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Chapter 12

Chapter 12. DNA Technology and Genomics. 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

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Chapter 12

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  1. Chapter 12 DNA Technology and Genomics

  2. 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

  3. 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

  4. 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

  5. LE 12-01-3 Bacterium Cell containing gene of interest Plasmid isolated DNA isolated Gene inserted into plasmid Bacterial chromosome Plasmid Recombinant DNA (plasmid) DNA Gene of interest Plasmid put into bacterial cell Recombinant bacterium Cell multiplies with gene of interest Copies of protein Copies of gene Clone of cells Gene for pest resistance inserted into plants Protein used to make snow form at higher temperature Gene used to alter bacteria for cleaning up toxic waste Protein used to dissolve blood clots in heart attack therapy

  6. 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

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

  8. Animation: Restriction Enzymes

  9. 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 • Isolate DNA from two sources • Cut both DNAs with the same restriction enzyme • Mix the DNAs, which join by basepairing

  10. Add DNA ligase to bond the DNA • Put plasmid into bacterium by transformation • Clone the bacterium Animation: Cloning a Gene

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

  12. 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)

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

  14. 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 • Cell transcribes genes • RNA splicing removes introns • Single-strand DNA created from RNA with reverse transcriptase • Enzymes added to break down RNA • Second DNA strand synthesized

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

  16. 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

  17. 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

  18. Video: Biotechnology Lab

  19. 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

  20. LE 12-08 Radioactive probe (DNA) A C C G T A Mix with single- stranded DNA from various bacterial (or phage) clones G Single-stranded DNA C A T G A T T C A T C C C A G A T G T C A C T G G A T A G T A T T C Base pairing indicates the gene of interest G C G G A A

  21. 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 • Isolate mRNA • Make cDNA from mRNA using reverse transcriptase • Apply cDNA (single-stranded) to wells • cDNA binds to corresponding gene; unbound cDNA is rinsed away; remaining DNA produces a glow

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

  23. 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

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

  25. 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

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

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

  28. Restriction fragments can be used as DNA probes to detect harmful alleles • Patterns of normal and harmful alleles identified • Banding patterns compared with probe

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

  30. 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

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

  32. 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

  33. 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

  34. 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

  35. 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

  36. LE 12-14 Initial DNA segment 1 4 8 2 Number of DNA molecules

  37. 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

  38. 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

  39. Repetitive DNA-nucleotide sequences present in many copies • Teleomeres found at chromosome ends • Transposons ("jumping genes") that can move about within the genome

  40. 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

  41. 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

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

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