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CHAPTER 12 DNA Technology PowerPoint® Lectures for Essential Biology, Third Edition – Neil Campbell, Jane Reece, and Eric Simon Essential Biology with Physiology, Second Edition – Neil Campbell, Jane Reece, and Eric Simon Lectures by Chris C. Romero Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Recombinant DNA Technology • Recombinant DNA technology is a set of techniques for combining genes from different sources into a single DNA molecule. – An organism that carries recombinant DNA is called a genetically modified (GM) organism. • Recombinant DNA technology is applied in the field of biotechnology. – Biotechnology uses various organisms to perform practical tasks. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Figure 12.2 From Humulin to Genetically Modified Foods • By transferring the gene for a desired protein product into a bacterium, proteins can be produced in large quantities. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Making Humulin • In 1982, the world’s first genetically engineered pharmaceutical product was produced. – Humulin, human insulin, was produced by genetically modified bacteria. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Unnumbered Figure p. 222 • Humulin was the first recombinant DNA drug approved by the FDA. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Figure 12.3 • DNA technology is also helping medical researchers develop vaccines. – A vaccine is a harmless variant or derivative of a pathogen. – Vaccines are used to prevent infectious diseases. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Genetically Modified (GM) Foods • Today, DNA technology is quickly replacing traditional plant-breeding programs. – In the United States today, roughly one-half of the corn crop and over three-quarters of the soybean and cotton crops are genetically modified. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings • Corn has been genetically modified to resist insect infestation. – This corn has been damaged by the European corn borer. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Figure 12.4 • “Golden rice” has been genetically modified to contain beta-carotene. – Our bodies use beta-carotene to make vitamin A. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Figure 12.5 Farm Animals and “Pharm” Animals • While transgenic plants are used today as commercial products, transgenic whole animals are currently only in the testing phase. • These transgenic sheep carry a gene for a human blood protein. – This protein may help in the treatment of cystic fibrosis. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Figure 12.6 • While transgenic animals are currently used to produce potentially useful proteins, none are yet found in our food supply. • It is possible that DNA technology will eventually replace traditional animal breeding. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Recombinant DNA Techniques • Bacteria are the workhorses of modern biotechnology. • To work with genes in the laboratory, biologists often use bacterial plasmids. – Plasmids are small, circular DNA molecules that are separate from the much larger bacterial chromosome. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Figure 12.7 • Plasmids can easily incorporate foreign DNA. • Plasmids are readily taken up by bacterial cells. – Plasmids then act as vectors, DNA carriers that move genes from one cell to another. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings • Recombinant DNA techniques can help biologists produce large quantities of a desired protein. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Figure 12.8 A Closer Look: Cutting and Pasting DNA with Restriction Enzymes • Recombinant DNA is produced by combining two ingredients: – A bacterial plasmid – The gene of interest • To combine these ingredients, a piece of DNA must be spliced into a plasmid. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings • This splicing process can be accomplished using restriction enzymes. – These enzymes cut DNA at specific nucleotide sequences. • These cuts produce pieces of DNA called restriction fragments – That may have “sticky ends” that are important for joining DNA from different sources. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Figure 12.9 Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Read background information and underline answers to: -What is recombinant DNA? -What is a plasmid? -What occurs at the origin of replication? -What are ampicillin and kanamycin? -What are pAMP and pKAN? -What role do bacteria play? http://www.sumanasinc.com/webcontent/animations /content/plasmidcloning.html Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Bacterial growth without plasmid: No antibiotics Ampicillin Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Kanamycin Ampicillin& Kanamycin Bacterial growth without plasmid: No antibiotics Ampicillin Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Kanamycin Ampicillin& Kanamycin Bacterial growth with plasmid: No antibiotics Ampicillin Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Kanamycin Ampicillin& Kanamycin Bacterial growth with plasmid: No antibiotics Ampicillin Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Kanamycin Ampicillin& Kanamycin Recombinant Paper Plasmid lab 1. pAMP = pKAN = 1A (amp) and 1B 2 A (kan) 2B and 1A + 1B (resistant to amp) 1B + 2B (no resistance) 1A + 2B (resistant to amp) 2A + 1B (no origin of rep) 1A + 2A (resistant to both) 2A + 2B (resistant to kan) Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Recombinant paper plasmid lab 2. No antibiotics + plasmid No antibiotics - plasmid Amp + plasmid Amp - plasmid Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Kan + plasmid Kan - plasmid Amp/Kan + plasmid Amp/Kan - plasmid Recombinant Paper Plasmid lab 3. pAMP = pKAN = 1A (amp) and 1B 2 A (kan) 2B and Would survive on one antibiotic but not the other! 