Chapter 9 Reading Notes

+Chapter 9 reading notes

Cloning Basics

  1. DNA is cut, put into new DNA so it can be copied, then it is copied in a host cell. Finally, the host cells are screened for evidence that they contain the new DNA.
  2. What is each of the following and what do they do?
    1. Endonuclease
    2. Restriction endonuclease
    3. Ligase
    4. DNA polymerase
  3. Reverse transcriptase
  4. What role does DNA methylase play in protecting host bacteria from restriction enzymes?
  5. What is a sticky end? Blunt end?
  6. Once DNA is cut, it needs to be put into a vector. Be able to describe:
    1. Plasmids
    2. Bacteriophage
    3. Bacterial Artificial Chromosomes
    4. Yeast Artificial Chromosomes
  7. What are the limitations of DNA size that can be cloned into each type of vector. How is each vector used to get the DNA into a host cell for replication? What are the advantages associated with each type of cloning vector?
  8. Explain how these screening methods are used to select clones that contain the DNA of interest: antibiotic resistance, lacZ gene.
  9. What are the key differences between an expression vector (one that will result in producing lots of a protein) and a vector that is used to generate a DNA library? Compare figure 9-11 and figure 9-4.

DNA Technologies

  1. What is site directed mutagenesis? What is it used for? How is it done?
  2. What is s DNA library? What is a cDNA library?
  3. What is a green fusion protein? What does it do? Why is it used? (See figure 9-15a)
  4. What is an epitope tag? (See figure 9-15b) There is more on “tags” on page 328 and 329.
  5. Be able to describe DNA fingerprinting. What is a rif lip? How much does the DNA vary from one person to the next?
  6. What percentage of the human genome is translated into protein? What does the other DNA “do”. Why is it there? How many genes do we have? Chapter 26 has information (for enquiring minds) on how more than one protein can be expressed from the same gene, page 1014.

More information is available on transposons in Chapter 25 on page 988, this leads you to read further about the diversity of immunoglobins on page 990. Each person can generate up to 15 million different IgGs.

Gene products

  1. What are the three levels of protein function?
  2. What information can be gleened from comparative genomics? (Figure 9-20)
  3. What experiments/techniques are used to determine cellular function?

Techniques

  1. What can a DNA array be used for? (Figure 9-22 and 9-23)
  2. Figure 9-25 shows a method for determining protein-protein interactions. How does this work?

Genetically modified organisms

Section 9-4 gives us insight into the exciting field of GMOs (Genetically modified organisms). This is another area of hot societal debate. Keep in mind that scientists have been using classical genetics via breeding to alter genomes, so one way to perceive GMO technology is that it is a quicker faster way to better tomatoes. As you read this section, think about the potential problems that could be created. How can scientists prevent these problems? Is the technology advanced to the level that allows researchers to know exactly what will happen when they move foreign DNA into an organism? What controls do we need? What are the benefits? Risks? Do a brief benefit vs risk analysis.

Figure 9-28 shows how two plasmids are used to transform a plant. The tomato plant on the left of figure 9-30 clearly shows a benefit. However, there is no mention of the risk to butterflies (they are needed for pollination), but the toxin affects them (if they eat the plant) as well as the harmful moths. Most butterfly larva do not eat tomatoes, but how do we make sure that this toxin gene does not move into a plant that they do eat?

Cloning Around

  1. What methods are used for mammalian cloning? What are the advantages and disadvantages? What are the problems?
  2. What is a knock out mouse?
  3. How is proteomics (it is a field, and is thus a singular noun) being used to develop new pharmaceuticals?
  4. Read about the role of the “orphan” receptor GPR14. It was an “orphan” until researchers were able to determine that it bound to a potent physiological peptide.
  5. Be sure to read box 9-2 on Human Gene Therapy.What is the major impediment. What caused the one patient do subsequently develop leukema?
  6. Examples of recombinant DNA products are shown in table 9-4. Which one do you think has had the most impact on human health?
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