Chapter 9 Mastering
Suppose the thermocycler is INCORRECTLY programmed to omit the 72∘C step in each cycle of an otherwise normal PCR run. Which of the following would most likely occur? DNA polymerase would synthesize DNA more slowly. DNA template strands would bind to each other. DNA polymerase would be active at 94∘C. Primers would NOT bind to DNA template strands.
DNA polymerase would synthesize DNA more slowly.
Foreign DNA can be inserted into cells using a variety of different methods. Which method involves the formation of microscopic pores in the cell's membrane? heat shock transformation protoplast fusion electroporation
electroporation
Which of the following is an application that uses PCR?
Sequencing a gene, diagnosing a disease, and providing enough DNA for cloning into another organism
What does a thermocycler do? Adds reagents to facilitate the PCR run Purifies DNA from a crude sample Subjects samples to temperature changes Monitors the synthesis of DNA
Subjects samples to temperature changes
PCR stands for
polymerase chain reaction.
If you have inserted a gene in the Ti plasmid, the next step in genetic engineering is transformation of an animal cell. inserting the Ti plasmid into Agrobacterium. inserting the Ti plasmid into a plant cell. transformation of E. coli with Ti plasmid. splicing T DNA into a plasmid.
inserting the Ti plasmid into Agrobacterium
What is the temperature used for the extension step? 72 °C 60 °C 94 °C
72 °C
An advantage of cDNA over genomic DNA is that it lacks introns. is very easy to isolate. can form very large DNA segments. lacks exons. contains selectable markers.
lacks introns.
The reaction catalyzed by reverse transcriptase is DNA → mRNA. mRNA → cDNA. tRNA → mRNA. mRNA → protein. DNA → DNA.
mRNA → cDNA.
Self-replicating DNA used to transmit a gene from one organism to another is a library. PCR. vector. Southern blot. clone.
vector
In recombinant DNA technology, a vector is a self-replicating segment of DNA, such as a plasmid or viral genome. T/F
true
GM crops have raised issues among communities in which they are produced. Which of the following are concerns raised by the public about genetically engineered crops? The Bt gene could ultimately make the plants pathogenic to humans. Genetically engineered crops could harm the economy. The Bt toxin could result in the death of non-pest species of insects. The Bt toxin could negatively affect the taste of the food that is produced from these crops.
-The Bt toxin could result in the death of non-pest species of insects. -The Bt toxin could negatively affect the taste of the food that is produced from these crops. -The Bt gene could ultimately make the plants pathogenic to humans.
What are the first five thermocycler steps in a PCR run?
1) 94 C for 1 minute 2) 55 C for 1 minute 3) 72 C for 1 Minute 4) 94 C for 1 minute 5) 55 C for 1 minute
In the figure, after digestion with the appropriate restriction enzyme, what is the smallest piece containing the entire ampicillin-resistance (amp) gene? A) 0.17 kilobase pairs B) 0.25 kbp C) 1.08 kbp D) 1.50 kbp E) 3.00 kbp
1.50 kbp
In which direction does DNA polymerase synthesize the new DNA strand? 5' to 3' Both 5' to 3' and 3' to 5' 3' to 5'
5' to 3'
Each step of the polymerase chain reaction is associated with a particular temperature. Primer annealing= DNA extension= DNA denaturation=
55 C 72 C 94 C
The following steps must be performed to make a bacterium produce human protein X. 1-Translation 2-Restriction enzyme 3-Prokaryotic transcription 4-DNA ligase 5-Transformation 6-Eukaryotic transcription 7-Reverse transcription Which of the following places the steps in the correct order?
6, 7, 2, 4, 5, 3, 1
You have a small gene that you wish replicated by PCR. After 3 replication cycles, how many double-stranded DNA molecules do you have? 2 thousands 4 16 8
8
What is the sequence of the temperatures of a typical PCR reaction? 72 °C, 60 °C, 94 °C 94 °C, 72 °C, 60 °C 72 °C, 94 °C, 60 °C 60 °C, 72 °C, 94 °C 94 °C, 60 °C, 72 °C
94 °C, 60 °C, 72 °C
The restriction enzyme EcoRI recognizes the sequence GAATTC. Which of the following is TRUE of DNA after it is treated with EcoRI? All of the DNA fragments will have single-stranded regions ending in AA. All of the DNA will have blunt ends. All of the DNA fragments will have single-stranded regions ending in G. All of the DNA will be circular. Some of the DNA will have single-stranded regions ending in AA and others will end in G.
All of the DNA fragments will have single-stranded regions ending in AA.
Which of the following best describes how recombinant DNA technology currently helps patients who do NOT produce adequate amounts of growth hormone (hGH)—a condition that otherwise leads to stunted growth? -Recombinant vectors are used to stimulate hGH production in these patients. -Bacteria now produce hGH. -Bacteria now produce rDNA coding for hGH. -Recombinant vectors now produce hGH.
Bacteria now produce hGH.
