Genetic Technologies: Amplifying and Modifying DNA for Specific Applications

1. Human Genetics Concepts and Applications Eighth Edition Powerpoint Lecture Outline Ricki Lewis Prepared by Dubear Kroening University of Wisconsin-Fox Valley

2. Chapter 19 Genetic Technologies: Amplifying, Modifying, and Monitoring DNA

3. 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 be considered including patent laws Figure 19.1

4. 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

5. Technology Timeline

6. 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

7. Uses of PCR Table 19.1

8. Figure 19.2

9. 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

10. 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

11. 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

12. Creating Recombinant DNA Molecules Figure 19.3a

13. Figure 19.3b

14. Figure 19.3c

15. 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

16. Plasmids Figure 19.4

17. Recombinant DNA Figure 19.5

18. 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. Figure 19.6

20. 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

21. 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

22. 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

23. 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

24. Table 19.3

25. 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

26. Making a Transgenic Plant Figure 19.7 May use Ti plasmids to obtain foreign DNA

27. 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

28. Table 19.4

29. Table 19.5

30. 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

31. Table 19.6

32. Figure 19.8

33. 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

34. 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

35. Figure 19.9

36. Figure 19.9

37. Figure 19.9

38. Table 19.7

39. Figure 19.10

40. Solving a Problem Figure 19.11