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Chapter 12

Chapter 12. 0. DNA Technology and Genomics. 0. 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. Investigator at one of the crime scenes (above), Narborough,

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Chapter 12

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  1. Chapter 12 0 DNA Technology and Genomics

  2. 0 • 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. Investigator at one of the crime scenes (above), Narborough, England (left) 0 • 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

  4. BACTERIAL PLASMIDS AND GENE CLONING 0 • 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. Plasmid isolated 1 DNA isolated 2 Gene inserted into plasmid 3 Plasmid put into bacterial cell 4 Cell multiplies with gene of interest 5 0 • Researchers can insert desired genes into plasmids, creating recombinant DNA • And insert those plasmids into bacteria Bacterium Cell containing gene of interest Plasmid Bacterial chromosome Recombinant DNA (plasmid) DNA Gene of interest Recombinant bacterium Copies of gene Copies of protein Clone of cells Gene for pest resistance inserted into plants Protein used tomake snow format highertemperature Figure 12.1 Gene used to alter bacteria for cleaning up toxic waste Protein used to dissolve bloodclots in heart attack therapy

  6. 0 • If the recombinant bacteria multiply into a clone • The foreign genes are also copied

  7. 0 • 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. Creating recombinant DNA using restriction enzymes and DNA ligase Restriction enzymerecognition sequence G A A T T C C T T A A G DNA 1 Restriction enzymecuts the DNA intofragments A AT TC G C T T A A G 2 Sticky end A AT TC G Addition of a DNAfragment fromanother source G 3 C T T A A Two (or more)fragments sticktogether bybase-pairing G A AT T C C T TA A G G A AT T C C T TA A G 4 DNA ligase pastes the strand 5 Figure 12.2 Recombinant DNA molecule

  9. 0 • 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. Isolate DNAfrom two sources 1 Cut both DNAswith the samerestriction enzyme 2 3 Mix the DNAs;they join bybase-pairing 4 Add DNA ligaseto bond the DNA covalently 5 Put plasmid into bacteriumby transformation 6 Clone the bacterium 0 • Cloning a gene in a bacterial plasmid E.coli Human cell Plasmid DNA Gene V Sticky ends Gene V Recombinant DNAplasmid Recombinant bacterium Figure 12.3 Bacterial clone carrying manycopies of the human gene

  11. Genome cut up withrestriction enzyme Recombinantplasmid Recombinantphage DNA or Phageclone Bacterialclone Phage library Plasmid library 0 • 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 Figure 12.4

  12. 1 Transcription 2 RNA splicing(removes introns) Isolation of mRNAfrom cell and additionof reverse transcriptase;synthesis of DNA strand 3 4 Breakdown of RNA Synthesis of secondDNA strand 5 0 • 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 Exon Exon Intron Intron Exon DNA ofeukaryoticgene RNA transcript mRNA Test tube Reverse transcriptase cDNA strand cDNA of gene(no introns) Figure 12.5

  13. Table 12.6 CONNECTION 0 • 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

  14. Figure 12.6 0 • Different organisms, including bacteria, yeast, and mammals • Can be used for this purpose

  15. CONNECTION 0 • 12.7 DNA technology is changing the pharmaceutical industry • DNA technology • Is widely used to produce medicines and to diagnose diseases

  16. Figure 12.7A 0 • Therapeutic hormones • In 1982, humulin, human insulin produced by bacteria • Became the first recombinant drug approved by the Food and Drug Administration

  17. 0 • Diagnosis and Treatment of Disease • DNA technology • Is being used increasingly in disease diagnosis

  18. Figure 12.7B 0 • Vaccines • DNA technology • Is also helping medical researchers develop vaccines

  19. RESTRICTION FRAGMENT ANALYSIS AND DNA FINGERPRINTING 0 • 12.8 Nucleic acid probes identify clones carrying specific genes • DNA technology methods • Can be used to identify specific pieces of DNA

  20. A T C C G A Radioactive probe (DNA) Mix with single- stranded DNA from various bacterial (or phage) clones Single-stranded DNA A T G C G C T T A T C G A T C C G A A G C C T T A T G C A T A G G T A G G C T A A Base pairing indicates the gene of interest 0 • 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 Figure 12.8

