Introduction

2. Introduction Biotechnology is the use or alteration of cells or biological molecules for specific applications A transgenic organism has DNA from different species Recombinant DNA comes from more than one type of organism Both are possible because of the universality of the genetic code

3. Introduction Figure 19.1 Mice containing the jellyfish gene for green fluorescent protein (GFP)

4. What is Patentable? To qualify for patent protection, a transgenic organism must be new, useful, and non-obvious Patent law has had to evolve to keep up with modern biotechnology A DNA sequence alone does not warrant patent protection - It must be useful as a tool for research or as a novel or improved product, such as a diagnostic test or drug

5. What is Patentable? A new problem for patenting DNA stems from the shift in focus of the entire field from a single-gene to a genome-wide approach Especially problematic are: - “Panels” of tests - “Direct-to-consumer” genetic tests that scan client’s DNA for many thousands of SNPs

6. The “Patent Thicket” Term used to describe the need to license patents for every SNP or snippet of DNA Suggested ways around this: - Allow DNA to be patented only for use in diagnostic tests - Exempt individuals from litigation if they use patented DNA sequences A broader action is to ban patenting any DNA or its encoded proteins

8. Amplifying DNA Polymerase chain reaction (PCR) - Works on DNA molecules outside of cells - Replicates sequence millions of times Recombinant DNA technology - Amplifies DNA within cells often using sequences from other organisms

9. PCR Consists of a repetition of three basic steps: 1. Denaturation: Heat is used to separate the two strands of target DNA 2. Annealing: Two short DNA primers bind to the DNA at a lower temperature 3. Extension: The enzyme Taq1 DNA polymerase adds bases to the primers All this is done in a thermal cycler Copies of DNA accumulate exponentially

10. PCR Figure 19.2

11. Table 19.1

12. Transcription-Mediated Amplification Copies target DNA into RNA and then uses RNA polymerase to amplify RNA Does not require temperature shifts Generates 100 to 1,000 copies per cycle, compared to PCR’s doubling - Can yield 10 billion copies of a selected sequence in 30 minutes

13. Recombinant DNA Technology Recombinant DNA technology is also known as gene cloning It began in 1975 when molecular biologists convened to discuss the safety and implications of this new technology However, it turned out to be safer than expected - It also spread to industry faster and in more diverse ways than imagined

14. Creating Recombinant DNA Molecules Manufacturing recombinant DNA requires restriction enzymes that cut donor and recipient DNA at the same sequence These enzymes cut DNA at sites that are palindromic The cutting action of many of these enzymes generate single-stranded extensions called “sticky ends”

15. Figure 19.3

16. Creating Recombinant DNA Molecules Another “tool” used is a cloning vector - Carries DNA from the cells of one species into the cells of another Commonly used vectors include: - Plasmids - Bacteriophages - Disabled retroviruses

17. Creating Recombinant DNA Molecules Cut DNA from donor and plasmid vector with the same restriction enzyme Mix to generate recombinant DNA molecule When such a modified plasmid is introduced into a bacterium, it is mass produced as the bacterium divides

18. Figure 19.4

19. 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 transcriptase to make DNA

20. Figure 19.5

21. Selecting Recombinant Molecules Three types of recipient cells can result from attempt to introduce a DNA molecule into a bacterial cell 1. Cells that lack plasmids 2. Cells with plasmids that do not contain foreign genes 3. Cells that contain plasmids with foreign genes

22. 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

23. Selecting For 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 so the bacterial colony that grows will be colorless

24. Applications of Recombinant DNA Recombinant DNA is used to: - Study the biochemical properties or genetic pathways of that protein - Mass-produce proteins (e.g., insulin) Sometimes conventional methods are still the better choice because of economics Textile industry can produce indigo dye in E. coli by genetically modifying genes of the glucose pathway and introducing genes from another bacterial species

25. Table 19.3

26. Transgenic Animals An even more efficient way to express some recombinant genes is in a body fluid of a transgenic animal Transgenic sheep, cows, and goats have all expressed human genes in their milk, - Clotting factors - Clot busters - Collagen - Antibodies

27. Several techniques are used to insert DNA into cells to create transgenic animals - Chemicals that open transient holes in plasma membrane - Liposomes that carry DNA into 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

28. Transgenic Animals Finally, an organism must be regenerated from the altered cell If the trait is dominant, the transgenic animal must express it in the appropriate tissue at the right time in development If the trait is recessive, crosses between heterozygotes may be necessary to yield homozygotes that express the trait

29. Animal Models Transgenic animals are far more useful as models of human diseases - Example: Inserting the mutant human beta globin gene that causes sickle-cell anemia into mice Drug candidates can be tested on these animal models before testing on humans - Will be abandoned if they cause significant side effects

30. Animal Models Transgenic animal models have limitations - Researchers cannot control where a transgene inserts, and how many copies do so - The level of gene expression necessary for a phenotype may differ in the model and humans - Animal models may not mimic the human condition exactly because of differences in development or symptoms

31. Animal Models Figure 19.6

32. Bioremediation Transgenic organisms can provide processes as well as products Bioremediation: The use of bacteria or plants to detoxify environmental pollutants Examples - Nickel-contaminated soils - Mercury-tainted soils - Trinitrotoluene (TNT) in land mines

33. Figure 19.7

34. Monitoring Gene Function Gene expression DNA microarrays (gene chips) are devices that detect and display the mRNAs in a cell A microarray is a piece of glass or plastic that is about 1.5 centimeters square Many small pieces of DNA of known sequence are attached to one surface, in a grid pattern In many applications, a sample from an abnormal situation is compared to a normal control

35. Monitoring Gene Function Messenger RNAs are extracted from the samples and cDNAs are made These are differentially-labeled and then applied to the microarray The pattern and color intensities of the spots indicate which genes are expressed A laser scanner detects and computer algorithms interpret the results

36. Monitoring Gene Function Figure 19.8

39. Table 19.7

40. Silencing DNA In some situations, silencing gene expression may be useful - Blocking transcription of oncogenes Three techniques can be used to control gene expression - RNA interference - Antisense sequences - Knockouts from gene targeting

41. RNA Interference Single-stranded RNAs can fold into short, double-stranded regions (hairpins) when the sequence is complementary Figure 19.9

42. RNA Interference Short, double-stranded RNAs sent into cells separate into single strands - One of these strands binds its complement in mRNA, preventing it from being translated This block in translation is RNA interference (RNAi) - The small RNAs that carry it out are called “small interfering RNAs” (siRNAs)

43. Antisense Sequences Antisense-induced exon skipping silences mutations that cause exons to be cut out of maturing mRNA Researchers introduce short, synthetic DNA molecules called morpholinos - Complementary or “antisense” to parts of the gene with the splicing mutation Morpholinos bind to their complement - Thus, the original mutation is bypassed, enabling production of full-length protein

44. Knockouts from Gene Targeting Gene targeting is a technique that uses homologous recombination to replace a normal DNA sequence with one that cannot be transcribed or translated - This silences gene expression by creating a “knockout” gene - Moreover, observing what happens (or not) can reveal the gene’s normal function A variation of the technique exchanges genes that have an altered function, producing a “knockin”

45. Knockouts from Gene Targeting Knockout mice are valuable in several ways: - Are more accurate models than transgenic mice - Populations are easily tested - Knockouts for several genes can be created to observe polygenic traits - Mice with diseases that humans also get can be observed