1. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings PowerPoint Lectures for Biology: Concepts and Connections, Fifth Edition – Campbell, Reece, Taylor, and Simon Lectures by Chris Romero Chapter 12 DNA Technology and Genomics

2. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings DNA and Crime Scene Investigations Many violent crimes go unsolved –For lack of enough evidence If biological fluids are left at a crime scene –DNA can be isolated from them

3. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings DNA fingerprinting is a set of laboratory procedures –That determines with near certainty whether two samples of DNA are from the same individual –That has provided a powerful tool for crime scene investigators Investigator at one of the crime scenes (above), Narborough, England (left)

4. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings BACTERIAL PLASMIDS AND GENE CLONING 12.1 Plasmids are used to customize bacteria: An overview Gene cloning is one application of DNA technology –Methods for studying and manipulating genetic material

5. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Researchers can insert desired genes into plasmids, creating recombinant DNA –And insert those plasmids into bacteria Bacterium Bacterial chromosome Plasmid 1 isolated 3 Gene inserted into plasmid 2 DNA isolated Cell containing gene of interest DNA Gene of interest Recombinant DNA (plasmid) 4 Plasmid put into bacterial cell Recombinant bacterium 5 Cell multiplies with gene of interest Copies of protein Copies of gene Clone of cells Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein used to dissolve blood clots in heart attack therapy Protein used to make snow form at higher temperature Figure 12.1

6. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings If the recombinant bacteria multiply into a clone –The foreign genes are also copied

7. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 12.2 Enzymes are used to “cut and paste” DNA The tools used to make recombinant DNA are –Restriction enzymes, which cut DNA at specific sequences –DNA ligase, which “pastes” DNA fragments together

8. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Creating recombinant DNA using restriction enzymes and DNA ligase Restriction enzyme recognition sequence G A A T T C C T T A A G DNA 1 2 3 4 C T T A A A AT TC G C T T A A Addition of a DNA fragment from another source Two (or more) fragments stick together by base-pairing G A AT T C C T TA A G G A AT T C C T TA A G 5 DNA ligase pastes the strand Restriction enzyme cuts the DNA into fragments Recombinant DNA molecule G G Sticky end G Figure 12.2

9. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 12.3 Genes can be cloned in recombinant plasmids: A closer look Bacteria take the recombinant plasmids from their surroundings –And reproduce, thereby cloning the plasmids and the genes they carry

10. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Cloning a gene in a bacterial plasmid 1 Isolate DNA from two sources 2 Cut both DNAs with the same restriction enzyme E.coli Plasmid DNA Gene V Sticky ends 3Mix the DNAs; they join by base-pairing 4 Add DNA ligase to bond the DNA covalently 5 Put plasmid into bacterium by transformation Gene V Recombinant DNA plasmid Recombinant bacterium 6 Clone the bacterium Bacterial clone carrying many copies of the human gene Human cell Figure 12.3

11. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 12.4 Cloned genes can be stored in genomic libraries Genomic libraries, sets of DNA fragments containing all of an organism’s genes –Can be constructed and stored in cloned bacterial plasmids or phages Recombinant plasmid Genome cut up with restriction enzyme Recombinant phage DNA or Bacterial clone Phage clone Phage library Plasmid library Figure 12.4

12. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 12.5 Reverse transcriptase helps make genes for cloning Reverse transcriptase can be used to make smaller, complementary DNA (cDNA) libraries –Containing only the genes that are transcribed by a particular type of cell Cell nucleus DNA of eukaryotic gene Exon Intron Exon Intron Exon 1Transcription 2 RNA splicing (removes introns) 3 Isolation of mRNA from cell and addition of reverse transcriptase; synthesis of DNA strand 4 Breakdown of RNA 5 Synthesis of second DNA strand RNA transcript mRNA Reverse transcriptase cDNA strand cDNA of gene (no introns) Test tube Figure 12.5

13. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings CONNECTION 12.6 Recombinant cells and organisms can mass- produce gene products Applications of gene cloning include –The mass production of gene products for medical and other uses Table 12.6

14. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Different organisms, including bacteria, yeast, and mammals –Can be used for this purpose Figure 12.6

15. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 12.7 DNA technology is changing the pharmaceutical industry DNA technology –Is widely used to produce medicines and to diagnose diseases CONNECTION

16. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Therapeutic hormones In 1982, humulin, human insulin produced by bacteria –Became the first recombinant drug approved by the Food and Drug Administration Figure 12.7A

17. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Diagnosis and Treatment of Disease DNA technology –Is being used increasingly in disease diagnosis

18. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Vaccines DNA technology –Is also helping medical researchers develop vaccines Figure 12.7B

19. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 12.8 Nucleic acid probes identify clones carrying specific genes DNA technology methods –Can be used to identify specific pieces of DNA RESTRICTION FRAGMENT ANALYSIS AND DNA FINGERPRINTING

20. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings A nucleic acid probe –Is a short, single-stranded molecule of radioactively labeled or fluorescently labeled DNA or RNA –Can tag a desired gene in a library Radioactive probe (DNA) Single-stranded DNA Mix with single- stranded DNA from various bacterial (or phage) clones Base pairing indicates the gene of interest A T C C G A A T G C G C T T A T C G A G C C T T A T G C A T A T C C G A A G G T A G G C T A A Figure 12.8

21. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 12.9 DNA microarrays test for the expression of many genes at once DNA microarray assays –Can reveal patterns of gene expression in different kinds of cells CONNECTION

22. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings DNA microarray 1 mRNA isolated Reverse transcriptase and fluorescent DNA nucleotides 2 cDNA made from mRNA 4Unbound cDNA rinsed away 3 cDNA applied to wells DNA microarray Each well contains DNA from a particular gene Actual size (6,400 genes) Nonfluorescent spot Fluorescent spot cDNA DNA of an expressed gene DNA of an unexpressed gene Figure 12.9

23. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 12.10 Gel electrophoresis sorts DNA molecules by size ++ – – Power source Gel Mixture of DNA molecules of different sizes Longer molecules Shorter molecules Completed gel Figure 12.10

24. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 12.11 Restriction fragment length polymorphisms can be used to detect differences in DNA sequences

25. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings How Restriction Fragments Reflect DNA Sequence Restriction fragment length polymorphisms (RFLPs) –Reflect differences in the sequences of DNA samples Crime sceneSuspect w x yy z Cut DNA from chromosomes C C G G G G C C A C G G T G C C C C G G G G C C C C G G G G C C Figure 12.11A

26. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings After digestion by restriction enzymes –The fragments are run through a gel – + Longer fragments Shorter fragments x w y z y 12 Figure 12.11B

27. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Using DNA Probes to Detect Harmful Alleles Radioactive probes –Can reveal DNA bands of interest on a gel

28. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Detecting a harmful allele using restriction fragment analysis 1 2 3 4 5 Restriction fragment preparation Gel electrophoresis Blotting Radioactive probe Detection of radioactivity (autoradiography) IIIIII IIIIII Restriction fragments Filter paper Probe Radioactive, single- stranded DNA (probe) Film I II III I II III Figure 12.11C

29. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 12.12 DNA technology is used in courts of law CONNECTION

30. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings DNA fingerprinting can help solve crimes Defendant’s blood Blood from defendant’s clothes Victim’s blood Figure 12.12A Figure 12.12B

31. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 12.13 Gene therapy may someday help treat a variety of diseases Gene therapy –Is the alteration of an afflicted individual’s genes CONNECTION Cloned gene (normal allele) 1 Insert normal gene into virus 2 Infect bone marrow cell with virus 3 Viral DNA inserts into chromosome 4 Inject cells into patient Bone marrow Bone marrow cell from patient Viral nucleic acid Retrovirus Figure 12.13

32. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Gene therapy –May one day be used to treat both genetic diseases and nongenetic disorders Unfortunately, progress is slow

33. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 12.14 The PCR method is used to amplify DNA sequences The polymerase chain reaction (PCR) –Can be used to clone a small sample of DNA quickly, producing enough copies for analysis 1248 Initial DNA segment Number of DNA molecules Figure 12.14

34. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Figure 12.15 GENOMICS CONNECTION 12.15 The Human Genome Project is an ambitious application of DNA technology The Human Genome Project, begun in 1990 and now largely completed, involved –Genetic and physical mapping of chromosomes, followed by DNA sequencing

35. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings The data are providing insight into –Development, evolution, and many diseases

36. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 12.16 Most of the human genome does not consist of genes The haploid human genome contains about 25,000 genes –And a huge amount of noncoding DNA

37. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Much of the noncoding DNA consists of repetitive nucleotide sequences –And transposons that can move about within the genome

38. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 12.17 The science of genomics compares whole genomes The sequencing of many prokaryotic and eukaryotic genomes –Has produced data for genomics, the study of whole genomes CONNECTION

39. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Besides being interesting themselves –Nonhuman genomes can be compared with the human genome Table 12.17

40. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Proteomics –Is the study of the full sets of proteins produced by organisms

41. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 12.18 Genetically modified organisms are transforming agriculture GENETICALLY MODIFIED ORGANISMS CONNECTION

42. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Recombinant DNA technology –Can be used to produce new genetic varieties of plants and animals, genetically modified (GM) organisms Agrobacterium tumefaciens DNA containing gene for desired trait Ti plasmid 1 Insertion of gene into plasmid using restriction enzyme and DNA ligase Recombinant Ti plasmid 2 Introduction into plant cells in culture 3 Regeneration of plant Plant with new trait T DNA carrying new gene within plant chromosome Plant cell T DNA Restriction site Figure 12.18A

43. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Transgenic organisms –Are those that have had genes from other organisms inserted into their genomes Figure 12.18B

44. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings A number of important crops and plants –Are genetically modified

45. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 12.19 Could GM organisms harm human health or the environment? Development of GM organisms –Requires significant safety measures CONNECTION Figure 12.19A

46. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings Genetic engineering involves risks –Such as ecological damage from GM crops Figure 12.19B

47. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 12.20 Genomics researcher Eric Lander discusses the Human Genome Project Genomics pioneer Eric Lander –Points out that much remains to be learned from the Human Genome Project CONNECTION Figure 12.20

48. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings 12.17 The science of genomics compares whole genomes The sequencing of many prokaryotic and eukaryotic genomes –Has produced data for genomics, the study of whole genomes

49. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings HOMEWORK Read Cohen’s work for plasmids and answer the questions on the following website http://www.stanford.edu/class/bio11n/html/week1 /papertopic.htm Read Villa-Komaroff et al. work and answer the questions on the following website http://www.stanford.edu/class/bio11n/html/week2 /papertopic.htm

50. Copyright © 2005 Pearson Education, Inc. Publishing as Benjamin Cummings READING http://www.dnaftb.org/dnaftb/