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 DNA fingerprinting has provided a powerful tool for crime scene investigators – DNA is isolated from biological fluids left at a crime scene – The technique determines with near certainty whether two samples of DNA are from the same individual DNA technology—methods for studying and manipulating genetic material—plays significant roles in many areas of society

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 Recombinant DNA technology: techniques for combining genes from different sources Gene cloning: production of multiple identical copies of gene-carrying DNA Genetic engineering: direct manipulation of genes for practical purposes Biotechnology: use of organisms or their components to make useful products

5. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Recombinant DNA technology uses plasmids, small, circular DNA molecules that replicate separately from a bacterial chromosome – Desired genes inserted into plasmids to form recombinant DNA – Plasmids inserted into bacteria – Foreign genes copied when recombinant bacteria multiply into a clone

6. LE 12-01-3 Bacterial chromosome Plasmid Bacterium Plasmid isolated Recombinant DNA (plasmid) Recombinant bacterium 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 Copies of protein Cell multiplies with gene of interest Protein used to make snow form at higher temperature Plasmid put into bacterial cell DNA Gene of interest Gene inserted into plasmid DNA isolated Cell containing gene of interest

7. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 12.2 Enzymes are used to "cut and paste" DNA DNA from two sources cut by restriction enzyme at specific restriction sites Resulting restriction fragments contain a double-stranded sequence of DNA with single-stranded "sticky ends" Fragments pair at their sticky ends by hydrogen bonding DNA ligase pastes the strand into a recombinant DNA molecule

8. LE 12-02 Restriction enzyme recognition sequence Restriction enzyme cuts the DNA into fragments Addition of a DNA fragment from another source DNA GCAATT T T C G G C C C G G A A A A A A T T T T A A T T Sticky end Two (or more) fragments stick together by base-pairing DNA ligase pastes the strand Recombinant DNA molecule G G G GC C C C AAAA A A TT TT TT TT AA C T TA AG

9. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Animation: Restriction Enzymes Animation: Restriction Enzymes

10. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 12.3 Genes can be cloned in recombinant plasmids: a closer look Bacteria take up recombinant plasmids from their surroundings and reproduce, thereby cloning the plasmids and the genes they carry 1.Isolate DNA from two sources 2.Cut both DNAs with the same restriction enzyme 3.Mix the DNAs, which join by basepairing

11. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 4.Add DNA ligase to bond the DNA 5.Put plasmid into bacterium by transformation 6.Clone the bacterium Animation: Cloning a Gene Animation: Cloning a Gene

12. LE 12-03 Isolate DNA from two sources Cut both DNAs with the same restriction enzyme Mix the DNAs; they join by base-pairing Add DNA ligase to bond the DNA covalently Recombinant DNA plasmid E. coli Plasmid Human cell Sticky ends Gene V DNA Gene V Put plasmid into bacterium by transformation Recombinant bacterium Clone the bacterium Bacterial clone carrying many copies of the human gene

13. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 12.4 Cloned genes can be stored in genomic libraries Genomic library – Set of cloned DNA fragments containing all of an organism's genes – Fragments can be constructed and stored in cloned bacterial plasmids (plasmid library) or phages (phage library)

14. LE 12-04 Recombinant plasmid Bacterial clone Plasmid library Phage clone Phage library Recombinant phage DNA Genome cut up with restriction enzyme or

15. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 12.5 Reverse transcriptase helps make genes for cloning Complementary DNA (cDNA), which contains only the genes that are transcribed by a particular type of cell, can be created using reverse transcriptase 1.Cell transcribes genes 2.RNA splicing removes introns 3.Single-strand DNA created from RNA with reverse transcriptase 4.Enzymes added to break down RNA 5.Second DNA strand synthesized

16. LE 12-05 Cell nucleus mRNA Test tube DNA of eukaryotic gene Reverse transcriptase cDNA strand cDNA of gene (no introns) RNA transcript Exon Intron Transcription RNA splicing (removes introns) Isolation of mRNA from cell and addition of reverse transcriptase; synthesis of DNA strand Breakdown of RNA Synthesis of second DNA strand

17. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings CONNECTION 12.6 Recombinant cells and organisms can mass-produce gene products Recombinant cells and organisms constructed by DNA technology are used to manufacture many useful products, chiefly proteins – Bacteria are usually the best vectors – Some eukaryotic cells are used Saccharomyces cerevisiae fungus for brewing and baking Mammalian cells for pharmaceuticals

20. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings CONNECTION 12.7 DNA technology is changing the pharmaceutical industry DNA technology is widely used to produce medicines and to diagnose diseases – Therapeutic hormones Example: humulin, human insulin produced by bacteria – Diagnosis and treatment of disease Example: analysis to identify HIV – Development of vaccines

23. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Video: Biotechnology Lab Video: Biotechnology Lab

24. RESTRICTION FRAGMENT ANALYSIS AND DNA FINGERPRINTING 12.8 Nucleic acid probes identify clones carrying specific genes Detecting genes depends on base pairing between the gene and a complementary sequence on another nucleic acid molecule A nucleic acid probe – Is a short, single-stranded molecule of radioactively or fluorescently labeled DNA or RNA – Can base pair with a desired gene in a library, thus tagging it Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

25. LE 12-08 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 A T A T C C CG G A A T C C G A A A A G G G G C T T G G G G C C C A A A T T T T C C T T A T

26. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings CONNECTION 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 1.Isolate mRNA 2.Make cDNA from mRNA using reverse transcriptase 3.Apply cDNA (single-stranded) to wells 4.cDNA binds to corresponding gene; unbound cDNA is rinsed away; remaining DNA produces a glow

