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Chapter 19: Recombinant DNA Technology Student Learning Objectives Upon the completion of this chapter you should be able to: 1. Know how restriction enzymes and vectors are used to create recombinant DNA molecules. 2. Understand the gene cloning procedure. 3. Know the various types of the polymerase chain reaction (PCR) technique. 4. Understand the concept of a library, and how libraries are screened for gene products. 5. Distinguish between the various methods used to detect and analyze genes and their products. 6. Know the techniques of DNA sequencing and site-directed mutagenesis. 19.1 Gene Cloning Using Vectors Overview Although recombinant DNA is present in any cell that undergoes crossing-over, sitedirected recombination, or has transposon activity, the ability to duplicate this outside the cell has only been possible since the early 1970s. However, since that time scientists have developed a variety of techniques and procedures to manipulate DNA and unlock the secrets of gene action. The first step for most scientists is to produce large numbers of copies of the gene of interest. This process is called cloning. While the term cloning is now typically associated with organismal cloning, such as sheep and humans, at the molecular level it simply means copying. In order to understand the cloning process, you have to first study how restriction enzymes are used to create recombinant DNA molecules (Figure 19.1 and Table 19.3). You can then focus your attention on Figure 19.2, which presents all the steps involved in gene cloning. The section ends with a discussion of how complementary DNA (cDNA) can be made from mRNA using the enzyme reverse transcriptase (Figure 19.3). Outline of Key Terms Restriction endonucleases (restriction enzymes) Palindromic sequences DNA ligase Recombinant vector Competent cells Transformation Complementary DNA (cDNA) Oligonucleotide Reverse transcriptase Recombinant DNA technology Recombinant DNA molecules Gene cloning Host cell Vector Plasmids R factors Origin of replication Selectable marker Viral vector 232 Focal Points Some uses of gene cloning (Table 19.1) The action of a restriction enzyme and production of recombinant DNA (Figure 19.1) Some restriction enzymes used in gene cloning (Table 19.3) The steps in gene cloning (Figure 19.2) Synthesis of cDNA (Figure 19.3) Exercises and Problems Questions 1 to 11 refer to the process of cloning. For each, select the correct definition for the term. _____ 1. Plasmids _____ 2. Restriction enzymes _____ 3. Selectable marker _____ 4. Vector _____ 5. Recircularized vector _____ 6. Viral vector _____ 7. Competent cells _____ 8. Transformation _____ 9. Transfection _____ 10. Hybrid vector _____ 11. Host cells a. A carrier for DNA in a cloning experiment. b. A vector that does not contain a piece of chromosomal DNA. c. Enzymes that recognize palindromic sequences in the DNA and cut them using endonuclease activity. d. Small circular pieces of extranuclear DNA. e. The process by which a bacterium receives the cloned DNA from the environment. f. Cells that harbor vectors. g. The process by which a virus introduces the cloned DNA into the cell. h. Cells that are capable of taking up DNA from the environment. i. A vector that contains a piece of chromosomal DNA. j. The use of a virus to carry the cloned DNA into a host cell. k. A gene contained within a vector that allows a researcher to choose bacterial colonies that contain only recombinant vectors. 233 For questions 12 to 16, place the following steps of a cloning experiment in order. The letter of the first step of the experiment is the answer for question 12, the second step is for Q13, etc. _____ 12. _____ 13. _____ 14. _____ 15. _____ 16. a. Mix the cut plasmid and chromosomal DNA together. b. Cut the plasmid and chromosomal DNA using the same restriction endonuclease. c. Treat the mixture with DNA ligase. d. Screen the colonies for those that indicate recombinant vectors. e. Plate cells on a media containing the substrate for the selectable marker. For questions 17 to 22, complete the sentence with the most appropriate term(s). 17. Restriction enzymes are also termed restriction ___________. 18. Restriction enzymes typically recognize sequences which are ________ (i.e.: identical when read 5′ to 3′ on both strands) 19. DNA that is made from RNA is termed ________ DNA. 20. The enzyme ________ can make a DNA copy from an RNA strand. 21. The enzyme ________ can make phosphodiester bonds between two DNA bases. 