These are homework exercises to accompany Nickle and Barrette-Ng's "Online Open Genetics" TextMap. Genetics is the scientific study of heredity and the variation of inherited characteristics. It includes the study of genes, themselves, how they function, interact, and produce the visible and measurable characteristics we see in individuals and populations of species as they change from one generation to the next, over time, and in different environments.
8.1 What information, and what reagents would you need to use PCR to detect HIV in a blood sample?
8.2 A 6.0 kbp PCR fragment flanked by recognition sites for the HindIII restriction enzyme is cut with HindIII then ligated with a 3kb plasmid vector that has also been cut with HindIII. This recombinant plasmid is transformed into E. coli. From one colony a plasmid is prepared and digested with HindIII.
a) When the product of the HindIII digestion is analyzed by gel electrophoresis, what will be the size of the bands observed?
b) What bands would be observed if the recombinant plasmid was cut with EcoRI, which has only one site, directly in the middle of the PCR fragment?
c) What bands would be observed if the recombinant plasmid was cut with both EcoRI and HindIII at the same time?
8.3 If you started with 10 molecules of double stranded DNA template, what is the maximum number of molecules you would you have after 10 PCR cycles?
8.4 What is present in a PCR tube at the end of a successful amplification reaction? With this in mind, why do you usually only see a single, sharp band on a gel when it is analyzed by electrophoresis?
8.5 A coat protein from a particular virus can be used to immunize children against further infection. However, inoculation of children with proteins extracted from natural viruses sometimes causes fatal disease, due to contamination with live viruses. How could you use molecular biology to produce an optimal vaccine?
8.6 How would cloning be different if there were no selectable markers?
8.7 Research shows that a particular form of cancer is caused by a 200bp deletion in a particular human gene that is normally 2kb long. Only one mutant copy is needed to cause the disease.
a) Explain how you would use Southern blotting to diagnose the disease.
b) How would any of the blots appear if you hybridized and washed at very low temperature?
8.8 Refer to question 8.7.
a) Explain how you would detect the presence of the same deletion using PCR, rather than a Southern blot.
b) How would PCR products appear if you annealed at very low temperature?
8.9 You have a PCR fragment for a human olfactory receptor gene (perception of smells). You want to know what genes a dog might have that are related to this human gene. How can you use your PCR fragment and genomic DNA from a dog to find this out? Do you think dogs have more or less of these genes?
8.10 You add ligase to a reaction containing a sticky-ended plasmid and sticky-ended insert fragment, which both have compatible ends. Unbeknownst to you, someone in the lab left the stock of ligase enzyme out of the freezer overnight and it degraded (no longer works). Explain in detail what will happen in your ligation experiment in this situation should you try and transform with it.