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Chapter 12 DNA Technology Power. Point® Lectures for Campbell Essential Biology, Fifth Edition, and Campbell Essential Biology with Physiology, Fourth Edition – Eric J. Simon, Jean L. Dickey, and Jane B. Lectures by Edward J. Zalisko Reece © 2013 Pearson Education, Inc.
Biology and Society: DNA, Guilt, and Innocence • DNA profiling is the analysis of DNA samples that can be used to determine whether the samples come from the same individual. • DNA profiling can therefore be used in courts to indicate if someone is guilty of a crime. © 2013 Pearson Education, Inc.
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Biology and Society: DNA, Guilt, and Innocence • DNA technology has led to other advances in the – creation of genetically modified crops and – identification and treatment of genetic diseases. © 2013 Pearson Education, Inc.
RECOMBINANT DNA TECHNOLOGY • Biotechnology – is the manipulation of organisms or their components to make useful products and – has been used for thousands of years to – make bread using yeast and – selectively breed livestock for desired traits. © 2013 Pearson Education, Inc.
RECOMBINANT DNA TECHNOLOGY • Biotechnology today means the use of DNA technology, techniques for – studying and manipulating genetic material, – modifying specific genes, and – moving genes between organisms. © 2013 Pearson Education, Inc.
RECOMBINANT DNA TECHNOLOGY • Recombinant DNA is constructed when scientists combine pieces of DNA from two different sources to form a single DNA molecule. • Recombinant DNA technology is widely used in genetic engineering, the direct manipulation of genes for practical purposes. © 2013 Pearson Education, Inc.
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Applications: From Humulin to Foods to “Pharm” Animals • By transferring the gene for a desired protein into a bacterium or yeast, proteins that are naturally present in only small amounts can be produced in large quantities. © 2013 Pearson Education, Inc.
Making Humulin • In 1982, the world’s first genetically engineered pharmaceutical product was sold. • Humulin, human insulin – was produced by genetically modified bacteria and – is used today by more than 4 million people with diabetes. • Today, humulin is continuously produced in gigantic fermentation vats filled with a liquid culture of bacteria. © 2013 Pearson Education, Inc.
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Making Humulin • DNA technology is used to produce medically valuable molecules, including – human growth hormone (HGH), – the hormone erythropoietin (EPO), which stimulates production of red blood cells, and – vaccines, harmless variants or derivatives of a pathogen used to prevent infectious diseases. © 2013 Pearson Education, Inc.
Genetically Modified (GM) Foods • Today, DNA technology is quickly replacing traditional breeding programs. • Scientists have produced many types of genetically modified (GM) organisms, organisms that have acquired one or more genes by artificial means. • A transgenic organism contains a gene from another organism, typically of another species. © 2013 Pearson Education, Inc.
Genetically Modified (GM) Foods • In the United States today, roughly half of the corn crop and more than three-quarters of the soybean and cotton crops are genetically modified. • Corn has been genetically modified to resist insect infestation, attack by an insect called the European corn borer. © 2013 Pearson Education, Inc.
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Genetically Modified (GM) Foods • Strawberry plants produce bacterial proteins that act as a natural antifreeze, protecting the plants from cold weather. • Potatoes and rice have been modified to produce harmless proteins derived from the cholera bacterium and may one day serve as edible vaccines. © 2013 Pearson Education, Inc.
“Pharm” Animals • A transgenic pig has been produced that carries a gene for human hemoglobin, which can be – isolated and – used in human blood transfusions. • In 2006, genetically modified pigs carried roundworm genes that produce proteins that convert less healthy fatty acids to omega-3 fatty acids. • However, unlike transgenic plants, no transgenic animals are yet sold as food. © 2013 Pearson Education, Inc.
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Recombinant DNA Techniques • Bacteria are the workhorses of modern biotechnology. • To work with genes in the laboratory, biologists often use bacterial plasmids, small, circular DNA molecules that replicate separately from the larger bacterial chromosome. © 2013 Pearson Education, Inc.
Figure 12. 7 Bacterial chromosome Remnant of bacterium Colorized TEM Plasmids
Recombinant DNA Techniques • Plasmids – can carry virtually any gene, – can act as vectors, DNA carriers that move genes from one cell to another, and – are ideal for gene cloning, the production of multiple identical copies of a gene-carrying piece of DNA. © 2013 Pearson Education, Inc.
Recombinant DNA Techniques • Recombinant DNA techniques can help biologists produce large quantities of a desired protein. © 2013 Pearson Education, Inc.
Figure 12. 8 Bacterial cell 1 2 Isolate plasmids. 3 Plasmid Isolate DNA. Cut both DNAs with same enzyme. Gene Other of interest genes 4 DNA fragments from cell Cell containing the gene of interest DNA Mix the DNA fragments and join them together. Gene of interest Recombinant DNA plasmids 5 Bacteria take up recombinant plasmids. Recombinant bacteria Bacterial clone 6 Clone the bacteria. 7 A gene for pest resistance is inserted into plants. A gene is used to alter bacteria for cleaning up toxic waste. Some uses of proteins Some uses of genes 8 Genes may be inserted into other organisms. Find the clone with the gene of interest. The gene and protein of interest are isolated from the bacteria. Bacteria produce proteins, which can be harvested and used directly. A protein is used to dissolve blood clots in heart attack therapy. A protein is used to prepare “stone-washed” blue jeans.
