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Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Powerpoint Lecture Outline Human Genetics Concepts and Applications Eighth Edition Ricki Lewis Prepared by Dubear Kroening University of Wisconsin-Fox Valley 19-1 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Chapter 19 Genetic Technologies: Amplifying, Modifying, and Monitoring DNA 19-2 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Biotechnology • Use or alteration of cells or biological molecules for specific application • Transgenic organisms are possible, but the genetic code is universal • Ethical and legal issues to Figure 19.1 be considered including patent laws 19-3 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Transgenic Animals • Have genetic modifications and carry that genetic alteration from other organisms in all of their cells • Recombinant DNA – bacteria making human insulin • Patenting issues 19-4 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Technology Timeline 19-5 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Amplifying DNA • Polymerase chain reaction (PCR) – Increases the amount of a DNA sequence in a tube – Replicates sequence millions of times • Recombinant DNA technology – Amplifies DNA within cells often using sequences from other organisms 19-6 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Uses of PCR Table 19.1 19-7 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Figure 19.2 19-8 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Transcription-Mediated Amplification • Copies target DNA into RNA and then uses RNA polymerase to amplify RNA • Does not require temperature shifts • Makes 10 billion copies in ½ hour 19-9 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Recombinant DNA • Recombinant DNA is a molecule that combines DNA from two sources, also known as gene cloning • Creates a new combination of genetic material • Human gene for insulin was placed in bacteria to make large quantities for diabetics • Genetically modified organisms are possible because of the universal nature of the genetic code • Safety concerns 19-10 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Creating Recombinant DNA Molecules • Cut DNA from donor and recipient with the same restriction enzymes • Cut DNA fragment is combined with a vector • Vector DNA moves and copies DNA fragment of interest • Vector cut with restriction enzymes • The complementary ends of the DNAs bind and ligase enzyme reattaches the sugar-phosphate backbone of the DNA 19-11 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Creating Recombinant DNA Molecules Figure 19.3a 19-12 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Figure 19.3b 19-13 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Figure 19.3c 19-14 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Vectors • Are DNA molecules that can be moved into and replicated in an organism • They are classified by – The organisms that replicated the vector – The size of DNA that can be inserted Table 19.2 19-15 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Plasmids Figure 19.4 19-16 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Recombinant DNA Figure 19.5 19-17 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Isolating Gene of Interest • Genomic library – Collections of recombinant DNA that contain pieces of the genome • DNA probe – Radioactively (or fluorescently) labeled gene fragments • cDNA library – Genomic library of protein encoding genes produced by extracting mRNA and using reverse trancriptase to make DNA 19-18 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Figure 19.6 19-19 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Selecting Recombinant Molecules • Three types of cells can result from attempt to introduce a DNA molecule into a bacterial cell: – Cells lack plasmid – Cells contain plasmid that do not contain foreign genes – Cells that contain plasmids with foreign genes 19-20 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Selecting for Cells with Vectors • Vectors are commonly engineered to carry antibiotic resistance genes • Host bacteria without a plasmid die in the presence of the antibiotic • Bacteria harboring the vector survive • Growing cells on media with antibiotics ensures that all growing cells must carry the vector 19-21 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Selecting Cells with Inserted DNA • The site of insertion of the DNA of interest can be within a color-producing gene on the vector • Insertion of a DNA fragment will disrupt the vector gene and there will be a lack of color 19-22 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Applications of Recombinant DNA Recombinant DNA is used to: • Study the biochemical properties or genetic pathways of that protein • Mass produce a particular protein (e.g., insulin) • Sometimes conventional methods are still the better choice • Textile industry can produce indigo dye in E. coli by genetically modifying genes of the glucose pathway and introducing genes from another bacterial species 19-23 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Table 19.3 19-24 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Transgenic Organisms • When recombinant DNA is applied to multicellular organisms, individuals must be bred to yield homozygous individuals • Plants may be produced by asexual reproduction (cuttings) • Different vectors and gene transfer techniques can be used 19-25 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Making a Transgenic Plant Figure 19.7 May use Ti plasmids to obtain foreign DNA 19-26 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Bt Insecticide gene • From bacterium Bacillus thuringiensis (bt) • Specifies a protein that destroys the stomach lining of certain insect larva • 2/3 of U.S. corn is transgenic for the bt gene 19-27 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Table 19.4 19-28 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Table 19.5 19-29 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Transgenic Animals • More difficult than plants • Several techniques to insert DNA – Chemicals to open holes in plasma membrane and liposomes carry DNA in cells – Electroporation–a brief jolt of electricity to open membrane – Microinjection – uses microscopic needles – Particle bombardment – a gun like device shoots metal particles coated with foreign DNA 19-30 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Table 19.6 19-31 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Figure 19.8 19-32 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Bioremediation • Transgenic organisms can provide process as well as products • Ability to detoxify pollutants • Examples – Hg-contaminated soils – GFP gene reveal locations of land mines 19-33 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Monitoring Gene Function • Gene Expression Profiling – Indicates genes transcribed • DNA Variation Screening – Detects mutations in Single Gene Polymorphisms (SNPs) • Microarray Comparative Genomic Hybridization – Deletions and amplifications of DNA sequences between cells or species 19-34 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Figure 19.9 19-35 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Figure 19.9 19-36 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Figure 19.9 19-37 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Table 19.7 19-38 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Figure 19.10 19-39 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Solving a Problem Figure 19.11 19-40