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8E: Techniques of Molecular Genetics (Exercises)

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  • 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.

    Study Questions:

    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.

    Chapter 8 - Answers

    8.1 You would need to know that HIV is an RNA-virus, but you should be able to detect the DNA pro-virus in infected white blood cells. You would have to be able to extract DNA from white blood cells, then use HIV-specific primers to see if HIV pro-virus DNA could be amplified. Thus, you would need to know some of the sequence of the HIV genome. You would probably want to compare your primers to the human genome sequence too, to make sure the primers are complementary only to HIV-DNA, but not human DNA. You would probably want to try to amplify some known HIV-free human DNA with the primers as a negative control, just to be sure that the primers were HIV-specific. And amplify the sequences from a known positive sample to know you can detect the sequences (positive control).

    For the PCR reaction, you would need primers (as mentioned), dNTPs, Taq polymerase, and other buffers or salts as required by the polymerase. You would need an agarose gel, ethidium bromide, and electrophoresis buffers to analyze the PCR products to detect a band in a control positive sample, have it absent in a negative sample, and then test your experimental to obtain a valid result.

    8.2 a) There will be a 6kb band (the insert) and a 3kb band (the plasmid vector)

    b) There would be a 9kb band.

    c) There would only be a 3kb band, which represents the plasmid, and both insert fragments.

    8.3 The amplification factor is 2n, where n is the number of cycles. So after 10 cycles, starting with 10 molecules, you would have 10 × 210 = 10,240 molecules.

    8.4 Present are polymerase, some dNTPs, and primers, as well as the original template, and PCR products. By far the most abundant products will be the one flanked at both ends by the primer sequences; these will be the only thing observed on the gel, since the template and other PCR products are present in much lower abundance and are usually not visualized.

    8.5 Identify the gene encoding the antigenic fragment of the virus. Clone this gene into E. coli and produce lots of recombinant protein, purify, and use as a vaccine.

    8.6 Without a selectable marker, you would have to individually test millions of bacterial colonies to find one that contained your cloned fragment. Furthermore, you could not keep the plasmid in the E. coli because the retention of the plasmid is dependent upon the antibiotic resistance selectable marker.

    8.7 a) Radioactively label a piece of DNA that hybridized to the gene, outside of the part of the gene contained in the deletion. Extract DNA from the suspect cancer cells of individuals, digest with a restriction enzyme (the best choice would be ones that cleave just outside the gene), and separate the DNA by electrophoresis. Southern blot the gel and probe with DNA complementary to the gene. Be sure the probe spans the 200bp deleted region. Wash at high stringency and expose to a sheet of X-ray film (or equivalent). Individuals with the deletion will have two bands, one at a lower position (200 bp lower) on the gel than those samples that do not have a deletion (or the cancer).

    b) You would probably get hybridization to extra bands, or even just a big smear, since the probe would hybridize non-specifically.

    8.8 a) Use PCR primers that flank the deletion. Extract DNA from cancer cell samples for use a template (one sample per reaction), and analyze the PCR products by gel electrophoresis.

    Cancer cells will have two bands, one full length, one 200bp shorter. Unaffected tissue will only have full length products.

    b) If the temperature was too low, the PCR products would probably appear as smears nearly the entire length of each lane, since the primers would bind to the genomic templates at many different positions and amplify fragments of many different lengths.

    8.9 Label the PCR fragment for use as a probe. Hybridize the probe to a Southern blot of dog DNA. Cut out and clone any bands that hybridize to the probe.

    Or, more recently, ignore the fragment and dog DNA sample, and take the sequence of the human olfactory receptor gene and BLAST it against the dog genome sequence. Compare the sequence output results to identify the dog olfactory receptor genes.

    More? Do the test.

    8.10 The complementary, sticky ends of the insert and plasmid vector will anneal together, but the non-functional ligase will not be able to covalently link the insert and vector together. Thus the almost-recombinant DNA will not be stable enough to be transformed, and thus unable to replicate -> no transformants.