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Chapter 20

Chapter 20. DNA Technology and Genomics. Overview: Understanding and Manipulating Genomes. Sequencing of the human genome was largely completed by 2003 DNA sequencing has depended on advances in technology, starting with making recombinant DNA

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Chapter 20

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  1. Chapter 20 DNA Technology and Genomics

  2. Overview: Understanding and Manipulating Genomes • Sequencing of the human genome was largely completed by 2003 • DNA sequencing has depended on advances in technology, starting with making recombinant DNA • In recombinant DNA, nucleotide sequences from two different sources, often two species, are combined in vitro into the same DNA molecule • Methods for making recombinant DNA are central to genetic engineering, the direct manipulation of genes for practical purposes

  3. DNA technology has revolutionized biotechnology, the manipulation of organisms or their genetic components to make useful products • An example of DNA technology is the microarray, a measurement of gene expression of thousands of different genes

  4. Concept 20.1: DNA cloning permits production of multiple copies of a specific gene or other DNA segment • To work directly with specific genes, scientists prepare gene-sized pieces of DNA in identical copies, a process called gene cloning

  5. DNA Cloning and Its Applications: A Preview • Most methods for cloning pieces of DNA in the laboratory share general features, such as the use of bacteria and their plasmids • Cloned genes are useful for making copies of a particular gene and producing a gene product

  6. LE 20-2 Cell containing gene of interest Bacterium Gene inserted into plasmid Bacterial chromosome Plasmid Gene of interest Recombinant DNA (plasmid) DNA of chromosome Plasmid put into bacterial cell Recombinant bacterium Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Protein expressed by gene of interest Gene of interest Copies of gene Protein harvested Basic research and various applications Basic research on gene Basic research on protein Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Human growth hor- mone treats stunted growth

  7. Using Restriction Enzymes to Make Recombinant DNA • Bacterial restriction enzymes cut DNA molecules at DNA sequences called restriction sites • A restriction enzyme usually makes many cuts, yielding restriction fragments • The most useful restriction enzymes cut DNA in a staggered way, producing fragments with “sticky ends” that bond with complementary “sticky ends” of other fragments • DNA ligase is an enzyme that seals the bonds between restriction fragments

  8. LE 20-3 Restriction site 5¢ 3¢ DNA 3¢ 5¢ Restriction enzyme cuts the sugar-phosphate backbones at each arrow. Sticky end DNA fragment from another source is added. Base pairing of sticky ends produces various combinations. Fragment from different DNA molecule cut by the same restriction enzyme One possible combination DNA ligase seals the strands. Recombinant DNA molecule

  9. Cloning a Eukaryotic Gene in a Bacterial Plasmid • In gene cloning, the original plasmid is called a cloning vector • A cloning vector is a DNA molecule that can carry foreign DNA into a cell and replicate there

  10. Producing Clones of Cells • Cloning a human gene in a bacterial plasmid can be divided into six steps: 1. Vector and gene-source DNA are isolated 2. DNA is inserted into the vector 3. Human DNA fragments are mixed with cut plasmids, and base-pairing takes place 4. Recombinant plasmids are mixed with bacteria 5. The bacteria are plated and incubated 6. Cell clones with the right gene are identified

  11. LE 20-4_1 Bacterial cell lacZ gene (lactose breakdown) Human cell Isolate plasmid DNA and human DNA. Restriction site ampR gene (ampicillin resistance) Bacterial plasmid Gene of interest Sticky ends Human DNA fragments Cut both DNA samples with the same restriction enzyme. Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids. Recombinant DNA plasmids

  12. LE 20-4_2 Bacterial cell lacZ gene (lactose breakdown) Human cell Isolate plasmid DNA and human DNA. Restriction site ampR gene (ampicillin resistance) Bacterial plasmid Gene of interest Sticky ends Human DNA fragments Cut both DNA samples with the same restriction enzyme. Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids. Recombinant DNA plasmids Introduce the DNA into bacterial cells that have a mutation in their own lacZ gene. Recombinant bacteria

