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Fig. 13-CO, p.330

Fig. 13-CO, p.330. Learning Objectives. 1. Purification & detection of nucleic acids. 2. Restriction Endonucleases 3. Cloning 4. Genetic Engineering 5. DNA libraries 6. PCR 7. DNA Fingerprinting. Gel Electrophoresis. Fig. 13-2, p.331.

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Fig. 13-CO, p.330

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  1. Fig. 13-CO, p.330

  2. Learning Objectives 1. Purification & detection of nucleic acids. 2. Restriction Endonucleases 3. Cloning 4. Genetic Engineering 5. DNA libraries 6. PCR 7. DNA Fingerprinting

  3. Gel Electrophoresis Fig. 13-2, p.331

  4. Separation of oligonucleotides by gel electrophoresis Fig. 13-1, p.331

  5. T An example of an autoradiography : The use of radioactive isotope 35S Fig. 13-3, p.332

  6. Hydrolysis of DNA by restriction endonucleases Fig. 13-4, p.333

  7. Production of recombinant DNA Fig. 13-5, p.334

  8. Table 13-1, p.335

  9. Restriction Endonucleases(r.e) • Enzymes produced by bacteria that hydrolyze the phosphodiester backbone of DNA at specific sequences • The sequences targeted by r.e are palindromes, • meaning their sequence reads the same on • both strands going in the same direction • 3. Most r.e cut DNA in a way that leaves sticky ends • that are very useful for recombining DNA from • different sources.

  10. p.335

  11. Dolly 1996-2003 In July, 2016, four identical clones of Dolly (Daisy, Debbie, Dianna and Denise) were alive and healthy at nine years old. p.331

  12. The cloning process that produced Dolly

  13. The cloning of a virus Fig. 13-6, p.336

  14. The cloning of cells Fig. 13-7, p.336

  15. The cloning of human DNA fragments with a viral vector Fig. 13-8, p.336

  16. Selecting for recombinant DNA in a bacterial plasmid Fig. 13-9, p.338

  17. DNA Plasmid Extra chromosomal self-replicating genetic elements of a bacterial cell & can be transferred from one strain of a bacterial species to another by cell-to-cell contact. p.338

  18. For A Successful Experiment When bacteria take up a plasmid, we say they have been transformed Bacteria are encouraged to take up foreign DNA by: 1.heat-shock the bacteria at 42 C. followed by placing them on ice. 2. Place them in an electric field “electroporation” Then selection through selectable markers on the plasmid.

  19. Plasmid pBR322 Fig. 13-10, p.339

  20. A vector cloning site containing multiple restriction sites Fig. 13-11, p.339

  21. Cloning with pU plasmids Fig. 13-12, p.340

  22. Clone selection via blue/white screening population Fig. 13-13, p.341

  23. Cloning • It refers to creating a genetically identical population • DND can be combined by using r.e that create sticky ends in the DNA. This rDNA has a target DNA sequence of interest • The target DNA sequence is carried in some type of vector, usually a bacterial plasmid or a virus • The target DNA sequence is inserted into a host organism & the natural doubling time of the organism is used to create many copies of the target DNA sequence • Organisms that are carrying the target DNA are identified through a process called selection, which often involves antibiotic resistance

  24. Synthesis of insulin in humans Fig. 13-15, p.345

  25. Production of recombinant human insulin Fig. 13-16, p.346

  26. Fusion Proteins and Fast Purifications . Affinity chromatography & fusion proteins combine to purify a protein efficiently p.348

  27. Transgenic tomato plant : Recombinant DNA methods have produced plants that resist defoliation by caterpillars, with longer shelf life. Fig. 13-18, p.349

  28. Genetic Engineering in Agriculture • Disease resistance e.g. corn & cotton • Nitrogen fixation • Frost-free plants e.g strawberries & potatoes • Tomatoes with a long shef life .deactivating the gene in tomato which produce ethylene. • Increased milk production giving cows bovine somatotropin (BST) “growth hormone”…. • Good predator attraction straw berry gene on mustard plants produces a chemical attractant for predator mites that eat the herbivorous spider mites.

  29. Genetic Engineering (g.e) • It is the process of inserting genes of interest into • specific organisms for either a medical or scientific • benefit • 2. Gene therapy is the process of inserting a missing • gene into an organism • 3. Bacteria are often used as the factories to produce a • protein from a cloned gene. This has led to the • production of human insulin & erythropoietin..etc. • 4. The gene must be cloned into an expression vector, • usually a plasmid with special features that allows it • to be transcribed & translated in a host cell. • 5. In agriculture g.e. is used to produce crops that are • resistant to insects or have long shelf lives…

  30. Steps involved in the construction of a DNA library Fig. 13-19, p.350

  31. DNA Library • It is a collection of clones of an entire genome • The genome is digested with r.e & the pieces are cloned into vectors & transformed into cell lines • Specific radioactive probes to a sequence of interest are reacted to filters that have copies of the bacterial colonies in the library. The probe binds to the sequence of interest, and the colony’s location can be seen by autoradiography • A cDNA library is constructed by using reverse transcriptase to make DNA from mRNA in a cell. This cDNA is then used to construct a library similar to a genomic DNA library.

  32. Screening a genomic library by colony hybridization or plaque hybridization Fig. 13-20, p.351

  33. Formation of cDNA Fig. 13-21, p.352

  34. Polymerase Chain Reaction (PCR) Fig. 13-22, p.353

  35. PCR • It is a sophisticated, automated technique for amplifying DNA from very small amounts of a sample. • The DNA to be amplified is mixed with specific primers, dATPs, dCTPs, dGTPs, dTTPs and a heat-stable form of DNA polymerase • The mixture undergoes 20 to 40 rounds of DNA polymerization via cycling the temp.so that the DNA strands separate, the primers anneal, and the polymerase fills in the DNA. Each cycle doubles the target DNA • The technique has revolutionized forensic science as DNA can be amplified from just a few cells and then the DNA analyzed and identified.

  36. The Southern blot Fig. 13-23, p.357

  37. The basis for restriction-fragment length polymorphism Fig. 13-24, p.359

  38. Paternity testing Fig. 13-25, p.360

  39. Localization of the gene associated with cyctic fibrosis On human chromosome 7 Restrictin fragment Length polymorphism (RFLP) Fig. 13-26, p.360

  40. DNA Fingerprinting 1. It is created by digesting DNA with r.e., separating the pieces on a gel & then visualizing some of the pieces by using labeled probes. 2. Differences in DNA patterns between different individuals are based on different base sequences of their DNA. These base sequences mean that different restriction sites will lead to different-length fragments.

  41. END L12

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