Which of the following best describes why a vector is used in genetic modification procedures? -Cells usually won't copy an isolated gene sequence. -The gene of interest must be isolated from adjacent genes. -The clone must be able to produce proteins from the rDNA containing the gene of interest. -The vector ensures that the clone remains pure.
Cells usually won't copy an isolated gene sequence
Which of the following methods could be used to identify the source of an outbreak? production of a recombinant protein reverse genetics DNA fingerprinting artificial selection
DNA fingerprinting
Which of the following attaches the target gene to a desired location? Restriction enzymes Plasmids DNA ligase Chromosomal DNA
DNA ligase
What provides the energy for DNA polymerization in a PCR reaction? DNA polymerase Deoxyribonucleoside triphosphates Template DNA Primers
Deoxyribonucleoside triphosphates
The Ti plasmid isolated from Agrobacterium can be used to insert DNA into any type of plant. T/F
False
Bt crops, including potatoes and cotton, are genetically engineered using laboratory techniques. Which of the following utilizes recombinant DNA technology to produce advantageous traits in the crops that are produced? Genetically engineered crops have natural characteristics that give them a genetic advantage. Genetically engineered crops have a genetic advantage because the parent strains have advantageous traits. Genetically engineered crops have an advantageous gene from another organism inserted into their genome. Genetically engineered crops naturally produce larger plants and bountiful products.
Genetically engineered crops have an advantageous gene from another organism inserted into their genome.
Why is DNA polymerase from Thermus aquatics ideal for PCR? It does not require energy to polymerize DNA. It does not require primers. It can withstand the high temperatures associated with PCR. It can synthesize DNA 5' to 3' and 3' to 5'.
It can withstand the high temperatures associated with PCR.
Which of the following statements about recombinant DNA technology is FALSE? It can be used to screen individuals for many different types of genetic diseases. It allows researchers to make protein products of a gene. It allows researchers to make many copies of a gene of interest. It has limited application because genes of interest cannot be moved from one type of cell to another.
It has limited application because genes of interest cannot be moved from one type of cell to another.
Which of the following is an advantage of using E. coli to make a human gene product? It cannot process introns. It does not secrete most proteins. Its genes are well known. Endotoxin may be in the product. Endotoxin may be in the product and it does not secrete most proteins.
Its genes are well known.
Which of the following is NOT a desired characteristic of DNA vectors used in gene cloning procedures? circular form of DNA or integrates into the host chromosome has a selectable marker self-replication may replicate in several species large size
Large size
You want to determine whether a person has a certain mutant gene. The process involves using a primer and a heat-stable DNA polymerase. This process is site-directed mutagenesis. transformation. translation. PCR. restriction mapping.
PCR
Why is PCR a valuable technique? -PCR creates large amounts of DNA from minute source quantities. -PCR separates DNA from crude mixtures of other biomolecules. -PCR stimulates transcription of genes (DNA). -PCR harvests small quantities of DNA.
PCR creates large amounts of DNA from minute source quantities.
Recombinant DNA techniques typically involve generating a clone. Why? -Recombining the clone produces the recombinant DNA. -A clone is used to get the gene of interest into a suitable cell. -Producing a clone generates many copies of the gene of interest. -A clone is generated when a cell takes up the vector.
Producing a clone generates many copies of the gene of interest
Which of the following are used to silence specific genes and hold promise for treating cancer or viral diseases, such as hepatitis B? reverse transcriptase PCR (rtPCR) RNA interference (RNAi) DNA fingerprinting tumor-inducing plasmids (Ti plasmids) complementary DNA (cDNA)
RNA interference (RNAi)
How do the strands separate during PCR? The DNA polymerase breaks the hydrogen bonds between the two strands. The primers separate the strands during the annealing step. The high heat of the denaturation step breaks the hydrogen bonds between the two strands. The cycling of the temperatures breaks the hydrogen bonds between the two strands.
The high heat of the denaturation step breaks the hydrogen bonds between the two strands.
What is a thermocycler? The process of cycling through the different temperatures of a PCR reaction 30 times The special DNA polymerase, used in a PCR reaction, that can tolerate the high temperatures The machine that controls the heat of the reaction, cycling between the different temperatures of the different steps during PCR The name for the DNA primers used in a PCR reaction
The machine that controls the heat of the reaction, cycling between the different temperatures of the different steps during PCR
A source of heat-stable DNA polymerase is Thermus aquaticus. Pseudomonas. Saccharomyces cerevisiae. Bacillus thuringiensis. Agrobacterium tumefaciens.
Thermus aquaticus.
Which of the following is NOT a property of useful vectors? - They always contain only one gene. - They must have properties that allow their survival in the host cell. - They must be able to self-replicate. - They must be small enough to allow them to be manipulated prior to injection.
They always contain only one gene.
What is the function of the primers in PCR? They provide energy for the DNA polymerization reactions. They provide a 3' end for the DNA polymerase. They polymerize free nucleotides to form the new DNA strands. They are the monomer building blocks from which the DNA strand is synthesized.
They provide a 3' end for the DNA polymerase.
