Chapter 19: DNA Technology

DNA Cloning

  1. Explain how advances in recombinant DNA technology have helped scientists study the eukaryotic genome.
  2. Describe the natural function of restriction enzymes.
  3. Explain how the creation of sticky ends by restriction enzymes is useful in producing a recombinant DNA molecule.
  4. Outline the procedures for cloning a eukaryotic gene in a bacterial plasmid.
  5. Describe the role of an expression vector.
  6. Explain how eukaryotic genes are cloned to avoid the problems associated with introns.
  7. Describe two advantages of using yeast cells instead of bacteria as hosts for cloning or expressing eukaryotic genes.
  8. Describe three techniques to aggressively introduce recombinant DNA into eukaryotic cells.
  9. Define and distinguish between genomic libraries using plasmids, phages, and cDNA.
  10. Describe the polymerase chain reaction (PCR) and explain the advantages and limitations of this procedure.

DNA Analysis and Genomics

  11. Explain how gel electrophoresis is used to analyze nucleic acids and proteins and to distinguish between two alleles of a gene.
  12. Describe the process of nucleic acid hybridization.
  13. Describe the Southern blotting procedure and explain how it can be used to detect and analyze instances of restriction fragment length polymorphism (RFLP).
  14. Explain how RFLP analysis facilitated the process of genomic mapping.
  15. List the goals of the Human Genome Project.
  16. Explain how linkage mapping, physical mapping, and DNA sequencing each contributed to the genome mapping project.
  17. Describe the alternate approach to whole-genome sequencing pursued by J. Craig Venter and the Celera Genomics company. Describe the advantages and disadvantages of public and private efforts.
  18. Describe the surprising results of the human genome project.
  19. Explain how the vertebrate genome, including that of humans, generates greater diversity than the genomes of invertebrate organisms.
  20. Describe what we have learned by comparing the human genome to that of other organisms.
  21. Explain the purposes of gene expression studies. Describe the use of DNA microarray assays and explain how they facilitate such studies.
  22. Explain how in vitro mutagenesis and RNA interference help to discover the functions of some genes.
  23. Define and compare the fields of proteomics and genomics.
  24. Explain the significance of single nucleotide polymorphisms in the study of the human genome.

Practical Applications of DNA Technology

  25. Describe how DNA technology can have medical applications in such areas as the diagnosis of genetic disease, the development of gene therapy, vaccine production, and the development of pharmaceutical products.
  26. Explain how DNA technology is used in the forensic sciences.
  27. Describe how gene manipulation has practical applications for environmental and agricultural work.
  28. Describe how plant genes can be manipulated using the Ti plasmid carried by Agrobacterium as a vector.
  29. Explain how DNA technology can be used to improve the nutritional value of crops and to develop plants that can produce pharmaceutical products.
  30. Describe the safety and ethical questions related to recombinant DNA studies and the biotechnology industry.

DNA CLONING

         DNA technology makes it possible to clone genes for basic research and commercial applications: an overview(pp. 376-377, FIGURE 20.1) DNA technology is a powerful set of techniques that enables biologists to manipulate and analyze DNA. It can help make useful new products and organisms.

 

         Restriction enzymes are used to make recombinant DNA (pp. 377-378, FIGURE 20.2) A variety of bacterial restriction enzymes recognize short, specific nucleotide sequences in DNA and cut the sequences at specific points on both strands to yield a set of double-stranded DNA fragments with single-stranded sticky ends. The sticky ends readily form base pairs with complementary single-stranded segments on other DNA molecules. The enzyme DNA ligase can seal the strands to produce recombinant DNA molecules.

