonies resulting from taking up the vectors containing our gene of interest. 9. If we plated our transformed culture on Complete+Kan media, what would each colony on the plates represent? Each colony would represent an original yeast cell that took up a vector from our library. 10. What media should we plate on to find the cells that took up a vector from the library that contains a wild-type Cys4 gene? We should plate on Cysteine dropout +Kan media. Kan will select for taking up the vector, while the absence of cysteine in the media will select for those cells that can now make their own cysteine, i.e. for those cells where complementation
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