22. A short strand of DNA is termed a(n) ________. 19.2 Polymerase Chain Reaction Overview The second section of this chapter presents the polymerase chain reaction (PCR). This technique is used to amplify DNA, without the aid of vectors or host cells. Each cycle of PCR involves three steps: denaturation, primer annealing, and primer extension (Refer to Figure 19.4). These steps are repeated many times, thereby generating millions of copies of the original DNA fragment. This section also presents recent “modifications” of PCR. These include reverse transcriptase PCR (Figure 19.6), which allows for the amplification of RNA, and real-time PCR (Figures 19.7 and 19.8), which is used to quantify the amount of DNA or mRNA in a sample. Outline of Key Terms Thermocycler Reverse transcriptase PCR (RT-PCR) Real-time PCR Cycle threshold method (Ct method) Polymerase chain reaction (PCR) Primers Template DNA Taq polymerase Annealing Primer extension 234 Focal Points The polymerase chain reaction (Figures 19.4 and 19.5) Reverse transcriptase PCR (Figure 19.6) Real-time PCR (Figures 19.7 and 19.8) Exercises and Problems Complete the following sentences with the most appropriate term(s). 1. The machine in which PCR is conducted is called the __________. 2. The enzyme that carries out PCR is called ________. 3. In PCR, the step where the primers bind to DNA is called ________. 4. _________ PCR allows for the amplification of RNA. 5. _________ PCR can be used to quantify the amount of a specific gene or mRNA in a sample. 19.3 DNA libraries and Blotting Methods Overview The third section of this chapter begins by examining the creation of a DNA library. Note how this can be achieved using either genomic DNA or cDNA (Figure 19.9). Many times researchers do not know what gene product they are looking for precisely, and therefore must screen the entire collection of the organism’s DNA for a gene of interest. The screening of a DNA library for gene(s) of interest is called colony hybridization (Figure 19.10). The section then discusses three blotting methods termed Southern, Northern, and Western, which are used to detect DNA, RNA, and protein, respectively. As you proceed through this section, notice the similarities and differences between the procedures. Outline of Key Terms Northern blotting Western blotting Antibody Antigen Epitope DNA library Genomic library cDNA library Colony hybridization DNA probe Southern blotting High stringency Low stringency Focal Points Construction and screening of a DNA library (Figures 19.9 and 19.10) Southern, Northern, and Western blotting methods (pages 458-461) 232 Exercises and Problems For questions 1 to 4, choose the correct definition for the term. _____ 1. Stringency _____ 2. Colony hybridization _____ 3. Genomic library _____ 4. cDNA library a. Uses a probe to screen a library for a gene of interest. b. The conditions at which a probe will hybridize with its target molecule. c. A collection of recombinant vectors that are derived from expressed RNA in a cell. d. A collection of recombinant vectors that use chromosomal DNA as the source. For questions 5 to 13, select the type of hybridization experiment that is indicated by the statement. Some questions may have more than one answer. a. Western blotting b. Southern blotting c. Northern blotting _____ 5. Identifies a specific gene from a collection of chromosomal DNA fragments. _____ 6. Identifies if a specific protein is present in the sample. _____ 7. Uses antibodies as the probe. _____ 8. Uses a gel to separate the molecules by size or molecular mass. _____ 9. This process may also be done by RT-PCR. _____ 10. May be used to identify gene families. _____ 11. Identifies a specific RNA from a collection of expressed RNAs. _____ 12. Frequently uses a radioactive label on the probe. _____ 13. Uses a fluorescent secondary antibody to detect presence of the primary antibody. 