Chapter 12. DNA Technology. Lectures by Edward J. Zalisko

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1 Chapter 12 DNA Technology PowerPoint Lectures for Campbell Essential Biology, Fifth Edition, and Campbell Essential Biology with Physiology, Fourth Edition Eric J. Simon, Jean L. Dickey, and Jane B. Reece Lectures by Edward J. Zalisko 1

2 RECOMBINANT DNA TECHNOLOGY Biotechnology is the manipulation of organisms or their components to make useful products Biotechnology today means the use of DNA technology, techniques for studying and manipulating genetic material, modifying specific genes, and moving genes between organisms. 2

3 RECOMBINANT DNA TECHNOLOGY Recombinant DNA is constructed when scientists combine pieces of DNA from two different sources to form a single DNA molecule. Recombinant DNA technology is widely used in genetic engineering, the direct manipulation of genes for practical purposes. 3

4 Making Humulin DNA technology is used to produce medically valuable molecules, including human growth hormone (HGH), the hormone erythropoietin (EPO), which stimulates production of red blood cells, and vaccines, harmless variants or derivatives of a pathogen used to prevent infectious diseases. 4

5 Genetically Modified (GM) Foods Today, DNA technology is quickly replacing traditional breeding programs. Scientists have produced many types of genetically modified (GM) organisms, organisms that have acquired one or more genes by artificial means. A transgenic organism contains a gene from another organism, typically of another species. 5

6 Genetically Modified (GM) Foods In the United States today, roughly half of the corn crop and more than three-quarters of the soybean and cotton crops are genetically modified. Corn has been genetically modified to resist insect infestation, attack by an insect called the European corn borer. 6

7 Genetically Modified (GM) Foods Golden rice 2 is a transgenic variety of rice that carries genes from daffodils and corn and could help prevent vitamin A deficiency and resulting blindness. 7

8 Pharm Animals A transgenic pig has been produced that carries a gene for human hemoglobin, which can be isolated and used in human blood transfusions. In 2006, genetically modified pigs carried roundworm genes that produce proteins that convert less healthy fatty acids to omega-3 fatty acids. However, unlike transgenic plants, no transgenic animals are yet sold as food. 8

9 Recombinant DNA Techniques To work with genes in the laboratory, biologists often use bacterial plasmids, small, circular DNA molecules that replicate separately from the larger bacterial chromosome. Colorized TEM Plasmids Bacterial chromosome 9

10 Recombinant DNA Techniques Plasmids can carry virtually any gene, can act as vectors, DNA carriers that move genes from one cell to another, and are ideal for gene cloning, the production of multiple identical copies of a gene-carrying piece of DNA. Recombinant DNA techniques can help biologists produce large quantities of a desired protein. 10

11 Animation: Cloning a Gene Right click slide / select Play 11

12 Figure 12.8 Bacterial cell 1 Isolate plasmids. 2 Isolate DNA. Cell containing the gene of interest Plasmid 3 Cut both DNAs with same enzyme. Gene of interest Other genes DNA fragments from cell DNA 4 Mix the DNA fragments and join them together. Gene of interest Recombinant DNA plasmids 5 Bacteria take up recombinant plasmids. Bacterial clone Recombinant bacteria 6 Clone the bacteria. 7 Find the clone with the gene of interest. A gene for pest resistance is inserted into plants. Some uses of genes Some uses of proteins A protein is used to dissolve blood clots in heart attack therapy. A gene is used to alter bacteria for cleaning up toxic waste. Genes may be inserted into other organisms. 8 The gene and protein of interest are isolated from the bacteria. Bacteria produce proteins, which can be harvested and used directly. A protein is used to prepare stone-washed blue jeans. 12

13 A Closer Look: Cutting and Pasting DNA with Restriction Enzymes Recombinant DNA is produced by combining two ingredients: - a bacterial plasmid and the gene of interest. To combine these ingredients, a piece of DNA must be spliced into a plasmid. This splicing process can be accomplished by using restriction enzymes, which cut DNA at specific nucleotide sequences (restriction sites), and producing fragments of DNA with sticky ends important for joining DNA from different sources.

14 Figure Recognition site (recognition sequence) for a restriction enzyme DNA 1 A restriction enzyme cuts the DNA into fragments. Restriction enzyme 2 A DNA fragment is added from another source. 3 Fragments stick together by base pairing. 4 DNA ligase joins the fragments into strands. DNA ligase Recombinant DNA molecule 14

15 A Closer Look: Obtaining the Gene of Interest How can a researcher obtain DNA that encodes a particular gene of interest? A collection of cloned DNA fragments that includes an organism s entire genome (a complete set of its genes) is called a genomic library. Another way to obtain a gene of interest is to use reverse transcriptase and synthesize the gene by using an mrna template. 15

16 Figure Radioactive probe (single-stranded DNA) Mix with single-stranded DNA from various bacterial clones Single-stranded DNA Base pairing indicates the gene of interest 16

