1. © 2010 Pearson Education, Inc. Lectures by Chris C. Romero, updated by Edward J. Zalisko PowerPoint ® Lectures for Campbell Essential Biology, Fourth Edition – Eric Simon, Jane Reece, and Jean Dickey Campbell Essential Biology with Physiology, Third Edition – Eric Simon, Jane Reece, and Jean Dickey Chapter 12 DNA Technology

2. © 2010 Pearson Education, Inc. Biology and Society  This is a DNA microarray - http://highered.mcgraw- hill.com/sites/0072556781/student_view0/chapter15/animation_quiz_2.htmlhttp://highered.mcgraw- hill.com/sites/0072556781/student_view0/chapter15/animation_quiz_2.html  —an incredibly powerful tool to explore gene expression in cells. Microarrays also have the potential to be used to diagnose and predict the onset of debilitating diseases such as cancer. There is much promise for this technology, but who has access to this information? Assume that the technology exists today that could screen your specific DNA genome for cancer risk.  In this scenario, do you think that insurance companies would be justified in requiring a DNA screening for cancer upon application for health or life insurance? Strongly Agree Strongly Disagree A. E. C. B. D.

3. Biology and Society: DNA, Guilt, and Innocence DNA profiling is the analysis of DNA samples that can be used to determine whether the samples come from the same individual. DNA profiling can therefore be used in courts to indicate if someone is: –Guilty –Innocent DNA technology has led to other advances in the: –Creation of genetically modified crops –Identification and treatment of genetic diseases © 2010 Pearson Education, Inc.

4. RECOMBINANT DNA TECHNOLOGY Biotechnology: –Is the manipulation of organisms or their components to make useful products –Has been used for thousands of years to –Make bread using yeast –Selectively breed livestock for desired traits Biotechnology today means the use of DNA technology, methods for: –Studying and manipulating genetic material –Modifying specific genes –Moving genes between organisms

5. © 2010 Pearson Education, Inc. Recombinant DNA is formed when scientists combine nucleotide sequences (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. By transferring the gene for a desired protein into a bacterium or yeast, proteins that are naturally present in only small amounts can be produced in large quantities – “Pharm”ing animals - http://youtu.be/XqphlQ_joN0 http://youtu.be/XqphlQ_joN0 Humulin, human insulin - http://youtu.be/IJ3u9fQURt0http://youtu.be/IJ3u9fQURt0 Human Growth Hormone - http://kisdwebs.katyisd.org/campuses/MRHS/teacherweb/hallk/Teacher%20Documents/AP%20Bi ology%20Materials/Genetics/Producing%20Human%20Growth%20Hormone/19_A01s.swf http://kisdwebs.katyisd.org/campuses/MRHS/teacherweb/hallk/Teacher%20Documents/AP%20Bi ology%20Materials/Genetics/Producing%20Human%20Growth%20Hormone/19_A01s.swf.

6. © 2010 Pearson Education, Inc. Genetically Modified (GM) Foods Today, DNA technology is quickly replacing traditional plant- breeding programs. Scientists have produced many types of genetically modified (GM) organisms - http://youtu.be/M_ztZGbLEJ0 organisms that have acquired one or more genes by artificial meanshttp://youtu.be/M_ztZGbLEJ0 A transgenic organism contains a gene from another organism, typically of another species - http://www.pbs.org/wgbh/harvest/etc/video.html http://www.pbs.org/wgbh/harvest/etc/video.html

7. © 2010 Pearson Education, Inc. Biology and Society  Do you think food products that are produced from GM organisms should be labeled to inform consumers? Strongly Agree Strongly Disagree A. E. C. B. D. Do a Web search on “Golden Rice” and you’ll be astounded at the diversity of opinion and information on this particular genetically modified (GM) organism. Agricultural scientists are increasingly turning to genetic engineering to develop new crop solutions, and yet, around the world GM crops are not universally accepted.