  13. LE 20-4_3 lacZ gene (lactose breakdown) Bacterial cell Human cell Isolate plasmid DNA and human DNA. Restriction site ampR gene (ampicillin resistance) Bacterial plasmid Gene of interest Sticky ends Human DNA fragments Cut both DNA samples with the same restriction enzyme. Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids. Recombinant DNA plasmids Introduce the DNA into bacterial cells that have a mutation in their own lacZ gene. Recombinant bacteria Plate the bacteria on agar containing ampicillin and X-gal. Incubate until colonies grow. Colony carrying recombinant plasmid with disrupted lacZ gene Colony carrying non- recombinant plasmid with intact lacZ gene Bacterial clone

  14. Identifying Clones Carrying a Gene of Interest • A clone carrying the gene of interest can be identified with a nucleic acid probe having a sequence complementary to the gene • This process is called nucleic acid hybridization • An essential step in this process is denaturation of the cells’ DNA, separation of its two strands

  15. LE 20-5 Colonies containing gene of interest Master plate Master plate Probe DNA Radioactive single-stranded DNA Solution containing probe Gene of interest Film Single-stranded DNA from cell Filter Filter lifted and flipped over Hybridization on filter A special filter paper is pressed against the master plate, transferring cells to the bottom side of the filter. The filter is treated to break open the cells and denature their DNA; the resulting single-stranded DNA molecules are treated so that they stick to the filter. The filter is laid under photographic film, allowing any radioactive areas to expose the film (autoradiography). After the developed film is flipped over, the reference marks on the film and master plate are aligned to locate colonies carrying the gene of interest.

  16. Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR) • The polymerase chain reaction, PCR, can produce many copies of a specific target segment of DNA • A three-step cycle—heating, cooling, and replication—brings about a chain reaction that produces an exponentially growing population of identical DNA molecules

  17. LE 20-7 5¢ 3¢ Target sequence Genomic DNA 3¢ 5¢ 5¢ 3¢ Denaturation: Heat briefly to separate DNA strands 3¢ 5¢ Annealing: Cool to allow primers to form hydrogen bonds with ends of target sequence Cycle 1 yields 2 molecules Primers Extension: DNA polymerase adds nucleotides to the 3¢ end of each primer New nucleo- tides Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence

  18. Concept 20.2: Restriction fragment analysis detects DNA differences that affect restriction sites • Restriction fragment analysis detects differences in the nucleotide sequences of DNA molecules • Such analysis can rapidly provide comparative information about DNA sequences

  19. Gel Electrophoresis and Southern Blotting • One indirect method of rapidly analyzing and comparing genomes is gel electrophoresis • This technique uses a gel as a molecular sieve to separate nuclei acids or proteins by size

  20. LE 20-8 Mixture of DNA molecules of differ- ent sizes Longer molecules Cathode Shorter molecules Power source Gel Glass plates Anode

  21. In restriction fragment analysis, DNA fragments produced by restriction enzyme digestion of a DNA molecule are sorted by gel electrophoresis • Restriction fragment analysis is useful for comparing two different DNA molecules, such as two alleles for a gene

  22. LE 20-9 Normal b-globin allele 175 bp 201 bp Large fragment Ddel Ddel Ddel Ddel Sickle-cell mutant b-globin allele 376 bp Large fragment Ddel Ddel Ddel Ddel restriction sites in normal and sickle-cell alleles of -globin gene Normal allele Sickle-cell allele Large fragment 376 bp 201 bp 175 bp Electrophoresis of restriction fragments from normal and sickle-cell alleles

  23. A technique called Southern blotting combines gel electrophoresis with nucleic acid hybridization • Specific DNA fragments can be identified by Southern blotting, using labeled probes that hybridize to the DNA immobilized on a “blot” of gel

