Download Chapter 19: Recombinant DNA Technology

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
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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.
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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
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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)
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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
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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)
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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
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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
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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
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