 

         Genes can be cloned in recombinant DNA vectors: a closer look (pp. 378-381, FIGURES 20.3-20.5) Plasmids can serve as vectors (carriers) to introduce foreign genes into host bacteria. Recombinant DNA is made by inserting restriction fragments from DNA containing a gene of interest into the vector DNA, which has been cut open by the same enzyme. Gene cloning results when the foreign genes replicate inside the host bacterial cell as part of the recombinant vector. Eukaryotic cells can also serve as host cells for gene cloning. Cell clones carrying the gene of interest can be identified with a radioactively labeled nucleic acid probe, which has a sequence complementary to the gene.

         Cloned genes are stored in DNA libraries (pp. 381-382, FIGURE 20.6) When the starting material for DNA (gene) cloning is an entire genome, the resulting collection of recombinant vector clones is called a genomic library. Alternatively, a cDNA (complementary DNA) library can be made by cloning DNA made in vitro by reverse transcription of all the mRNA produced by a particular kind of cell. Libraries of cDNA are especially useful for working with eukaryotic genes (whose introns are not present in the cDNA versions) and for studying gene expression.

         The polymerase chain reaction (PCR) clones DNA entirely in vitro (pp. 382-383, FIGURE 20.7) For quickly making many copies of a particular segment of DNA, this method uses primers that bracket the desired sequence and a heat-resistant DNA polymerase.

DNA ANALYSIS AND GENOMICS

         Restriction fragment analysis detects DNA differences that affect restriction sites (pp. 383-386, FIGURES 20.8-20.10) Gel electro-phoresis makes it possible to separate and isolate DNA restriction fragments of different lengths. Restriction fragment length polymorphisms (RFLPs) are differences in DNA sequence on homologous chromosomes that result in different patterns of restriction fragment lengths. These patterns are visualized as bands on gel electrophoresis. Specific fragments can be identified by Southern blotting, using labeled probes that hybridize to the DNA stuck to a "blot" of the gel. RFLPs are prevalent genetic markers, present throughout eukaryotic noncoding DNA. RFLP analysis has many applications, including genetic mapping and diagnosis of genetic disorders.

         Entire genomes can be mapped at the DNA level (pp. 386-389, FIGURES 20.11-20.13) An international research effort, the Human Genome Project involves linkage mapping, physical mapping, and DNA sequencing of the human genome and the genomes of other organisms. An alternative approach starts with sequencing of random DNA fragments, relying especially heavily on computer power to assemble the sequences. The human genome is thought to have 30,000 to 40,000 genes, fewer than once thought.

 

         Genome sequences provide clues to important biological questions (pp. 389-393, FIGURE 20.14) Genome sequences are helping researchers find new genes, probe details of gene organization and control, and answer questions about evolution. DNA microarrays allow researchers to compare patterns of gene expression in different tissues and under different conditions. Genomics is the systematic study of entire genomes; proteomics is the systematic study of all the proteins encoded by a genome. Single nucleotide polymorphisms (SNPs) provide useful markers for studying human genetic variation.

PRACTICAL APPLICATIONS OF DNA TECHNOLOGY

         DNA technology is reshaping medicine and the pharmaceutical industry (pp. 393-395; FIGURES 20.15, 20.16) Medical applications of DNA technology include diagnostic tests for genetic and other diseases; safer, more effective vaccines; the large-scale production of many new, and some previously scarce, pharmaceutical products; and the prospect of treating or even curing certain genetic disorders.

         DNA technology offers forensic, environmental, and agricultural applications (pp. 395-399; FIGURES 20.17-20.20) DNA "fingerprints" obtained from RFLP or STR analysis of tissue found at the scenes of violent crimes provide evidence in trials; such fingerprints are also useful in parenthood disputes. Genetic engineering can modify the metabolism of microorganisms so that they can be used to extract minerals from the environment or degrade waste materials. In agriculture, transgenic plants and animals are being designed to improve food productivity and quality.

 

         DNA technology raises important safety and ethical questions (p. 399) Several U.S. government agencies are responsible for setting policies about and regulating recombinant DNA technology. The potential benefits of genetic engineering must be carefully weighed against the potential hazards of creating products or developing procedures that are harmful to humans or the environment.