8. © 2010 Pearson Education, Inc. Recombinant DNA techniques can help biologists produce large quantities of a desired protein by: –Cloning genes - http://highered.mcgraw- hill.com/sites/0072556781/student_view0/chapter14/animation_quiz_1.ht mlhttp://highered.mcgraw- hill.com/sites/0072556781/student_view0/chapter14/animation_quiz_1.ht ml –That taps the property of genetic recombination in bacteria - http://highered.mcgraw- hill.com/sites/0072556781/student_view0/chapter13/animation_quiz_1.ht ml http://highered.mcgraw- hill.com/sites/0072556781/student_view0/chapter13/animation_quiz_1.ht ml –Using restriction enzymes - http://highered.mcgraw- hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/007243 7316/120078/bio37.swf::Restriction%20Endonucleaseshttp://highered.mcgraw- hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/007243 7316/120078/bio37.swf::Restriction%20Endonucleases

9. Plasmid Bacterial cell Isolate plasmids. Some uses of genes Gene for pest resistance Gene for toxic-cleanup bacteria Genes may be inserted into other organisms. Find the clone with the gene of interest. The gene and protein of interest are isolated from the bacteria. Clone the bacteria. Recombinant bacteria Bacterial clone Gene of interest Recombinant DNA plasmids Bacteria take up recombinant plasmids. Harvested proteins may be used directly. Some uses of proteins Protein for “stone-washing” jeans DNA Cell containing the gene of interest Protein for dissolving clots Isolate DNA. DNA fragments from cell Cut both DNAs with same enzyme. Gene of interest Other genes Mix the DNAs and join them together. Figure 12.8-8

10. © 2010 Pearson Education, Inc. Concept Check Restriction enzymes are useful tools for cutting DNA fragments. What do you think is the function of restriction enzymes in their normal bacterial environment? a.Restriction enzymes remove and recycle old mRNAs. b.Restriction enzymes cut up DNA taken from the environment and used as a nutrient source. c.Restriction enzymes remove the excess DNA that results from DNA replication. d.Restriction enzymes cut invading viral DNA sequences at specific sequences, disrupting the viral DNA genes.

11. Recognition sequence for a restriction enzyme Restriction enzyme Sticky end Sticky end DNA ligase Recombinant DNA molecule A DNA fragment is added from another source. A restriction enzyme cuts the DNA into fragments. Fragments stick together by base pairing. DNA ligase joins the fragments into strands. Figure 12.9-4

12. © 2010 Pearson Education, Inc. Interpreting Data The image to the right represents a simulated electrophoresis gel run of a restriction analysis of λ virus DNA. Both EcoR1 (E) and BamH1 (B) cut λ DNA into six fragments. Why aren’t the fragments aligned together on the gel? a.The enzymes cut at random sites each time. b.The enzymes each cut at unique restriction sites that create differently sized fragments. c.The two enzymes create equal- sized fragments but the gel cannot resolve the difference.

13. © 2010 Pearson Education, Inc. A Closer Look: Obtaining the Gene of Interest How can a researcher obtain DNA that encodes a particular gene of interest? –A “shotgun” approach yields millions of recombinant plasmids carrying many different segments of foreign DNA. –A collection of cloned DNA fragments that includes an organism’s entire genome (a complete set of its genes) is called a genomic library - http://www.sumanasinc.com/webcontent/animations/content/dnalibra ry.html http://www.sumanasinc.com/webcontent/animations/content/dnalibra ry.html Once a genomic library is created, the bacterial clone containing the desired gene is identified using a specific sequence of radioactive nucleotides matching those in the desired gene, called a nucleic acid probe.

14. Cell nucleus DNA of eukaryotic gene RNA transcript mRNA Test tube cDNA of gene without introns Reverse transcriptase cDNA strand being synthesized Transcription Introns removed and exons spliced together Isolation of mRNA from cell and addition of reverse transcriptase Synthesis of cDNA strand Synthesis of second DNA strand by DNA polymerase Exon Intron Exon Intron Figure 12.11-5 http://highered.mcgraw- hill.com/olcweb/cgi/plugi npop.cgi?it=swf::535::53 5::/sites/dl/free/0072437 316/120078/bio_h.swf::c DNA cDNA Libraries

15. © 2010 Pearson Education, Inc. DNA PROFILING AND FORENSIC SCIENCE Lighter side – A Biotechnology Lab - http://youtu.be/lCblncsE8wQ http://youtu.be/lCblncsE8wQ DNA profiling: –Can be used to determine if two samples of genetic material are from a particular individual –Has rapidly revolutionized the field of forensics, the scientific analysis of evidence from crime scenes To produce a DNA profile, scientists compare genetic markers, sequences in the genome that vary from person to person.

16. © 2010 Pearson Education, Inc. Biology and Society Do you think that states should create and maintain DNA databases similar to fingerprint databases? Strongly Agree Strongly Disagree A. E. C. B. D. DNA technology has revolutionized forensic science. Some states have required DNA samples for all convicted felons. Some consider this to be an infringement on personal rights and freedoms. Several unsolved cases have been solved with this type of evidence and some convicted persons have been exonerated with DNA evidence.

17. © 2010 Pearson Education, Inc. 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 –Trace the evolutionary history of organisms

18. The Polymerase Chain Reaction (PCR) The polymerase chain reaction (PCR): –http://highered.mcgraw- hill.com/sites/0072556781 /student_view0/chapter14/ animation_quiz_6.htmlhttp://highered.mcgraw- hill.com/sites/0072556781 /student_view0/chapter14/ animation_quiz_6.html –Is a technique to copy quickly and precisely any segment of DNA and –Can generate enough DNA, from even minute amounts of blood or other tissue, to allow DNA profiling © 2010 Pearson Education, Inc. Initial DNA segment Number of DNA molecules 1 248 Figure 12.15

19. © 2010 Pearson Education, Inc. Short Tandem Repeat (STR) Analysis How do you test if two samples of DNA come from the same person? DNA Fingerprinting - http://highered.mcgraw- hill.com/sites/dl/free/0072835125/126997/animation40.htmlhttp://highered.mcgraw- hill.com/sites/dl/free/0072835125/126997/animation40.html Repetitive DNA: –Makes up much of the DNA that lies between genes in humans and –Consists of nucleotide sequences that are present in multiple copies in the genome

20. Crime scene DNA Suspect’s DNA Same number of short tandem repeats Different numbers of short tandem repeats STR site 1STR site 2 AGAT GATA Figure 12.16 Short tandem repeats (STRs) are: –Short sequences of DNA –Repeated many times, tandemly (one after another), in the genome STR analysis: –Is a method of DNA profiling –Compares the lengths of STR sequences at certain sites in the genome

21. © 2010 Pearson Education, Inc. http://highered.mcgraw- hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free /0072437316/120078/bio20.swf::Restriction%20Fragment%20Le ngth%20Polymorphismshttp://highered.mcgraw- hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free /0072437316/120078/bio20.swf::Restriction%20Fragment%20Le ngth%20Polymorphisms Gel electrophoresis - http://www.sumanasinc.com/webcontent/animations/content/gelel ectrophoresis.html http://www.sumanasinc.com/webcontent/animations/content/gelel ectrophoresis.html may also be used for RFLP analysis, in which DNA molecules are exposed to a restriction enzyme, which produces fragments that are compared and made visible by gel electrophoresis. RFLP Analysis

22. © 2010 Pearson Education, Inc. GENOMICS Genomics is the science of studying complete sets of genes (genomes) - http://youtu.be/hSL4_SpeJUE.http://youtu.be/hSL4_SpeJUE –The first targets of genomics were bacteria. –As of 2009, the genomes of nearly one thousand species have been published, including: –Baker’s yeast –Mice –Fruit flies –Rice

23. The Process of Science: Can Genomics Cure Cancer? Observation: A few patients responded quite dramatically to a new drug, gefitinib, which: –Targets a protein called EGFR found on the surface of cells that line the lungs –Is used to treat lung cancer Question: Are genetic differences among lung cancer patients responsible for the differences in gefitinib’s effectiveness? © 2010 Pearson Education, Inc.

24. Hypothesis: Mutations in the EGFR gene were causing the different responses to gefitinib. Prediction: DNA profiling that focuses on the EGFR gene would reveal different DNA sequences in the tumors of responsive patients compared with the tumors of unresponsive patients.

25. © 2010 Pearson Education, Inc. Experiment: The EGFR gene was sequenced in the cells extracted from the tumors of: –Five patients who responded to the drug –Four who did not Results: The results were quite striking. –All five tumors from gefitinib-responsive patients had mutations in EGFR. –None of the other four tumors did.

26. © 2010 Pearson Education, Inc. 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

27. Normal human gene isolated and cloned Normal human gene inserted into virus Virus injected into patient with abnormal gene Healthy person Harmless virus (vector) Virus containing normal human gene Bone marrow Bone of person with disease Figure 12.24-3 http://www.sumanasinc.com/scienceinfocus/sif_ genetherapy.html

28. Evolution Connection: Profiling the Y Chromosome 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. DNA profiling of the Y chromosome has revealed that: –Nearly 10% of Irish men were descendants of Niall of the Nine Hostages, a warlord who lived during the 5th century –The Lemba people of southern Africa are descended from ancient Jews –8% of males currently living in central Asia may be descended from Genghis Khan © 2010 Pearson Education, Inc.