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Chapter 12

Chapter 12. 0. DNA Technology and Genomics. 0. DNA and Crime Scene Investigations Many violent crimes go unsolved 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 • 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: set of laboratory procedures • determines w/near certainty whether 2 samples of DNA are from same individual • powerful tool for CSIs

  4. BACTERIAL PLASMIDS AND GENE CLONING 0 12.1 Plasmids are used to customize bacteria: An overview • Gene cloning is 1 application • DNA technology • Methods for studying & manipulating genetic material

  5. 0 • Researchers can insert desired genes into plasmids, creating recombinant DNA • And insert those plasmids into bacteria Figure 12.1

  6. Bacterium Plasmid isolated 1 DNA isolated 2 Gene inserted into plasmid 3 Plasmid put into bacterial cell 4 Cell multiplies with gene of interest 5 Cell containing gene of interest 0 Bacterial chromosome Plasmid DNA Recombinant DNA (plasmid) Gene of interest Recombinant bacterium Gene for pest Resistance inserted into plants Copies of gene Copies of protein Clone of cells Protein used to make snow form at highertemperature Figure 12.1 Gene used to alter bacteria for cleaning up toxic waste Protein used to dissolve bloodclots in heart attack therapy

  7. 0 • If the recombinant bacteria multiply into a clone • The foreign genes are also copied

  8. 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

  9. G A A T T C C T T A A G DNA 1 Restriction enzymerecognition sequence • Creating recombinant DNA using REs & DNA ligase Restriction enzymecuts the DNA intofragments A AT TC G C T T A A G 2 Sticky end Addition of a DNAfragment fromanother source A AT TC G 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 RecombinantDNA molecule Figure 12.2

  10. 0 12.3 Genes can be cloned in recombinant plasmids: A closer look • Bacteria take the recombinant plasmids from their surroundings • reproduce • cloning the plasmids and the genes they carry

  11. 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 DNA Plasmid Gene V Sticky ends Gene V Recombinant DNA plasmid Recombinant bacterium Figure 12.3 Bacterial clone carrying manycopies of the human gene

  12. Table 12.6 CONNECTION 0 12.6 Recombinant cells and organisms can mass-produce gene products • Applications of gene cloning include • medical and other uses

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

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

  15. Figure 12.7A 0 Therapeutic hormones • In 1982, humulin, human insulin produced by bacteria • 1st recombinant drug approved by FDA

  16. 0 Diagnosis and Treatment of Disease • DNA technology • Is being used increasingly in disease diagnosis

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

  18. pGLO LAB

  19. Mixture of DNA molecules of different sizes – – Longer molecules Power source Gel Shorter molecules Completed gel + + Figure 12.10 RESTRICTION FRAGMENT ANALYSIS AND DNA FINGERPRINTING 0 12.10 Gel electrophoresis sorts DNA molecules by size

  20. 0 12.11 Restriction fragment length polymorphisms can be used to detect differences in DNA sequences

  21. 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

  22. 1 2 – Longer fragments z x w y y Shorter fragments + Figure 12.11B 0 • After digestion by restriction enzymes • fragments are run through a gel

  23. 0 Using DNA Probes to Detect Harmful Alleles • Radioactive probes • Can reveal DNA bands of interest on a gel

  24. 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

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

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

  27. 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

  28. Cloned gene (normal allele) 1 Insert normal gene into virus Infect bone marrow cell with virus 2 3 Viral DNA inserts into chromosome Inject cells into patient 4 Gene Therapy 0 Retrovirus Viral nucleic acid Bone marrow cell from patient Bone marrow Figure 12.13

  29. 0 • Gene therapy • May one day be used to treat both genetic diseases and nongenetic disorders • Unfortunately, progress is slow

  30. 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

  31. 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 • Genetic and physical mapping of chromosomes, followed by DNA sequencing

  32. 0 • The data are providing insight into • Development, evolution, and many diseases

  33. 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

  34. 0 • Much of the noncoding DNA consists of repetitive nucleotide sequences • And transposons that can move about within the genome

  35. CONNECTION 0 12.17 The science of genomics compares whole genomes • The sequencing of many prokaryotic and eukaryotic genomes

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

  37. 0 • Proteomics • Is the study of the full sets of proteins produced by organisms

  38. GENETICALLY MODIFIED ORGANISMS:CONNECTION 0 12.18 Genetically modified organisms are transforming agriculture

  39. 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

  40. Figure 12.18B 0 • Transgenic organisms • have had genes from other organisms inserted into their genomes

  41. 0 • A number of important crops and plants • Are genetically modified

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

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

  44. 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

  45. 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

  46. 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

  47. 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

  48. 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

  49. 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

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