18. Molecular Genetic Tools

Genetic Cloning

Open Question
In this chapter we focused on how specific DNA sequences can be copied, identified, characterized, and sequenced. At the same time, we found many opportunities to consider the methods and reasoning underlying these techniques. From the explanations given in the chapter, what answers would you propose to the following fundamental questions?

What steps make PCR a chain reaction that can produce millions of copies of a specific DNA molecule in a matter of hours without using host cells?
Open Question
In this chapter we focused on how specific DNA sequences can be copied, identified, characterized, and sequenced. At the same time, we found many opportunities to consider the methods and reasoning underlying these techniques. From the explanations given in the chapter, what answers would you propose to the following fundamental questions?

In a recombinant DNA cloning experiment, how can we determine whether DNA fragments of interest have been incorporated into plasmids and, once host cells are transformed, which cells contain recombinant DNA?
Open Question
What purpose do the bla and lacZ genes serve in the plasmid vector pUC18?
Open Question
The human genome is 3×10⁹bp in length.How many fragments would be predicted to result from the complete digestion of the human genome with the following enzymes: Sau3A (˘GATC), BamHI (G˘GATCC), EcoRI (G˘AATTC), and NotI (GC˘GGCCGC)?
Open Question
The human genome is 3×10⁹bp in length.How would your initial answer change if you knew that the average GC content of the human genome was 40%?
Open Question
Write a short essay or sketch a diagram that provides an overview of how recombinant DNA techniques help geneticists study genes.
Open Question
Write a short essay that summarizes the impacts that genomic applications are having on society and discuss which of the ethical issues presented by these applications is the most daunting to society.
Open Question
What roles do restriction enzymes, vectors, and host cells play in recombinant DNA studies? What role does DNA ligase perform in a DNA cloning experiment? How does the action of DNA ligase differ from the function of restriction enzymes?
Open Question
Why are most recombinant human proteins produced in animal or plant hosts instead of bacterial host cells?
Open Question
One of the major causes of sickness, death, and economic loss in the cattle industry is Mannheimia haemolytica, which causes bovine pasteurellosis, or shipping fever. Noninvasive delivery of a vaccine using transgenic plants expressing immunogens would reduce labor costs and trauma to livestock. An early step toward developing an edible vaccine is to determine whether an injected version of an antigen (usually a derivative of the pathogen) is capable of stimulating the development of antibodies in a test organism. The following table assesses the ability of a transgenic portion of a toxin (Lkt) of M. haemolytica to stimulate development of specific antibodies in rabbits.Immunogen Injected Antibody Production in SerumLkt50*—saline extract +Lkt50*—column extract +Mock injection -*Lkt50 is a smaller derivative of Lkt that lacks all hydrophobic regions. indicates at least 50 percent neutralization of toxicity of Lkt; indicates no neutralization activity.Source: Modified from Lee et al. (2001). Infect. and Immunity 69:5786–5793.With regards to development of a usable edible vaccine, what work remains to be done?
Open Question
One of the major causes of sickness, death, and economic loss in the cattle industry is Mannheimia haemolytica, which causes bovine pasteurellosis, or shipping fever. Noninvasive delivery of a vaccine using transgenic plants expressing immunogens would reduce labor costs and trauma to livestock. An early step toward developing an edible vaccine is to determine whether an injected version of an antigen (usually a derivative of the pathogen) is capable of stimulating the development of antibodies in a test organism. The following table assesses the ability of a transgenic portion of a toxin (Lkt) of M. haemolytica to stimulate development of specific antibodies in rabbits.Immunogen Injected Antibody Production in SerumLkt50*—saline extract +Lkt50*—column extract +Mock injection -*Lkt50 is a smaller derivative of Lkt that lacks all hydrophobic regions. indicates at least 50 percent neutralization of toxicity of Lkt; indicates no neutralization activity.Source: Modified from Lee et al. (2001). Infect. and Immunity 69:5786–5793.What general conclusion can you draw from the data?
Open Question
The human insulin gene contains a number of sequences that are removed in the processing of the mRNA transcript. In spite of the fact that bacterial cells cannot excise these sequences from mRNA transcripts, explain how a gene like this can be cloned into a bacterial cell and produce insulin.
Open Question
Although many cloning applications involve introducing recombinant DNA into bacterial host cells, many other cell types are also used as hosts for recombinant DNA. Why?
Open Question
The human genome is 3×10⁹bp. You wish to design a primer to amplify a specific gene in the genome. In general, what length of oligonucleotide would be sufficient to amplify a single unique sequence? To simplify your calculation, assume that all bases occur with an equal frequency.
Open Question
Using DNA sequencing on a cloned DNA segment, you recover the nucleotide sequence shown below. Does this segment contain a palindromic recognition sequence for a restriction enzyme? If so, what is the double-stranded sequence of the palindrome, and what enzyme would cut at this sequence? (Consult Figure 20.1 for a list of restriction sites.)

CAGTATGGATCCCAT