PS_2_older_key - Biology 115 Winter 2005 January 21 Problem...

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Biology 115, Winter 2005 January 21 Problem Set #2 answers 1. True or False. If false, give your reasoning. A. Cosmid vectors can carry larger inserts than phage vectors. True B. YACs suffer from the drawback that they are low copy number vectors. True C. Phage and cosmid vectors share the advantage of allowing very efficient transfer of DNA into bacteria. True D. The key feature of an expression vector is its ability to be maintained in more than one type of host cell (for example both bacteria and yeast). No, that describes a shuttle vector (i.e. you can shuttle between two hosts). An expression vector allow a gene to be expressed, i.e. it has a promoter ( an perhaps other features like a start codon or transcription terminator). E. In a cDNA library, every gene in the genome is represented. In a genomic DNA library, only a fraction of genes are represented. False, the opposite is true. Only expressed genes are found in a cDNA library. F. Polylinkers are flexible hinge regions of proteins that separate domains of protein function encoded by different exons. False, polylinkers are closely spaced collections of unique restriction sites in a plasmid to facilitate cloning of inserts. 2. What feature of Taq polymerase makes it ideal for use in PCR? Answer: It is thermostabile and can therefore withstand repeated cycles of heating and cooling (which are necessary to melt DNA strands so that oligodeoxynucleotide primers can anneal to template DNA and allow a new cycle of DNA synthesis). 3. You discover a bacterial protein, ozonase, that beaks down ozone. You decide to clone the gene encoding the ozonase protein. a. You don't have any idea of what the amino acid sequence of the ozonase protein might be, but you do have a method for detecting the presence or absence of this protein in bacterial cells, and you also have a large quantity of the purified ozonase protein. How would you clone the gene encoding ozonase? What other reagents do you need for your strategy to work?
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Answer: You could: a. Screen a bacterial expression library for the presence or absence of the ozonase protein. You would need to have bacterial cells that don’t express ozonase so that you could detect transformants that do express it. b. Use your purified protein to make an anti-ozonase antibody and screen an expression library. Again, you would need recipient cells that do not normally express ozonase. Note: you are using a bacterial host to clone a bacterial gene, encoding a bacterial protein. Therefore, you do not need a cDNA clone of the ozonase gene. Furthermore, if you have the appropriate bacterial strains available to you, then you do not even need an expression library. You can just use a genomic DNA library (for example, in a plasmid vector) from ozonase expressing cells, and depend on finding an insert with both the ozonase open reading frame and the promoter that normally drives its transcription. b.
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PS_2_older_key - Biology 115 Winter 2005 January 21 Problem...

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