Chp. 13 An Introduction to Genetic Technology 13.1 What Are Clones?

1. Chp. 13 An Introduction to Genetic Technology 13.1 What Are Clones? • Clones • Genetically identical molecules, cells, or organisms all derived from a single ancestor • Cloning • The production of identical copies of molecules, cells, or organisms from a single ancestor

2. Cloning Higher Plants and Animals • Development of methods for cloning higher plants and animals represents a significant advance in genetic technology • Improving crops • Producing domestic animals

3. Animals Can Be Cloned by Several Methods • Embryo splitting • After in vitro fertilization, early embryonic cells are divided and grown into clones • Nuclear transfer (cell fusion) • Enucleated eggs are fused with embryonic or adult cells and grown into clones • Dolly the sheep

4. Cloning by Nuclear Fusion Fig. 13-1, p. 294

5. Dolly the Sheep • Dolly was cloned by fusion of a nucleus from an adult cell with an enucleated egg. Fig. 13-2, p. 294

6. Cloning by Nuclear Injection Fig. 13-3, p. 295

7. Keep In Mind • Cloned plants and animals are used in research, agriculture, and medicine

8. 13.2 Cloning Genes Is a Multistep Process • Recombinant DNA technology and the cloning of DNA molecules has revolutionized laboratory research, heath care and the food we eat. • Recombinant DNA technology: a series of techniques in which DNA fragments are linked to self-replicating vectors to create recombinant DNA molecules, which are replicated in host cells.

9. Why is DNA Cloning Important? • DNA clones are used to find genes, map them, and transfer them between species • Cloning technology is used to find carriers of genetic disorders, perform gene therapy, and create disease-resistant plants

10. DNA Cloning Requires Three Things • A way to cut DNA at specific sites to produce consistent sizes • A carrier molecule to hold DNA for cloning at for transfer to a host • A host cell where the DNA can be copied

11. DNA Can Be Cut at Specific SitesUsing Restriction Enzymes • Bacteria produce restriction enzymes to help protect themselves from viral infections • Restriction enzymes • Bacterial enzymes that cut DNA at specific sites

12. The Restriction Enzyme EcoRI • The recognition and cutting site for EcoRI Fig. 13-4, p. 296

13. Several Restriction Enzymesand Cutting Sites Fig. 13-5, p. 296

14. Vectors are Carriers of DNA to be Cloned • Linking DNA segments produced by restriction-enzymes with vectors (plasmids or engineered viral chromosomes) produces recombinant DNA • Vectors • Self-replicating DNA molecules used to transfer foreign DNA segments between host cells

15. Restriction Sites on Plasmid pBR322 Fig. 13-6, p. 297

16. Cloning Recombinant DNA • Recombinant DNA molecules are transferred into host cells; cloned copies are produced as the host cells grow and divide • Most common host cell: the bacterium E. coli • Cloned DNA molecules can be recovered from the host cells and purified for further use

17. Creating Recombinant DNA Molecules Fig. 13-7, p. 297

18. Steps in the Process of Cloning • DNA is cut with a restriction enzyme • Fragments produced end in specific sequences • Fragments are mixed with vector molecules cut by the same enzyme • DNA ligase joins recombinant DNA molecules

19. Steps in the Process of Cloning • Plasmid vectors with inserted DNA fragments are transferred into bacterial cells • Recombinant plasmids replicate and produce many clones of the recombinant DNA molecule • Colonies carrying cloned recombinant DNA molecules are identified, collected, and grown • Host cells are broken open and recombinant plasmids are extracted

20. Colonies of Bacteria on Petri Plates • Each colony is a clone, descended from a single cell Fig. 13-8, p. 298

21. 13.3 Cloned Libraries • A collection of cloned DNA sequences from one source is a library • Genomic library • Chromosomal library • Expressed sequence library • Libraries are resources for gene studies

22. Exploring Genetics Asilomar: Scientists Get Involved • An international conference was held at Asilomar, California, to consider the possible dangers of recombinant DNA technology • In 1976, guidelines were set in place for experiments using recombinant bacteria • New guidelines were published in 1982 • No experiments are currently prohibited

23. 13.4 Finding a Specific Clone in a Library • Clones for specific genes can be recovered from a library by using probes to screen the library • Probe • A labeled nucleic acid used to identify a complementary region in a clone or genome by hybridization

24. A DNA Probe Fig. 13-9, p. 299

25. Using a Probe to Identify a Specific Gene in a Genome Library

26. Individual bacterial cells from a DNA library are spread over the surface of a solid growth medium. The cells divide and form colonies—clusters of millions of genetically identical daughter cells. 1 Fig. 13-10a, p. 300