Figure 12. 8 c Some uses of genes Genes for cleaning up toxic waste Genes may be inserted into other organisms. Gene for pest resistance Some uses of proteins 8 The gene and protein of interest are isolated from the bacteria. Harvested proteins may be used directly. Protein for dissolving clots Protein for “stone-washing” jeans
A Closer Look: Cutting and Pasting DNA with Restriction Enzymes • Recombinant DNA is produced by combining two ingredients: 1. a bacterial plasmid and 2. the gene of interest. • To combine these ingredients, a piece of DNA must be spliced into a plasmid. © 2013 Pearson Education, Inc.
A Closer Look: Cutting and Pasting DNA with Restriction Enzymes • This splicing process can be accomplished by – using restriction enzymes, which cut DNA at specific nucleotide sequences (restriction sites), and – producing pieces of DNA called restriction fragments with “sticky ends” important for joining DNA from different sources. © 2013 Pearson Education, Inc.
Figure 12. 9 -4 Recognition site (recognition sequence) for a restriction enzyme DNA 1 A restriction enzyme cuts the Restriction enzyme DNA into fragments. ye tick S nd Stic ky e 2 nd A DNA fragment is added from another source. 3 Fragments stick together by base pairing. 4 DNA ligase joins the fragments into strands. DNA ligase Recombinant DNA molecule
A Closer Look: Obtaining the Gene of Interest • Another approach is to – use an automated DNA-synthesizing machine and – synthesize a gene of interest from scratch. © 2013 Pearson Education, Inc.
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DNA PROFILING AND FORENSIC SCIENCE • DNA profiling – can be used to determine if two samples of genetic material are from a particular individual and – has rapidly revolutionized the field of forensics, the scientific analysis of evidence from crime scenes. • To produce a DNA profile, scientists compare sequences in the genome that vary from person to person. © 2013 Pearson Education, Inc.
Figure 12. 13 -3 1 DNA isolated 2 DNA amplified 3 DNA compared Crime scene Suspect 1 Suspect 2
Investigating Murder, Paternity, and Ancient DNA • DNA profiling can be used to – test the guilt of suspected criminals, – identify tissue samples of victims, – resolve paternity cases, – identify contraband animal products, and – trace the evolutionary history of organisms. © 2013 Pearson Education, Inc.
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DNA Profiling Techniques The Polymerase Chain Reaction (PCR) • The polymerase chain reaction (PCR) – is a technique to copy quickly and precisely a specific segment of DNA and – can generate enough DNA, from even minute amounts of blood or other tissue, to allow DNA profiling. © 2013 Pearson Education, Inc.
Figure 12. 15 Initial DNA segment 1 2 4 8 Number of DNA molecules
Gel Electrophoresis • DNA analysis – compares the lengths of DNA fragments and – uses gel electrophoresis, a method for sorting macromolecules—usually proteins or nucleic acids —primarily by their – electrical charge and – size. © 2013 Pearson Education, Inc.
Figure 12. 17 -3 Mixture of DNA fragments of different sizes Band of longest (slowest) fragments Power source Band of shortest (fastest) fragments
Gel Electrophoresis • The DNA fragments are visualized as “bands” on the gel. • The differences in the locations of the bands reflect the different lengths of the DNA fragments. © 2013 Pearson Education, Inc.
Figure 12. 18 Amplified crime scene DNA Amplified suspect’s DNA Longer fragments Shorter fragments
GENOMICS • Genomics is the study of complete sets of genes (genomes). – The first targets of genomics research were bacteria. – As of 2011, – the genomes of more than 1, 700 species have been published and – more than 8, 000 are in progress. © 2013 Pearson Education, Inc.
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The Human Genome Project • Begun in 1990, the Human Genome Project was a massive scientific endeavor to – determine the nucleotide sequence of all the DNA in the human genome and – identify the location and sequence of every gene. © 2013 Pearson Education, Inc.
The Human Genome Project • At the completion of the project, – more than 99% of the genome had been determined to 99. 999% accuracy, – about 3 billion nucleotide pairs were identified, – about 21, 000 genes were found, and – about 98% of the human DNA was identified as noncoding. © 2013 Pearson Education, Inc.
The Human Genome Project • The Human Genome Project can help map the genes for specific diseases such as – Alzheimer’s disease and – Parkinson’s disease. © 2013 Pearson Education, Inc.
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Tracking the Anthrax Killer • In October 2001, – a Florida man died after inhaling anthrax and – by the end of the year, four other people had also died from anthrax. © 2013 Pearson Education, Inc.