  21. CONNECTION 0 • 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

  22. mRNA isolated 1 4 Unbound cDNA rinsed away cDNA applied to wells 3 cDNA made from mRNA 2 0 • DNA microarray DNA microarray Actual size (6,400 genes) Each well contains DNA from a particular gene Reverse transcriptase and fluorescent DNA nucleotides Nonfluorescent spot Fluorescent spot cDNA DNA of an expressed gene DNA of an unexpressed gene Figure 12.9

  23. Mixture of DNA molecules of different sizes – – Longer molecules Power source Gel Shorter molecules Completed gel + + Figure 12.10 0 • 12.10 Gel electrophoresis sorts DNA molecules by size

  24. 0 • 12.11 Restriction fragment length polymorphisms can be used to detect differences in DNA sequences

  25. Crime scene Suspect w C G Cut A T C G C G G C G C G C G C z x C G C G Cut Cut C G C G G C G C y y G C G C DNA from chromosomes 0 • How Restriction Fragments Reflect DNA Sequence • Restriction fragment length polymorphisms (RFLPs) • Reflect differences in the sequences of DNA samples Figure 12.11A

  26. 1 2 – Longer fragments z x w y y Shorter fragments + Figure 12.11B 0 • After digestion by restriction enzymes • The fragments are run through a gel

  27. 0 • Using DNA Probes to Detect Harmful Alleles • Radioactive probes • Can reveal DNA bands of interest on a gel

  28. Restriction fragment preparation 1 I II III Restriction fragments Gel electrophoresis 2 I II III Blotting 3 Filter paper Radioactive probe 4 Radioactive, single- stranded DNA (probe) Probe I Detection of radioactivity (autoradiography) 5 II III Film I II III 0 • Detecting a harmful allele using restriction fragment analysis Figure 12.11C

  29. CONNECTION 0 • 12.12 DNA technology is used in courts of law

  30. Blood from defendant’s clothes Defendant’s blood Victim’s blood Figure 12.12B 0 • DNA fingerprinting can help solve crimes Figure 12.12A

  31. 1 Insert normal gene into virus Infect bone marrow cell with virus 2 3 Viral DNA inserts into chromosome Inject cells into patient 4 CONNECTION 0 • 12.13 Gene therapy may someday help treat a variety of diseases • Gene therapy • Is the alteration of an afflicted individual’s genes Cloned gene (normal allele) Viral nucleic acid Retrovirus Bone marrow cell from patient Bone marrow Figure 12.13

  32. 0 • Gene therapy • May one day be used to treat both genetic diseases and nongenetic disorders • Unfortunately, progress is slow

  33. Initial DNA segment 1 2 4 8 Number of DNA molecules 0 • 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 Figure 12.14

  34. Figure 12.15 GENOMICS CONNECTION 0 • 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. 0 • The data are providing insight into • Development, evolution, and many diseases

  36. 0 • 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. 0 • Much of the noncoding DNA consists of repetitive nucleotide sequences • And transposons that can move about within the genome

  38. CONNECTION 0 • 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

  39. Table 12.17 0 • Besides being interesting themselves • Nonhuman genomes can be compared with the human genome

  40. 0 • Proteomics • Is the study of the full sets of proteins produced by organisms

  41. GENETICALLY MODIFIED ORGANISMSCONNECTION 0 • 12.18 Genetically modified organisms are transforming agriculture

  42. 1 2 3 Introduction into plant cells in culture Insertion of gene into plasmid using restriction enzyme and DNA ligase Regeneration of plant 0 • 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 Plant cell Ti plasmid Recombinant Ti plasmid T DNA carrying new gene within plant chromosome T DNA Plant with new trait Restriction site Figure 12.18A

  43. Figure 12.18B 0 • Transgenic organisms • Are those that have had genes from other organisms inserted into their genomes

  44. 0 • A number of important crops and plants • Are genetically modified

  45. Figure 12.19A CONNECTION 0 • 12.19 Could GM organisms harm human health or the environment? • Development of GM organisms • Requires significant safety measures

  46. Figure 12.19B 0 • Genetic engineering involves risks • Such as ecological damage from GM crops

  47. Figure 12.20 CONNECTION 0 • 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

  48. 0 • 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. 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. READING • http://www.dnaftb.org/dnaftb/

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