27. A DNA-binding filter pressed onto the surface of the growth medium will bind some cells from each colony. 2 Fig. 13-10b, p. 300

28. The filter is soaked in a solution that ruptures the cells and releases their DNA. The DNA clings to the paper in spots mirroring the distribution of colonies. 3 Fig. 13-10c, p. 300

29. A radioactive probe is added to the liquid. The probe hybridizes with (sticks to) only the spots of DNA that contain complementary base sequences. 4 Fig. 13-10d, p. 300

30. 5 Here, one radioactive spot darkens X-ray film. The position of the spot on the film is compared to the positions of all the original bacterial colonies. Cells from the colony that made the spot are cultured, and the DNA they contain is harvested. Fig. 13-10e, p. 300

31. 13.5 A Revolution in Cloning: The Polymerase Chain Reaction • Polymerase chain reaction (PCR) • A method for amplifying DNA segments using cycles of denaturation, annealing to primers, and DNA polymerase-directed DNA synthesis • PCR copies a DNA molecule without restriction enzymes, vectors, or host cells • Faster and easier than conventional cloning

32. First Step in PCR: Denaturation 1. DNA is heated to break the hydrogen bonds between the two polynucleotide strands • Two single-stranded DNA molecules serve as templates

33. Second Step in PCR: Annealing 2. Short nucleotide sequences (primers for DNA replication) are mixed with the DNA and bind to complementary regions on single-stranded DNA • Takes place at lower temperature • Primers are 20-30 nucleotides long, synthesized in the laboratory

34. Third Step in PCR: DNA Synthesis 3. The heat-stable enzyme Taq polymerase is added to synthesize a complementary DNA strand • These three steps make up one PCR cycle

35. The Polymerase Chain Reaction (PCR)

36. PCR starts with a fragment of double-stranded DNA. 1 The DNA is heated to 90°–94°C to unwind it. The single strands will become templates. 2 The reaction mixture contains primers designed to base-pair with complementary sequences at the end of the DNA to be copied. 3 Fig. 13-11a, p. 301

37. The mixture is cooled. Lowering the temperature promotes base-pairing between primers and the DNA strands to be copied. 4 Fig. 13-11b, p. 301

38. DNA polymerases recognize the primers and assemble complementary sequences to make new strands. This doubles the number of identical DNA fragments. 5 Fig. 13-11c, p. 301

39. The mixture is heated again. Raising the temperature makes all of the double-stranded DNA fragments unwind. 6 Fig. 13-11d, p. 301

40. The mixture is cooled. Lower temperature promotes base-pairing between more primers added to the mixture and the single strands. 7 Fig. 13-11e, p. 301

41. 8 DNA polymerase again doubles the number of identical fragments. Repeating the sequence of reactions doubles the number of DNA fragments each time. Billions of fragments are rapidly synthesized, as in the rows of PCR systems below that are copying human DNA. Fig. 13-11f, p. 301

42. PCR Doubles the Amount of DNA With Each Cycle Table 13-1, p. 302

43. Many Uses for PCR • DNA to be amplified by PCR does not have to be purified and can be present in small amounts • Used in clinical diagnosis, forensics, conservation • Samples can be small or old (insects in amber) Fig. 13-12, p. 302

44. 13.6 Analyzing Cloned Sequences • Cloned sequences are characterized in several ways, including Southern blotting and DNA sequencing • Southern blot • A method for transferring DNA fragments from a gel to a membrane filter, developed by Edwin Southern for use in hybridization experiments

45. Southern Blotting Can Be Used to Analyze Cloned Sequences • Southern blotting uses gel electrophoresis to separate DNA fragments • DNA fragments migrate through the gel from a negative pole to a positive pole • Small fragments move faster than large fragments • Radioactive probes identify blotted bands

46. Separation of Restriction Fragmentsby Gel Electrophoresis Fig. 13-13, p. 303

47. Southern Blotting Technique Fig. 13-14a, p. 304

48. DNA Sequencing is one form of genome analysis • DNA sequencing • A technique for determining the nucleotide sequence of a fragment of DNA • Basic method used in Human genome project and other genome projects • The Sanger method of DNA sequencing is the most commonly used method and can be automated

49. Sanger Sequencing uses Dideoxynucleotides Fig. 13-16, p. 305

50. Automated DNA Sequencing Fig. 13-15, p. 305