DNA Technology and Genomics - Chapter 12 Instructor Guide PDF

CHAPTER 12 DNA Technology and Genomics Chapter Objectives Opening Essay Explain why DNA technology is important. Gene Cloning 12.1 Explain how plasmids are used in gene cloning. 12.2 Explain how restriction enzymes are used to “cut and paste” DNA into...

CHAPTER 12 DNA Technology and Genomics Chapter Objectives Opening Essay Explain why DNA technology is important. Gene Cloning 12.1 Explain how plasmids are used in gene cloning. 12.2 Explain how restriction enzymes are used to “cut and paste” DNA into plasmids. 12.3 Explain how plasmids, phages, and BACs are used to construct genomic libraries. 12.4 Explain how a cDNA library is constructed and how it is different from genomic libraries constructed using plasmids or phages. 12.5 Explain how a nucleic acid probe can be used to identify a specific gene. Genetically Modified Organisms 12.6 Explain how different organisms are used to mass-produce proteins of human interest. 12.7 Explain how DNA technology has helped to produce insulin, growth hormone, and vaccines. 12.8 Explain how genetically modified (GM) organisms are transforming agriculture. 12.9 Describe the risks posed by the creation and culturing of GM organisms and the safeguards that have been developed to minimize these risks. DNA Profiling 12.11 Describe the basic steps of DNA profiling. 12.12 Explain how PCR is used to amplify DNA sequences. 12.13 Explain how gel electrophoresis is used to sort DNA and proteins. Lecture Outline I. Introduction A. DNA technology 1. has rapidly revolutionized the field of forensics, 2. permits the use of gene cloning to produce medical and industrial products, 146 Copyright © 2012 Pearson Education, Inc.  3. allows for the development of genetically modified organisms for agriculture, 4. permits the investigation of historical questions about human family and evolutionary relationships, and 5. is invaluable in many areas of biological research. II. Gene Cloning A. 12.1 Genes can be cloned in recombinant plasmids 1. Biotechnology is the manipulation of organisms or their components to make useful products. 2. For thousands of years, humans have a. used microbes to make wine and cheese and b. selectively bred stock, dogs, and other animals. 3. DNA technology is the set of modern techniques used to study and manipulate genetic material. 4. Genetic engineering involves manipulating genes for practical purposes. a. Gene cloning leads to the production of multiple, identical copies of a gene- carrying piece of DNA. b. Recombinant DNA is formed by joining nucleotide sequences from two different sources. i. One source contains the gene that will be cloned. ii. Another source is a gene carrier called a vector. iii. Plasmids (small, circular DNA molecules independent of the bacterial chromosome) are often used as vectors. 5. Steps in cloning a gene a. Plasmid DNA is isolated. b. DNA containing the gene of interest is isolated. c. Plasmid DNA is treated with a restriction enzyme that cuts in one place, opening the circle. d. DNA with the target gene is treated with the same enzyme and many fragments are produced. e. Plasmid and target DNA are mixed and associate with each other. f. Recombinant DNA molecules are produced when DNA ligase joins plasmid and target segments together. g. The recombinant plasmid containing the target gene is taken up by a bacterial cell. h. The bacterial cell reproduces to form a clone, a group of genetically identical cells descended from a single ancestral cell. B. 12.2 Enzymes are used to “cut and paste” DNA 1. Restriction enzymes cut DNA at specific sequences. a. Each enzyme binds to DNA at a different restriction site. b. Many restriction enzymes make staggered cuts that produce restriction fragments with single-stranded ends called “sticky ends.” c. Fragments with complementary sticky ends can associate with each other, forming recombinant DNA. 2. DNA ligase joins DNA fragments together. C. 12.3 Cloned genes can be stored in genomic libraries 1. A genomic library is a collection of all of the cloned DNA fragments from a target genome. 2. Genomic libraries can be constructed with different types of vectors: 147 INSTRUCTOR GUIDE FOR CAMPBELL BIOLOGY: CONCEPTS & CONNECTIONS Copyright © 2012 Pearson Education, Inc.  a. plasmid library: genomic DNA is carried by plasmids, b. bacteriophage (phage) library: genomic DNA is incorporated into bacteriophage DNA, c. bacterial artificial chromosome (BAC) library: specialized plasmids that can carry large DNA sequences. D. 12.4 Reverse transcriptase can help make genes for cloning 1. Complementary DNA (cDNA) can be used to clone eukaryotic genes. a. In this process, mRNA from a specific cell type is the template. b. Reverse transcriptase produces a DNA strand from mRNA. c. DNA polymerase produces the second DNA strand. 2. Advantages of cloning with cDNA include the ability to a. study genes responsible for specialized characteristics of a particular cell type and b. obtain gene sequences i. that are smaller in size, ii. easier to handle, and iii. do not have introns. E. 12.5 Nucleic acid probes identify clones carrying specific genes 1. Nucleic acid probes bind very selectively to cloned DNA. a. Probes can be DNA or RNA sequences complementary to a portion of the gene of interest. b. A probe binds to a gene of interest by base pairing. c. Probes are labeled with a radioactive isotope or fluorescent tag for detection. 2. One way to screen a gene library is as follows: a. Bacterial clones are transferred to filter paper. b. Cells are broken apart and the DNA is separated into single strands. c. A probe solution is added and any bacterial colonies carrying the gene of interest will be tagged on the filter paper. d. The clone carrying the gene of interest is grown for further study. III. Genetically Modified Organisms A. 12.6 Recombinant cells and organisms can mass-produce gene products 1. Recombinant cells and organisms constructed by DNA technologies are used to manufacture many useful products, chiefly proteins. 2. Bacteria are often the best organisms for manufacturing a protein product because bacteria a. have plasmids and phages available for use as gene-cloning vectors, b. can be grown rapidly and cheaply, c. can be engineered to produce large amounts of a particular protein, and d. often secrete the proteins directly into their growth medium. 3. Yeast cells a. are eukaryotes, b. have long been used to make bread and beer, c. can take up foreign DNA and integrate it into their genomes, d. have plasmids that can be used as gene vectors, and e. are often better than bacteria at synthesizing and secreting eukaryotic proteins. 4. Mammalian cells must be used to produce proteins with chains of sugars. Examples include a. human erythropoietin (EPO), which stimulates the production of red blood cells, Copyright © 2012 Pearson Education, Inc. CHAPTER 12 DNA Technology and Genomics 148  b. factor VIII to treat hemophilia, and c. tissue plasminogen activator (TPA) used to treat heart attacks and strokes. 5. Pharmaceutical researchers are currently exploring the mass production of gene products by a. whole animals or b. plants. 6. Recombinant animals a. are difficult and costly to produce and b. must be cloned to produce more animals with the same traits. B. 12.7 CONNECTION: DNA technology has changed the pharmaceutical industry and medicine 1. Products of DNA technology are already in use. a. Therapeutic hormones produced by DNA technology include i. insulin to treat diabetes and ii. human growth hormone to treat dwarfism. b. DNA technology is used to i. test for inherited diseases, ii. detect infectious agents such as HIV, and iii. produce vaccines, harmless variants (mutants) or derivatives of a pathogen that stimulate the immune system. C. 12.8 CONNECTION: Genetically modified organisms are transforming agriculture 1. Genetically modified (GM) organisms contain one or more genes introduced by artificial means. 2. Transgenic organisms contain at least one gene from another species. 3. The most common vector used to introduce new genes into plant cells is a. a plasmid from the soil bacterium Agrobacterium tumefaciens and b. called the Ti plasmid. 4. GM plants are being produced that a. are more resistant to herbicides and pests and b. provide nutrients that help address malnutrition. 5. GM animals are being produced with improved nutritional or other qualities. D. 12.9 Genetically modified organisms raise concerns about human and environmental health 1. Scientists use safety measures to guard against production and release of new pathogens. 2. Concerns related to GM organisms include the potential a. introduction of allergens into the food supply and b. spread of genes to closely related organisms. 3. Regulatory agencies are trying to address the a. safety of GM products, b. labeling of GM produced foods, and c. safe use of biotechnology. IV. DNA Profiling A. 12.11 The analysis of genetic markers can produce a DNA profile 1. DNA profiling is the analysis of DNA fragments to determine whether they come from the same individual. DNA profiling 149 INSTRUCTOR GUIDE FOR CAMPBELL BIOLOGY: CONCEPTS & CONNECTIONS Copyright © 2012 Pearson Education, Inc.  a. compares genetic markers from noncoding regions that show variation between individuals and b. involves amplifying (copying) of markers for analysis. B. 12.12 The PCR method is used to amplify DNA sequences 1. Polymerase chain reaction (PCR) is a method of amplifying a specific segment of a DNA molecule. 2. PCR relies upon a pair of primers that are a. short, b. chemically synthesized, single-stranded DNA molecules, and c. complementary to sequences at each end of the target sequence. 3. PCR a. is a three-step cycle that b. doubles the amount of DNA in each turn of the cycle. 4. The advantages of PCR include a. the ability to amplify DNA from a small sample, b. obtaining results rapidly, and c. a reaction that is highly sensitive, copying only the target sequence. C. 12.13 Gel electrophoresis sorts DNA molecules by size 1. Gel electrophoresis can be used to separate DNA molecules based on size as follows: a. A DNA sample is placed at one end of a porous gel. b. Current is applied and DNA molecules move from the negative electrode toward the positive electrode. c. Shorter DNA fragments move through the gel matrix more quickly and travel farther through the gel. d. DNA fragments appear as bands, visualized through staining or detecting radioactivity or fluorescence. e. Each band is a collection of DNA molecules of the same length. Chapter Guide to Teaching Resources  The general genetic engineering challenge discussed in Module 12.1 begins with the need to insert a gene of choice into a plasmid. This process is very similar to film or video editing. What do we need to do to insert a minute of one film into another? We will need techniques to a) cut and remove the minute of film to be inserted, b) a way to cut the new film apart, and c) a way to insert the new minute. In general, this is also like removing one boxcar from one train, and transferring the boxcar to another train. Students can become confused by the details of gene cloning through misunderstanding this basic editing relationship. (12.1)  The authors note the origin of the name restriction enzymes. In nature, these enzymes protect bacterial