19.4 Methods for Analyzing DNA- and RNA-binding Proteins Overview In chapter 22, we will discuss the technique of chromatin immunoprecipitation, which is often used to identify where histones bind to the DNA (look ahead to Figure 22.2). This section examines two additional methods used to study interactions between proteins and nucleic acid: 1) the gel-retardation assay (Figure 19.14), which is used to determine if a protein binds to a specific DNA or RNA molecule; and 2) the DNase I footprinting technique (Figure 19.15), which can show the specific regions of DNA that are bound by a protein. Outline of Key Terms Gel retardation assay Gel mobility shift assay DNase I footprinting 233 Focal Points Gel retardation assay (Figure 19.14) DNase I footprinting experiment (Figure 19.15) Exercises and Problems For questions 1 to 5, use the following key. Letters may be used more than once. a. Gel retardation assay b. DNase I footprinting c. Both A and B d. Neither A nor B _____ 1. Involves electrophoresis _____ 2. Uses a protein that can cut a nucleic acid molecule _____ 3. Uses oligonucleotide primers _____ 4. Examines protein-DNA interactions _____ 5. Examines protein-RNA interactions 19.5 DNA Sequencing and Site-directed Mutagenesis Overview The final section of this chapter examines the techniques of DNA sequencing and site-directed mutagenesis. DNA sequencing allows researchers to determine the base sequence of DNA. Technological advances, mostly as a result of the Human Genome Project, have made DNA sequencing easy and inexpensive. An overview of the traditional and the automated dideoxy sequencing methods is provided. Site-directed mutagenesis allows researchers to produce a mutation at a particular site in cloned DNA. This mutant can then be introduced into a living organism to observe the effects on gene expression and the phenotype of the organism. Outline of Key Terms Site-directed mutagenesis DNA sequencing Dideoxy sequencing Dideoxyribonucleotides (ddNTPs) Chain termination Sequencing ladder Automated sequencing Focal Points DNA sequencing (Figures 19.16 and 19.17) Site-directed mutagenesis (Figure 19.18) 236 Exercises and Problems Match each of the following terms to its correct statement. _____ 1. Chain termination _____ 2. Dideoxy sequencing _____ 3. Site-directed mutagenesis _____ 4. Dideoxyribonucleotides _____ 5. Automated sequencing _____ 6. Sequencing ladder a. Synthetic nucleotides used in sequencing reactions. b. A dideoxy sequencing reaction that uses fluorescent labels. c. The change in a specific base of a cloned DNA segment. d. The process by which a dideoxyribonucleotide stops DNA replication. e. The pattern of bands produced by traditional dideoxy sequencing reactions. f. The most common method of DNA sequencing. Chapter Quiz 1. A thermocycler is used for ________. a. dideoxy sequencing b. PCR reactions c. DNA footprinting d. colony hybridization 2. Which of the following can be used as a vector for gene cloning? a. plasmid b. virus c. Both A and B d. Neither A nor B 3. Which of the following is able to identify specific areas of DNA and cut them using an endonuclease activity? a. PCR b. Northern blotting c. dideoxy sequencing d. restriction enzymes 4. _______ blotting can detect the presence of a specific protein from a cell. a. Southern b. Northern c. Western 5. _______ blotting can be used to screen for gene families. a. Southern b. Northern c. Western 237 6. A collection of recombinant vectors is called a _______. a. Southern blot b. clone c. antigen d. library 7. cDNA is made using an enzyme called ________. a. dideoxyribonuclease b. integrase c. reverse transcriptase d. restriction endonuclease 8. R factors are an example of ________. a. host cells b. viral vectors c. restriction enzymes d. plasmids e. none of the above 9. Which of the following is NOT a step in a cloning experiment? a. Plasmid and chromosomal DNA is cut using a restriction enzyme. b. The plasmid and chromosomal DNA is mixed. c. Taq polymerase is added to combine the DNA fragments. d. The DNA is mixed with bacterial cells and plated. e. All of the above are correct and are part of a cloning experiment. 10. What is the purpose of the technique of site-directed mutagenesis? a. To alter the sequence of a DNA segment b. To determine the sequence of a DNA segment c. To alter the expression of a gene d. To determine if a protein binds to a gene’s promoter Answer Key for Study Guide Questions This answer key provides the answers to the exercises and chapter quiz for this chapter. Answers in parentheses ( ) represent possible alternate answers to a problem, while answers marked with an asterisk (*) indicate that the response to the question may vary. 19.1 12. b 13. a 14. c 15. e 16. d 17. endonucleases 18. palindromic 19. complementary (cDNA) 20. reverse transcriptase 21. DNA ligase 22. oligonucleotide 1. d 2. c 3. k 4. a 5. b 6. j 7. h 8. e 9. g 10. i 11. f 238 19.2 1. thermocycler 2. Taq polymerase 3. annealing 4. reverse transcriptase 5. real-time 19.3 1. b 2. a 3. d 4. c 5. b 6. a 7. a 8. a b c 9. c 10. b 11. c 12. b c 13. a 19.4 1. c 2. b 3. d 4. c 5. a 19.5 1. d 2. f 3. c 4. a 5. b 6. e Quiz 1. b 2. c 3. d 4. c 5. a 6. d 7. c 8. d 9. c 10. a 376