17 Figure Cell nucleus Exon Intron Exon DNA of eukaryotic gene 1 Intron Exon Transcription RNA transcript mrna 2 Introns removed and exons spliced together Test tube 3 Isolation of mrna from cell and addition of reverse transcriptase Reverse transcriptase 4 Synthesis of cdna strand cdna strand being synthesized cdna of gene without introns 5 Synthesis of second DNA strand by DNA polymerase 17

18 DNA PROFILING AND FORENSIC SCIENCE DNA profiling can be used to determine if two samples of genetic material are from a particular individual and has rapidly revolutionized the field of forensics, the scientific analysis of evidence from crime scenes. To produce a DNA profile, scientists compare sequences in the genome that vary from person to person. 18

19 Figure DNA isolated Crime scene Suspect 1 Suspect 2 2 DNA amplified 3 DNA compared 19

20 Investigating Murder, Paternity, and Ancient DNA DNA profiling can be used to test the guilt of suspected criminals, identify tissue samples of victims, resolve paternity cases, identify contraband animal products, and trace the evolutionary history of organisms. 20

21 DNA Profiling Techniques The Polymerase Chain Reaction (PCR) The polymerase chain reaction (PCR) is a technique to copy quickly and precisely a specific segment of DNA and can generate enough DNA, from even minute amounts of blood or other tissue, to allow DNA profiling. Then, gel electrophoresis, a method for sorting of DNA fragments, is used, primarily by their electrical charge and size.

22 Figure Initial DNA segment Number of DNA molecules 22

23 Blast Animation: DNA Fingerprinting Select Play 23

24 Blast Animation: Gel Electrophoresis Select Play 24

25 Figure Mixture of DNA fragments of different sizes Band of longest (slowest) fragments Power source Band of shortest (fastest) fragments 25

26 GENOMICS AND PROTEOMICS Genomics is the study of complete sets of genes (genomes). The first targets of genomics research were bacteria. As of 2011, the genomes of more than 1,700 species have been published and more than 8,000 are in progress. 26

27 Table

28 The Human Genome Project Begun in 1990, the Human Genome Project was a massive scientific endeavor to determine the nucleotide sequence of all the DNA in the human genome and identify the location and sequence of every gene. At the completion of the project, more than 99% of the genome had been determined to % accuracy, about 3 billion nucleotide pairs were identified, about 21,000 genes were found, and about 98% of the human DNA was identified as noncoding. 28

29 Proteomics Success in genomics has given rise to proteomics, the systematic study of the full set of proteins found in organisms. To understand the functioning of cells and organisms, scientists are studying when and where proteins are produced and how they interact. 29

30 HUMAN GENE THERAPY Human gene therapy is a recombinant DNA procedure, seeks to treat disease by altering the genes of the afflicted person, and often replaces or supplements the mutant version of a gene with a properly functioning one. 30

31 Figure Normal human gene 1 An RNA version of a normal human gene is inserted into a harmless RNA virus. RNA genome of virus Healthy person 2 Inserted human RNA Bone marrow cells of the patient are infected with the virus. 3 Viral DNA carrying the human gene inserts into the cell s chromosome. Bone marrow cell from the patient Bone marrow Bone of person with disease 4 The engineered cells are injected into the patient. 31

32 The Controversy over Genetically Modified Foods GM strains account for a significant percentage of several staple crops in the United States. Advocates of a cautious approach are concerned that crops carrying genes from other species might harm the environment, GM foods could be hazardous to human health, and/or transgenic plants might pass their genes to close relatives in nearby wild areas. 32

33 The Controversy over Genetically Modified Foods Negotiators from 130 countries (including the United States) agreed on a Biosafety Protocol that requires exporters to identify GM organisms present in bulk food shipments and allows importing countries to decide whether the shipments pose environmental or health risks. 33

34 Evolution Connection: The Y Chromosome as a Window on History Barring mutations, the human Y chromosome passes essentially intact from father to son. By comparing Y DNA, researchers can learn about the ancestry of human males.

35 Evolution Connection: The Y Chromosome as a Window on History DNA profiling of the Y chromosome has revealed that nearly 16 million men currently living may be descended from Genghis Khan, nearly 10% of Irish men were descendants of Niall of the Nine Hostages, a warlord who lived during the 1400s, and the Lemba people of southern Africa are descended from ancient Jews. 35

36 Figure 12.UN01 DNA isolated from two sources and cut by same restriction enzyme Plasmid (vector) Gene of interest (could be obtained from a library or synthesized) Recombinant DNA Transgenic organisms Useful products 36

37 Figure 12.UN02 Crime scene Suspect 1 Suspect 2 DNA Polymerase chain reaction (PCR) amplifies STR sites Longer DNA fragments Gel Shorter DNA fragments DNA fragments compared by gel electrophoresis (Bands of shorter fragments move faster toward the positive pole.) 37

38 Figure 12.UN03 RNA version of a normal human gene Virus with RNA genome Bone marrow A normal human gene is transcribed and translated in a patient, potentially curing the genetic disease permanently 38