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Chapter 20 DNA Technology and Genomics PowerPoint Lectures for Biology, Seventh Edition Neil Campbell and Jane Reece Lectures by Chris Romero Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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 Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings • 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 Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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 Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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 Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 20-2 Bacterium Gene inserted into plasmid Bacterial chromosome Cell containing gene of interest Plasmid Recombinant DNA (plasmid) Gene of interest Plasmid put into bacterial cell DNA of chromosome Recombinant bacterium Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest Gene of interest Protein expressed by gene of interest Copies of gene Basic research on gene Gene for pest resistance inserted into plants Protein harvested Basic research and various applications Gene used to alter bacteria for cleaning up toxic waste Protein dissolves blood clots in heart attack therapy Basic research on protein Human growth hormone treats stunted growth 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 Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 20-3 Restriction site DNA 5 3 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 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 Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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 Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 20-4_1 Bacterial cell Isolate plasmid DNA and human DNA. lacZ gene (lactose breakdown) Human cell Restriction site ampR gene (ampicillin resistance) Cut both DNA samples with the same restriction enzyme. Bacterial plasmid Gene of interest Sticky ends Human DNA fragments Mix the DNAs; they join by base pairing. The products are recombinant plasmids and many nonrecombinant plasmids. Recombinant DNA plasmids LE 20-4_2 Bacterial cell Isolate plasmid DNA and human DNA. lacZ gene (lactose breakdown) Human cell Restriction site ampR gene (ampicillin resistance) Cut both DNA samples with the same restriction enzyme. Bacterial plasmid Gene of interest Sticky ends Human DNA fragments 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 LE 20-4_3 Bacterial cell Isolate plasmid DNA and human DNA. lacZ gene (lactose breakdown) Human cell Restriction site ampR gene (ampicillin resistance) Cut both DNA samples with the same restriction enzyme. Bacterial plasmid Gene of interest Sticky ends Human DNA fragments 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 nonrecombinant plasmid with intact lacZ gene Colony carrying recombinant plasmid with disrupted lacZ gene Bacterial clone 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 Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 20-5 Master plate Filter Master plate Probe DNA Radioactive single-stranded DNA Solution containing probe Colonies containing gene of interest Gene of interest Single-stranded DNA from cell Film 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. 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 Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 20-7 5 3 Target sequence Genomic DNA Denaturation: Heat briefly to separate DNA strands Cycle 1 yields 2 molecules Annealing: Cool to allow primers to form hydrogen bonds with ends of target sequence Extension: DNA polymerase adds nucleotides to the 3 end of each primer Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence 3 5 5 3 3 5 Primers New nucleotides 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 Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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 Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 20-8 Cathode Power source Mixture of DNA molecules of different sizes Shorter molecules Gel Glass plates Anode Longer molecules • 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 Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 20-9 Normal b-globin allele 175 bp Ddel 201 bp Ddel Large fragment Ddel Ddel Sickle-cell mutant b-globin allele 376 bp Ddel Large fragment Ddel Ddel Ddel restriction sites in normal and sickle-cell alleles of b-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 • 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 Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 20-10 Restriction fragments DNA + restriction enzyme I II III Heavy weight Nitrocellulose paper (blot) Gel Sponge I Normal b-globin allele II Sickle-cell III Heterozygote allele Preparation of restriction fragments. Radioactively labeled probe for b-globin gene is added to solution in a plastic bag I II III Paper towels Alkaline solution Gel electrophoresis. Probe hydrogenbonds to fragments containing normal or mutant b-globin Blotting. I II Fragment from sickle-cell b-globin allele Paper blot Hybridization with radioactive probe. III Film over paper blot Fragment from normal b-globin allele Autoradiography. 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 Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Concept 20.5: The practical applications of DNA technology affect our lives in many ways • Many fields benefit from DNA technology and genetic engineering Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Medical Applications • One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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 Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 20-15 RFLP marker DNA Restriction sites Disease-causing allele Normal allele 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 Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 20-16 Cloned gene Insert RNA version of normal allele into retrovirus. Viral RNA Retrovirus capsid Let retrovirus infect bone marrow cells that have been removed from the patient and cultured. Viral DNA carrying the normal allele inserts into chromosome. Bone marrow cell from patient Inject engineered cells into patient. Bone marrow Pharmaceutical Products • Some pharmaceutical applications of DNA technology: – Large-scale production of human hormones and other proteins with therapeutic uses – Production of safer vaccines Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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 Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings LE 20-17 Defendant’s blood (D) Blood from defendant’s clothes Victim’s blood (V) 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 Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Agricultural Applications • DNA technology is being used to improve agricultural productivity and food quality Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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 Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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 Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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 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 Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings