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AP Biology Reading Guide – Chapter 13 Chapter 13 – Biotechnology The following websites offer wonderful tutorials and animations on these techniques, if you would like to explore more about a technique or topic. http://www.dnaftb.org/dnaftb/1/concept/ http://www.dnalc.org/ddnalc/resources/animations.html http://www.hhmi.org/biointeractive/vlabs/index.html (I especially recommend the bacterial identification lab module!!) __________________________________________________________________________________ OBJECTIVES Describe the natural function of restriction enzyme. Describe how restriction enzymes and gel electrophoresis are used to isolate DNA fragments. Explain how RFLP analysis and PCR can be applied to the diagnosing genetic disorders and forensics. Explain how advances in recombinant DNA technology have helped scientists study the eukaryotic genome. Describe how restriction enzymes and gel electrophoresis are used to isolate DNA fragments. Explain how the creation of sticky ends by restriction enzymes is useful in producing a recombinant DNA molecule. Outline the procedures for producing plasmid vectors. Explain how cloning vectors are used in recombinant DNA technology. List and describe the two major sources of genes for cloning. Describe the function of reverse transcriptase in retroviruses and explain how they are useful in recombinant DNA technology. Explain the importance of DNA synthesis and sequencing to modern studies of eukaryotic genomes. Describe how bacteria can be induced to produce eukaryotic gene products. List and describe four complementary approaches used to map the human genome. Describe how recombinant DNA technology can have medical applications such as diagnosis of genetic disease, development of gene therapy, vaccine production, and development of pharmaceutical products. Describe how gene manipulation has practical applications for agriculture. __________________________________________________________________________________ SUMMARY DNA Cloning – Producing Recombinant DNA o DNA technology makes it possible to clone genes for basic research and commercial applications o Restriction enzymes are used to make recombinant DNA o Genes can be cloned in recombinant DNA vectors o Cloned genes are stored in DNA libraries o PCR can be used to clone a single gene or an entire genome Detecting a Particular DNA Sequence Page 1 of 6 AP Biology Reading Guide – Chapter 13 o o o Restriction fragment analysis detects DNA differences that affect restriction sites RFLP analysis is the application of RE site differences PCR can be used to identify if a particular DNA sequence is present or conduct haplotype analysis (determine someone’s genotype) o Southern blotting uses a small, complementary DNA sequence as a probe to detect the presence of a particular DNA sequence and examine differences in DNA sequence between individuals o DNA sequencing allows for the determination of the primary DNA sequence o A combination of these techniques can be used to map entire genomes and identify genetic disorders Practical Applications of DNA Technology o DNA technology is reshaping medicine and the pharmaceutical industry o DNA technology offers forensic, environmental, and agricultural applications o DNA technology raises important safety and ethical questions __________________________________________________________________________________ 13.1 – RECOMBINANT DNA 1. What is recombinant DNA? 2. What are restriction enzymes? How do restriction enzymes recognize a restriction site? 3. What is the normal purpose of restriction enzymes in bacteria? prevent the restriction enzymes from digesting its own DNA? 4. How does the bacterium What is a palindrome? Why is this term used in relation to restriction enzymes? 5. How can restriction enzymes be used to create a physical map of a DNA molecule? (Try Question 3 on p.248 – Understand Concept) AGAROSE GEL ELECTROPHORESIS. 6. Which electrode do DNA fragments migrate towards (the anode or cathode)? Why? 7. In what order to DNA fragments separate by gel electrophoresis? Page 2 of 6 AP Biology Reading Guide – Chapter 13 8. What type of information can be obtained from DNA electrophoresis? RFLP ANALYSIS (SEE P. 247, APPLY THE CONCEPT) 9. What is DNA fingerprinting (also called RFLP analysis)? What is the basis for RFLP analysis? 10. Describe how RFLPs and gel electrophoresis can be used to diagnose a genetic mutation such as sickle cell anemia (fig 15.20). 13.2 - CONSTRUCTING RECOMBINANT DNA. 11. What is the purpose of DNA cloning? 12. How could you specifically select for only those bacterial cells that successfully incorporated the recombinant plasmid? 13. Describe the process involved in inserting genes from one kind of organism into cells of another kind of organism. Be sure to describe how the vector, REs and DNA ligase are all involved. 14. What characteristics should a vector exhibit? Page 3 of 6 AP Biology Reading Guide – Chapter 13 15. Why are plasmids the most common vector used in recombinant DNA? 16. How can we be sure the recombinant plasmid taken in by the bacteria actually contains the gene we inserted? (hint: reporter gene) 13.3 – SOURCES OF DNA FRAGMENTS FOR CLONING 17. What is the DNA sequence lacking introns called? 18. How might you produce a DNA gene sequence from a human (or any eukaryote) that lacks introns? 13.4 – APPLICATIONS OF BIOTECHNOLOGY 19. What is an expression vector? What must an expression vector contain? 20. List some examples of biotechnology products produced using a) Transgenic bacteria b) Transgenic plants c) Transgenic animals Page 4 of 6 AP Biology Reading Guide – Chapter 13 21. Summarize the ethical and other objections that have been raised against recombinant DNA studies, and give practical and research applications of recombinant DNA. Below are two free response questions taken from actual AP Biology exams. While I may not ask you to submit these for grading – I am expecting you to work through these problems. If you understand and can complete these questions, you are well on your way to understand the material from this chapter. 1. The human genome illustrates both continuity and change. a. Describe the essential features of each of the procedures/techniques below. For each of the procedures/techniques, explain how its application contributes to understanding genetics. The use of a bacterial plasmid to clone and sequence a human gene Polymerase chain reaction Restriction fragment length polymorphism analysis b. All humans are nearly identical genetically in coding sequences and have many proteins that are identical in structure and function. Nevertheless, each human has a unique DNA fingerprint. Explain this apparent contradiction. Page 5 of 6 AP Biology Reading Guide – Chapter 13 2. The diagram below shows a segment of DNA with a total length of 4,900 base pairs. The arrows indicate reaction sites for two restriction enzymes (enzyme X and enzyme Y). Enzyme X DNA Segment Length (bas e pairs) 400 500 Enzyme Y Enzyme Enzyme X 1,200 X 1,300 1,500 (A) Explain how the principles of gel electrophoresis allow for the separation of DNA fragments. (B) Describe the results you would expect from electrophoretic separation of fragments from the following treatments of the DNA segment above. Assume that the digestion occurred under appropriate conditions and went to completion. I. DNA digested with only enzyme X II. DNA digested with only enzyme Y III. DNA digested with enzyme X and enzyme Y combined IV. Undigested DNA (C) Explain both of the following: (1). The mechanism of action of restriction enzymes (2). The different results you would expect if a mutation occurred at the recognition site for enzyme Y. Page 6 of 6