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 Chapter 20 DNA Technology and Genomics

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

3. 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

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

5. 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

6. 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

7. LE 20-2 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

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

9. LE 20-3 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

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

11. 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

12. LE 20-4_1 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

13. LE 20-4_2 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

14. LE 20-4_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

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

16. LE 20-5 Master plate Filter Solution containing probe Filter lifted and flipped over Radioactive single-stranded DNA Probe DNA Gene of interest Single-stranded DNA from cell Film Hybridization on filter Master plate Colonies containing gene of interest 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.

17. 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 A three-step cycle—heating, cooling, and replication—brings about a chain reaction that produces an exponentially growing population of identical DNA molecules

18. 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

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

20. 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

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

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

23. LE 20-9 Normal  -globin allele 175 bp201 bpLarge fragment Sickle-cell mutant  -globin allele 376 bpLarge fragment 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

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

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

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

27. 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

28. 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

29. 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

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

31. 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

32. 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

33. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Pharmaceutical Products Some pharmaceutical applications of DNA technology: – Large-scale production of human hormones and other proteins with therapeutic uses – Production of safer vaccines

34. 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

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

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

37. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Agricultural Applications DNA technology is being used to improve agricultural productivity and food quality

38. 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

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

40. 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

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

42. 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