Tracking the Anthrax Killer • In 2008, investigators – completed a whole-genome analysis of the spores used in the attack, – found four unique mutations, and – traced the mutations to a single flask at an Army facility. © 2013 Pearson Education, Inc.
Figure 12. 21 Envelope containing anthrax spores Colorized SEM Anthrax spore
Tracking the Anthrax Killer • Although never charged, an army research scientist suspected in the case committed suicide in 2008, and the case remains officially unsolved. © 2013 Pearson Education, Inc.
Tracking the Anthrax Killer • The anthrax investigation is just one example of the new field of bioinformatics, the application of computational tools to molecular biology. Additional examples include – evidence that a Florida dentist transmitted HIV to several patients, – tracing the West Nile virus outbreak in 1999 to a single natural strain of virus infecting birds and people, and – determining that our closest living relative, the chimpanzee (Pan troglodytes), shares 96% of our genome. © 2013 Pearson Education, Inc.
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HUMAN GENE THERAPY • Human gene therapy – is a recombinant DNA procedure, – seeks to treat disease by altering the genes of the afflicted person, and – often replaces or supplements the mutant version of a gene with a properly functioning one. © 2013 Pearson Education, Inc.
Figure 12. 24 Normal human gene 1 An RNA version of a normal human gene is inserted into a harmless RNA virus. RNA genome of virus Inserted human RNA Healthy person 2 Bone marrow cells of the patient are infected with the virus. 3 Viral DNA carrying the human gene inserts into the cell’s chromosome. Bone marrow cell from the patient Bone marrow 4 The engineered cells are injected into the patient. Bone of person with disease
HUMAN GENE THERAPY • Severe combined immunodeficiency (SCID) is – a fatal inherited disease and – caused by a single defective gene that prevents the development of the immune system. • SCID patients quickly die unless treated with – a bone marrow transplant or – gene therapy. © 2013 Pearson Education, Inc.
HUMAN GENE THERAPY • From 2000 to 2011, gene therapy has cured 22 children with inborn SCID. • However, there have been some serious side effects. Four of the children developed leukemia, which proved fatal to one. © 2013 Pearson Education, Inc.
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 and – transfer of cancer genes into infectious bacteria and viruses. © 2013 Pearson Education, Inc.
SAFETY AND ETHICAL ISSUES • Strict laboratory safety procedures have been designed to – protect researchers from infection by engineered microbes and – prevent microbes from accidentally leaving the laboratory. © 2013 Pearson Education, Inc.
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The Controversy over Genetically Modified Foods • GM strains account for a significant percentage of several staple crops in the United States. • Advocates of a cautious approach are concerned that – crops carrying genes from other species might harm the environment, – GM foods could be hazardous to human health, and/or – transgenic plants might pass their genes to close relatives in nearby wild areas. © 2013 Pearson Education, Inc.
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The Controversy over Genetically Modified Foods • In the United States, all projects are evaluated for potential risks by a number of regulatory agencies, including the – Food and Drug Administration, – Environmental Protection Agency, – National Institutes of Health, and – Department of Agriculture. © 2013 Pearson Education, Inc.
Ethical Questions Raised by DNA Technology • DNA technology raises legal and ethical questions —few of which have clear answers. – Should genetically engineered human growth hormone be used to stimulate growth in HGHdeficient children? – Should we try to eliminate genetic defects in our children and their descendants? – Should people use mail-in kits that can tell healthy people their relative risk of developing various diseases? © 2013 Pearson Education, Inc.
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Ethical Questions Raised by DNA Technology • DNA technologies raise many complex issues that have no easy answers. • We as a society and as individuals must become educated about DNA technologies to address the ethical questions raised by their use. © 2013 Pearson Education, Inc.
Evolution Connection: The Y Chromosome as a Window on History • Barring mutations, the human Y chromosome passes essentially intact from father to son. • By comparing Y DNA, researchers can learn about the ancestry of human males. © 2013 Pearson Education, Inc.
Evolution Connection: The Y Chromosome as a Window on History • DNA profiling of the Y chromosome has revealed that – nearly 16 million men currently living may be descended from Genghis Khan, – nearly 10% of Irish men were descendants of Niall of the Nine Hostages, a warlord who lived during the 1400 s, and – the Lemba people of southern Africa are descended from ancient Jews. © 2013 Pearson Education, Inc.
Figure 12. UN 01 DNA isolated from two sources and cut by same restriction enzyme Gene of interest (could be obtained from a library or synthesized) Plasmid (vector) Recombinant DNA Transgenic organisms Useful products
Figure 12. UN 02 Crime scene Suspect 1 Suspect 2 DNA Polymerase chain reaction (PCR) amplifies STR sites Longer DNA fragments Gel Shorter DNA fragments compared by gel electrophoresis (Bands of shorter fragments move faster toward the positive pole. )
Figure 12. UN 03 RNA version of a normal human gene Virus with RNA genome Bone marrow A normal human gene is transcribed and translated in a patient, potentially curing the genetic disease permanently