1. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings PowerPoint Lectures for Biology, Seventh Edition Neil Campbell and Jane Reece Lectures by Chris Romero Frontiers of Biotechnology

2. 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 In broad terms, biotechnology is the manipulation of organisms or their components to make useful products

3. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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 – Techniques for gene cloning are used to prepare multiple identical copies of pieces of DNA. – 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

4. Bacterium Bacterial chromosome Plasmid Gene inserted into plasmid Cell containing gene of interest Gene of interest DNA of chromosome Recombinant DNA (plasmid) 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 Protein harvested Gene of interest Copies of gene Basic research on gene Basic research on protein Basic research and various applications 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

5. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Using Restriction Enzymes to Make Recombinant DNA Bacterial restriction enzymes protect bacteria from the DNA of phages or other bacteria by cutting up foreign DNA in a process called restriction. – 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

6. LE 20-2 Restriction site DNA 5 3 3 5 Restriction enzyme cuts the sugar-phosphate backbones at each arrow. One possible combination 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 DNA ligase seals the strands. Recombinant DNA molecule Sticky end

7. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings The Techniques of Gene Cloning Cloning a human gene in a bacterial plasmid can be divided into five steps: 1.Isolate a gene of interest, for example, the gene for human insulin 2.Insert the gene into a bacterial plasmid 3.Insert the plasmid into a vector, a cell that will carry the plasmid, such as a bacterium. To accomplish this, a bacterium must be made competent (be able to take up the plasmid) 4.Clone the gene. As the bacteria reproduce themselves by fission, the plasmid and the selected gene are also being cloned. Millions of copies of the gene are being produced. 5.Identify the bacteria that contain the selected gene and harvest it from the bacteria.

8. LE 20-3_3 Isolate plasmid DNA and human DNA. 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. Bacterial cell lacZ gene (lactose breakdown) Human cell Restriction site amp R gene (ampicillin resistance) Bacterial plasmid Gene of interest Sticky ends Human DNA fragments 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 non- recombinant plasmid with intact lacZ gene Colony carrying recombinant plasmid with disrupted lacZ gene Bacterial clone

9. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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 without using cells. – A three-step cycle—heating, cooling, and replication—brings about a chain reaction that produces an exponentially growing population of identical DNA molecules

10. LE 20-7 Genomic DNA Target sequence 5 3 3 5 5 3 3 5 Primers Denaturation: Heat briefly to separate DNA strands 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 1 yields 2 molecules New nucleo- tides Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence

11. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Concept 20.2: Restriction Fragment Analysis Restriction fragment analysis detects differences in the nucleotide sequences of DNA molecules – Such analysis can rapidly provide comparative information about DNA sequences 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

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

13. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Concept 20.3: Entire genomes can be mapped at the DNA level The most ambitious mapping project to date has been the sequencing of the human genome – Officially begun as the Human Genome Project in 1990, the sequencing was largely completed by 2003 Scientists have also sequenced genomes of other organisms, providing insights of general biological significance

14. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Concept 20.4: Genome sequences provide clues to important biological questions In genomics, scientists study whole sets of genes and their interactions – Genomics is yielding new insights into genome organization, regulation of gene expression, growth and development, and evolution

15. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Identifying Protein-Coding Genes in DNA Sequences Computer analysis of genome sequences helps identify sequences likely to encode proteins The human genome contains about 25,000 genes, but the number of human proteins is much larger Comparison of sequences of “new” genes with those of known genes in other species may help identify new genes

16. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

17. Concept 20.5: The practical applications of DNA technology affect our lives in many ways Many fields benefit from DNA technology and genetic engineering – One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic 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

18. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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

19. LE 20-16 Cloned gene Retrovirus capsid Bone marrow cell from patient Inject engineered cells into patient. 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. Viral DNA carrying the normal allele inserts into chromosome. Bone marrow

20. 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

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

22. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Environmental Cleanup & Agricultural Productivity 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 DNA technology is being used to improve agricultural productivity and food quality

23. 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

24. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Applications of Genetic Engineering Genetic engineering has spurred the growth of biotechnology, a new industry that is changing the way we interact with the living world. – Transgenic bacteria now produce a host of important substances useful for health & industry. Human insulin, growth hormone, and clotting factor are now produced by transgenic bacteria. – Transgenic animals have been used to study genes and improve the food supply. Such animals often grow faster and produce less fatty meat. – Transgenic plants are an important part of our food supply. Many transgenic plants produce a natural insecticide, so the crops do not have to be sprayed with pesticides.

25. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings

27. Cloning

28. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings 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