1A + 1B (resistant to amp) 1A + 2B (resistant to amp) 2A + 2B (resistant to kan) 2A + 1B (resistant to kan, but no origin of rep) Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Recombinant Paper Plasmid Lab 4. The most important reason is so that scientists can identify bacteria that have been transformed. - Can select only bacteria that have the gene of interest! Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings A Closer Look: Obtaining the Gene of Interest • How can a researcher obtain DNA that encodes a particular gene of interest? • The “shotgun” approach is one way to synthesize a gene of interest. – Millions of recombinant plasmids containing different segments of foreign DNA are produced. – This collection is called a genomic library. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings • Once a genomic library is created, biologists must identify the bacterial clone containing the desired gene. – A specific sequence of radioactive nucleotides matching those in the desired gene can be created. – This type of labeled nucleic acid molecule is called a nucleic acid probe. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Figure 12.10 • Reverse transcriptase is another method for synthesizing a gene of interest. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Figure 12.11 Thursday, May 16th 1. Quick Check 2. Dr. Shephard: Guilty or Innocent? 3. Notes: DNA Fingerprinting and Electrophoresis (pg 30-31) Homework: page 6** ** Different from website Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings gel electrophoresis mapping your genome23 and me website Dr. Shephard Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings DNA Profiling and Forensic Science • DNA technology has rapidly revolutionized the field of forensics. – Forensics is the scientific analysis of evidence from crime scenes. • DNA profiling can be used to determine whether or not two samples of genetic material are from a particular individual. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Figure 12.12 Murder, Paternity, and Ancient DNA • DNA profiling (fingerprinting) – Has become a standard criminology tool. – Has been used to identify victims of the September 11, 2001, World Trade Center attack. – Can be used in paternity cases. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings • DNA fingerprinting is also used in evolutionary research – To study ancient pieces of DNA, such as that of Cheddar Man. – Found in a cave near Cheddar, England – Direct ancestor of local teacher Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Figure 12.13 DNA Fingerprinting Techniques The Polymerase Chain Reaction (PCR) • The polymerase chain reaction (PCR) is a technique by which any segment of DNA can be copied quickly and precisely. – Through PCR, scientists can obtain enough DNA from even minute amounts of blood or other tissue to allow DNA profiling. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Figure 12.14 Short Tandem Repeat (STR) Analysis • How do you prove that two samples of DNA come from the same person? Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings • Short tandem repeats (STRs) – Are repetitive sequences of DNA that are repeated various times in the genome. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings • Scientists use STR analysis – To compare the number of repeats between different samples of DNA. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings • Once a set of DNA fragments is prepared, – The next step in STR analysis is to determine the lengths of these fragments. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Figure 12.15 Gel Electrophoresis • Gel Electrophoresis – Can be used to separate the DNA fragments obtained from different sources. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Friday, April 17th 1. Review DNA Fingerprinting and Electrophoresis and page 6 2. Electrophoresis practice: page 12-13 3. DNA GOES to the Races: page 8-9 ** above is homework if not completed in class*** Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings gel electrophoresis mapping your genome23 and me website Dr. Shephard Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Figure 12.12 Figure 12.16 • The DNA fragments are visualized as “bands” on the gel. – The bands of different DNA samples can then be compared. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Figure 12.17 • One common application of gel electrophoresis is RFLP analysis, – In which DNA molecules to be compared are exposed to a series of restriction enzymes. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Figure 12.18 Genomics and Proteomics • Genomics is the science of studying whole genomes. – The first targets of genomics were bacteria. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Table 12.1 The Human Genome Project • In 1990, an international consortium of government-funded researchers began the Human Genome Project. – The goal of the project was to sequence the human genome. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings • Sequencing of the human genome presented a major challenge. – It is very large. – Only a small amount of our total DNA is contained in genes that code for proteins. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings • The Human Genome Project – Can help map specific disease genes such as Parkinson’s disease. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Figure 12.20 Genome-Mapping Techniques • The Human Genome Project proceeded through several stages, – During which preliminary maps were created and refined. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings • The whole-genome shotgun method – Involves sequencing DNA fragments from an entire genome and reassembling them in a single stage. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Figure 12.21 The Process of Science: Can Genomics Cure Cancer? • In 2003, the Food and Drug Administration – Approved the drug gefinitib for the treatment of lung cancer. • Unfortunately, gefinitib is ineffective for many patients. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings • A 2004 study found that genetic differences among patients affected their response to the drug, – Exhibiting how genomics can affect the treatment of disease. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Figure 12.22 Proteomics • Success in genomics has given rise to proteomics, – The systematic study of the full set of proteins found in organisms. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Human Gene Therapy • Human gene therapy is a recombinant DNA procedure that seeks to treat disease by altering the genes of the afflicted person. – The mutant version of a gene is replaced or supplemented with a properly functioning one. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Figure 12.23 Treating Severe Combined Immunodeficiency • SCID is a fatal inherited disease caused by a single defective gene. – The gene prevents the development of the immune system. – SCID patients quickly die unless treated with a bone marrow transplant. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings • Since the year 2000, – Gene therapy has successfully cured 22 children with inborn SCID. • Unfortunately, three of the children developed leukemia and one of them died. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Safety and Ethical Issues • As soon as scientists realized the power of DNA technology, they began to worry about potential dangers such as: – The creation of hazardous new pathogens – The transfer of cancer genes into infectious bacteria and viruses Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings • Strict laboratory safety procedures have been designed to protect researchers from infection by engineered microbes. – Procedures have also been designed to prevent microbes from accidentally leaving the laboratory. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Figure 12.24 The Controversy over Genetically Modified Foods • GM strains account for a significant percentage of several agricultural crops in the United States. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Figure 12.25 • Advocates of a cautious approach have some concerns: – Crops carrying genes from other species might harm the environment. – GM foods could be hazardous to human health. – Transgenic plants might pass their genes to close relatives in nearby wild areas. Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings • Negotiators from 130 countries (including the United States) agreed on a Biosafety Protocol. – The protocol requires exporters to identify GM organisms present in bulk food shipments. • Several U.S. regulatory agencies evaluate biotechnology projects for potential risks: – Department of Agriculture – Food and Drug Administration – Environmental Protection Agency – National Institutes of Health Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Ethical Questions Raised by DNA Technology • Should genetically engineered human growth hormone be used to stimulate growth in HGHdeficient children? Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Figure 12.26 • Genetic engineering of gametes and zygotes has been accomplished in lab animals. – Should we try to eliminate genetic defects in our children? – Should we interfere with evolution in this way? • Advances in genetic fingerprinting raise privacy issues. • What about the information obtained in the Human Genome Project? – How do we prevent genetic information from being used in a discriminatory manner? Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings Evolution Connection: Genomes Hold Clues to Evolution • Genome data has confirmed evolutionary connections. • Comparisons of completed genome sequences strongly support the theory that there are three fundamental domains of life: – Bacteria – Archaea – Eukaryotes Copyright © 2007 Pearson Education Inc., publishing as Pearson Benjamin Cummings