  24. LE 20-10 Heavy weight Restriction fragments DNA + restriction enzyme Nitrocellulose paper (blot) I I I Gel Sponge Paper towels I Normal -globin allele I Sickle-cell allele I Heterozygote Alkaline solution Preparation of restriction fragments. Gel electrophoresis. Blotting. Probe hydrogen- bonds to fragments containing normal or mutant -globin I I I Radioactively labeled probe for -globin gene is added to solution in a plastic bag I I I Fragment from sickle-cell -globin allele Film over paper blot Fragment from normal -globin allele Paper blot Hybridization with radioactive probe. Autoradiography.

  25. Restriction Fragment Length Differences as Genetic Markers • Restriction fragment length polymorphisms (RFLPs, or Rif-lips) are differences in DNA sequences on homologous chromosomes that result in restriction fragments of different lengths • A RFLP can serve as a genetic marker for a particular location (locus) in the genome • RFLPs are detected by Southern blotting

  26. Concept 20.5: The practical applications of DNA technology affect our lives in many ways • Many fields benefit from DNA technology and genetic engineering

  27. Medical Applications • One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases

  28. Diagnosis of Diseases • Scientists can diagnose many human genetic disorders by using PCR and primers corresponding to cloned disease genes, then sequencing the amplified product to look for the disease-causing mutation • Even when a disease gene has not been cloned, presence of an abnormal allele can be diagnosed if a closely linked RFLP marker has been found

  29. LE 20-15 RFLP marker DNA Disease-causing allele Restriction sites Normal allele

  30. Human Gene Therapy • Gene therapy is the alteration of an afflicted individual’s genes • Gene therapy holds great potential for treating disorders traceable to a single defective gene • Vectors are used for delivery of genes into cells • Gene therapy raises ethical questions, such as whether human germ-line cells should be treated to correct the defect in future generations

  31. LE 20-16 Cloned gene Insert RNA version of normal allele into retrovirus. Viral RNA Let retrovirus infect bone marrow cells that have been removed from the patient and cultured. Retrovirus capsid Viral DNA carrying the normal allele inserts into chromosome. Bone marrow cell from patient Bone marrow Inject engineered cells into patient.

  32. Pharmaceutical Products • Some pharmaceutical applications of DNA technology: • Large-scale production of human hormones and other proteins with therapeutic uses • Production of safer vaccines

  33. Forensic Evidence • DNA “fingerprints” obtained by analysis of tissue or body fluids can provide evidence in criminal and paternity cases • A DNA fingerprint is a specific pattern of bands of RFLP markers on a gel • The probability that two people who are not identical twins have the same DNA fingerprint is very small • Exact probability depends on the number of markers and their frequency in the population

  34. LE 20-17 Defendant’s blood (D) Blood from defendant’s clothes Victim’s blood (V)

  35. Environmental Cleanup • Genetic engineering can be used to modify the metabolism of microorganisms • Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials

  36. Agricultural Applications • DNA technology is being used to improve agricultural productivity and food quality

  37. Animal Husbandry and “Pharm” Animals • Transgenic organisms are made by introducing genes from one species into the genome of another organism • Transgenic animals may be created to exploit the attributes of new genes (such as genes for faster growth or larger muscles) • Other transgenic organisms are pharmaceutical “factories,” producers of large amounts of otherwise rare substances for medical use

  38. Genetic Engineering in Plants • Agricultural scientists have endowed a number of crop plants with genes for desirable traits • The Ti plasmid is the most commonly used vector for introducing new genes into plant cells

  39. LE 20-19 Agrobacterium tumefaciens Ti plasmid Site where restriction enzyme cuts T DNA DNA with the gene of interest Recombinant Ti plasmid Plant with new trait

  40. Safety and Ethical Questions Raised by DNA Technology • Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures • Most public concern about possible hazards centers on genetically modified (GM) organisms used as food

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