27. LE 12-09 mRNA isolated cDNA made from mRNA cDNA applied to wells Unbound cDNA rinsed away Each well contains DNA from a particular gene Fluorescent spot DNA microarray Nonfluorescent spot cDNA DNA of an expressed gene DNA of an unexpressed gene Reverse transcriptase and fluorescent DNA nucelotides Actual size (6,400 genes)

28. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 12.10 Gel electrophoresis sorts DNA molecules by size Gel electrophoresis uses a gel as a molecular sieve to separate nucleic acids by size or electrical charge – Longer macromolecules move through the gel more slowly than shorter macromolecules, resulting in a pattern of bands on the gel

29. LE 12-10 Mixture of DNA molecules of different sizes Power source Gel Completed gel Longer molecules Shorter molecules

30. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 12.11 Restriction fragment length polymorphisms can be used to detect differences in DNA sequences Differences in DNA sequences on homologous chromosomes produce sets of restriction fragments that differ between individuals – Are called restriction fragment length polymorphisms (RFLPs) – Are of different lengths and will migrate different distances in an electrophoretic gel – Can be used as genetic markers

31. LE 12-11a Crime scene Suspect Cut w x z C G C C C G G G G G C A G C C T yy G G G C C C C G C C C C G G G G DNA from chromosomes

32. LE 12-11b Longer fragments 1 x Shorter fragments w y y z 2

33. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Restriction fragments can be used as DNA probes to detect harmful alleles – Patterns of normal and harmful alleles identified – Banding patterns compared with probe

34. LE 12-11c-3 Restriction fragment preparation Gel electrophoresis Restriction fragments  Blotting Filter paper Probe Radioactive, single- stranded DNA (probe) Radioactive probe         Film Detection of radioactivity (autoradiography)

35. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings CONNECTION 12.12 DNA technology is used in courts of law Forensic science is the scientific analysis of evidence for criminal and other legal investigations DNA fingerprinting requires only about 1,000 cells – Radioactive probes mark electrophoresis bands that contain certain markers – Produces a specific pattern of bands to compare to those of accused person – Highly reliable because odds of two people having identical DNA fingerprints are extremely small

36. LE 12-12a Defendant’s blood Blood from defendant’s clothes Victim’s blood

38. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings CONNECTION 12.13 Gene therapy may someday help treat a variety of diseases Gene therapy is the alteration of an afflicted individual's genes – Where a disorder is due to a single gene, it is sometimes possible to replace the defective gene with a normal allele – To be permanent, the normal allele must be transferred to cells that multiply throughout a person's life, such as bone marrow cells

39. LE 12-13 Cloned gene (normal allele ) Insert normal gene into virus Viral nucleic acid Retrovirus Infect bone marrow cell with virus Viral DNA inserts into chromosome Bone marrow cell from patient Bone marrow Inject cells into patient

40. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Gene therapy – May one day be used to treat both genetic diseases and nongenetic disorders, but progress is slow – Raises both technical and ethical issues

41. 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 quickly clone a very large number of DNA copies for analysis – DNA sample mixed with DNA polymerase, nucleotide monomers, other ingredients – Mixture exposed to cycles of heating to separate the DNA strands – During each cycle, DNA replicates, doubling the amount

42. LE 12-14 Initial DNA segment Number of DNA molecules 1 2 48

43. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings GENOMICS CONNECTION 12.15 The Human Genome Project is an ambitious application of DNA technology The Human Genome Project was begun in 1990 and is now largely completed – Initially involved three stages: genetic (linkage) and physical mapping of chromosomes, followed by DNA sequencing – Superseded by "shotgun" approach, going directly to stage 3 The data are providing insight into development, evolution, and many diseases

45. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 12.16 Most of the human genome does not consist of genes Surprisingly, the haploid human genome contains only about 25,000 genes About 97% of the human genome consists of noncoding DNA – Gene-control sequences – Introns – Noncoding DNA located between genes

46. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings – Repetitive DNA-nucleotide sequences present in many copies Teleomeres found at chromosome ends Transposons ("jumping genes") that can move about within the genome

47. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings CONNECTION 12.17 The science of genomics compares whole genomes Genomics is the study of whole sets of genes and their interactions – As of 2005, the genomes of about 150 species had been sequenced – Besides being interesting in themselves, nonhuman genomes provide understanding of the human genome Proteomics is the study of the full protein sets encoded by genomes

49. GENETICALLY MODIFIED ORGANISMS CONNECTION 12.18 Genetically modified organisms are transforming agriculture Recombinant DNA technology can produce new varieties of plants and animals for use in agriculture – Genetically modified (GM) organisms have acquired genes by artificial means – Transgenic organisms have had genes from other organisms inserted into their genomes A number of important crop plants are genetically modified using the Ti plasmid Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

50. LE 12-18a Agrobacterium tumefaciens T DNA Restriction site DNA containing gene for desired trait Recombinant Ti plasmid Ti plasmid Insertion of gene into plasmid using restriction enzyme and DNA ligase Introduction into plant cells in culture Plant cell Regeneration of plant Plant with new trait T DNA carrying new gene within plant chromosome

51. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Transgenic animals have also been engineered to be pharmaceutical "factories"

53. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings CONNECTION 12.19 Could GM organisms harm human health or the environment? Scientists have developed safety guidelines to minimize the risks involved in genetic engineering – Laboratory safety procedures – Organisms altered so they cannot live outside the lab Exported GM organisms must be identified Today most concern focuses on transgenic crop plants passing their genes to wild relatives

56. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings CONNECTION 12.20 Genomics researcher Eric Lander discusses the Human Genome Project Dr. Eric Lander founded the Broad Institute of MIT and Harvard – Uses genomics to develop new methods to investigate and treat diseases The Human Genome Project – Results will give researchers the opportunity to examine the human genome from a "big picture" approach – Revolutionizing evolutionary biology