cells against foreign DNA. Thus, these enzymes “restrict” the invasion of foreign genetic material. (12.2)  A genomic library of the sentence you are now reading would be all of the sentence fragments that made up the sentence. One could string together all of the words of this first sentence, without spaces between letters, and then conduct a word processing edit, placing a space between any place where the letter “e” is followed by the letter “n.” The resulting fragments of this original sentence would look like this and would be similar to a genomic library. Copyright © 2012 Pearson Education, Inc. CHAPTER 12 DNA Technology and Genomics 150  -Age nomiclibraryofthese nte nceyouare nowreadingwouldbeallofthese nte ncefragme ntsthatmadeupthese nte nce. (12.2–12.3)  A cDNA library is a way to learn what portion of the genome is active at any given time in a cell’s life. In a very general way, it is like looking at the list of books checked out at a school library (assuming that the checked-out books are being used). (12.4)  Some Internet search programs rely upon a methodology similar in one way to the use of a nucleic acid probe. For example, if you want to find the lyrics to a particular song, but do not know the song title or artist, you might search the Internet using a unique phrase from the song. The search engine will scan millions of web pages to identify those sites containing that particular phrase. However, unlike a nucleic acid probe, you would search for the song by using a few of the lyrics. A nucleic acid probe search uses a sequence complementary to the desired sequence. (12.5) Genetically Modified Organisms (12.6–12.9) Student Misconceptions and Concerns  The genetic engineering of organisms can be controversial, creating various degrees of social unease and resistance. Yet, many debates about scientific issues are confused by misinformation. This provides an opportunity for you to assign students to take a position on such issues and support their arguments with accurate research. Students might debate whether a food or drug made from GM/transgenic organisms should be labeled as such, or discuss the risks and advantages of producing GM organisms. (12.6–12.10)  The fact that the technologies described in this chapter can be used to swap genes between prokaryotes and eukaryotes reveals the fundamental similarities in genetic mechanisms shared by all forms of life. This very strong evidence of common descent is evidence of evolution that may be missed by your students. (12.6–12.10)  Roundup Ready Corn, a product of the agricultural biotechnology corporation Monsanto, is resistant to the herbicide Roundup. The general strategy for farmers is to spray fields of Roundup Ready corn with the herbicide Roundup, killing weeds but not the corn. A search of the Internet will quickly reveal the controversy associated with this and other genetically modified organisms (GMO), which can encourage interesting discussions and promote critical thinking skills. Module 12.9 discusses some of the issues related to the concerns over the use of GM organisms. (12.8–12.9) equilibrium = equ ilibri um (three fragments of three, six, and two letters) equalibrium = equalibri um (two fragments of nine and two letters)  Students might assume that the term junk DNA implies that these noncoding regions of DNA are useless. This might be a good time to note the old saying, absence of evidence is not evidence of absence. Our current inability to understand the role(s) of noncoding DNA does not mean that these regions have no significance.  Students might know that humans have 23 pairs of chromosomes. Consider asking them how many different types of chromosomes are found in humans. Some will not have realized that there are 24 types, 22 autosomes plus X and Y sex chromosomes. (12.18) 151 INSTRUCTOR GUIDE FOR CAMPBELL BIOLOGY: CONCEPTS & CONNECTIONS Copyright © 2012 Pearson Education, Inc. Key Terms biotechnology genomics restriction site clone Human Genome Project reverse transcriptase complementary DNA (HGP) short tandem repeat (cDNA) nucleic acid probe (STR) DNA ligase plasmid single nucleotide DNA profiling polymerase chain reaction polymorphism (SNP) DNA technology (PCR) STR analysis forensics primers telomeres gel electrophoresis proteomics Ti plasmid gene cloning recombinant DNA transgenic organism gene therapy repetitive DNA transposable element genetic engineering restriction enzyme vaccine genetically modified restriction fragment length vector (GM) organism polymorphism (RFLP) whole-genome shotgun genomic library restriction fragments method Word Roots bio- = life; -tech- = skill or art (biotechnology: the manipulation of organisms or their components to make useful products) electro- = electricity (gel electrophoresis: a technique for separating and purifying macromolecules, including DNA, by using an electrical charge to stimulate their migration through a gel-based matrix) gen- = produce (genetic engineering: the direct manipulation of genes for practical purposes) liga- = bound, tied (DNA ligase: an enzyme that catalyzes the covalent bonding of adjacent DNA nucleotides in DNA replication) poly- = many (polymerase chain reaction: a technique used to obtain many copies of a DNA molecule or part of a DNA molecule, involving use of the enzyme DNA polymerase) proteo- = proteins (proteomics: the study of whole sets of proteins and their interactions) telos- = an end (telomere: the repetitive DNA at each end of a eukaryotic chromosome) trans- = across (transgenic organism: an organism that contains genes from a different species) Copyright © 2012 Pearson Education, Inc. 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