After the 94∘C step, why must the thermocycler reduce the temperature to 55∘C? To allow primers to bind to the DNA template strands To allow the DNA template strands to bind to each other To optimize DNA polymerase activity To separate the DNA template strands
To allow primers to bind to the DNA template strands
In general, how might recombinant DNA technology be used to prevent a genetic disorder caused by a mutation in a single gene? To insert a desirable gene To insert a desirable gene, remove an undesirable gene, or replace a defective gene with a functioning gene To replace a defective gene with a working gene To remove an undesirable gene
To insert a desirable gene, remove an undesirable gene, or replace a defective gene with a functioning gene
Which of the following best describes the purpose of primers in PCR? -To provide a template for free nucleotides -To separate double-stranded DNA into single strands -To provide a structure from which DNA can be synthesized -To activate DNA polymerase to replicate DNA
To provide a structure from which DNA can be synthesized
One of the first commercial successes of recombinant DNA technology was the production of human insulin using genetically engineered E. coli. T/F
True
The Bt toxin derived from Bacillus thuringiensis has been introduced into some crop plants to make them resistant to insect destruction. T/F
True
Why is baker's yeast useful for expressing genetically engineered genes? -The yeast cells do not secrete their protein products. -Yeast cells are eukaryotic and so would likely be successful in expressing eukaryotic genes. -Many inducible promoters from the yeast genome have been cloned, such as the lac operon. -The yeast cells are best suited for making human products for medical use.
Yeast cells are eukaryotic and so would likely be successful in expressing eukaryotic genes.
Which of the following best describes a clone in the context of genetic modification procedures? -a vector, once it contains a copy of the gene of interest -a culture of genetically identical cells -an identical copy of the gene of interest -a cell that is genetically identical to its parent
a culture of genetically identical cells
A restriction fragment is A restriction fragment is a segment of tRNA. cDNA. a gene. a segment of DNA. a segment of mRNA.
a segment of DNA.
The Human Genome Project, which was completed in 2003, was focused on -determining all of the proteins encoded by the human genome. -cloning all of the genes of the human genome. -determining the nucleotide sequence of the entire human genome. -finding a cure for all human genetic disorders. -identifying all of the genes in the human genome.
determining the nucleotide sequence of the entire human genome.
The use of an antibiotic-resistance gene on a plasmid used in genetic engineering makes The use of an antibiotic-resistance gene on a plasmid used in genetic engineering makes replica plating possible. the recombinant cell dangerous. direct selection possible. the recombinant cell unable to survive. All of the answers are correct.
direct selection possible.
In the figure, the bacteria transformed with the recombinant plasmid and plated on media containing ampicillin and X-gal will form white, ampicillin-sensitive colonies. not grow. form blue, ampicillin-sensitive colonies. form blue, ampicillin-resistant colonies. form white, ampicillin-resistant colonies.
form white, ampicillin-resistant colonies.
The use of "suicide" genes in genetically modified organisms is designed to provide a means to eliminate non-modified organisms. kill the modified organisms before they are released in the environment. provide for resistance of the modified organisms to pesticides. delete genes necessary for modified organism's growth. prevent the growth of the modified organisms in the environment.
prevent the growth of the modified organisms in the environment.
Assume you have discovered a cell that produces a lipase that works in cold water for a laundry additive. You can increase the efficiency of this enzyme by changing one amino acid. This is done by enrichment. selective breeding. selection. site-directed mutagenesis. irradiating the cells.
site-directed mutagenesis.
PCR can be used to identify an unknown bacterium because DNA can be electrophoresed. the RNA primer is specific. DNA polymerase will replicate DNA. all cells have DNA. all cells have RNA.
the RNA primer is specific.
Which of the following processes is NOT involved in making cDNA? reverse transcription transcription RNA processing to remove introns translation
translation
Biotechnology involves the: use of animal cells to make vaccines. use of microorganisms to make desired products, the use of animal cells to make vaccines, and the development of disease-resistant crop plants. development of disease-resistant crop plants. use of microorganisms to make desired products and the use of animal cells to make vaccines. use of microorganisms to make desired products.
use of microorganisms to make desired products, the use of animal cells to make vaccines, and the development of disease-resistant crop plants.
Why would a recombinant DNA molecule be inserted into a host cell? It can be copied, transcribed, and translated into a desired protein. Restriction enzymes can only be used inside of a cell. Plasmids cannot be isolated outside of a host cell. It can protect the recombinant DNA.
It can be copied, transcribed, and translated into a desired protein.
In PCR, it is important to use Taq DNA polymerase, as opposed to other DNA polymerases. This is because Taq is capable of synthesizing DNA _________. at 94∘C from user-provided DNA and primers at 55∘C after exposure to 94∘C
after exposure to 94∘C
Restriction enzymes are bacterial enzymes that destroy phage DNA. bacterial enzymes that splice DNA. viral enzymes that destroy host DNA. animal enzymes that splice RNA.
bacterial enzymes that destroy phage DNA.
Which of the following pairings of recombinant DNA techniques and applications does NOT match? PCR: making many copies of a segment of DNA genetic modification of yeast: production of purified insulin gene therapy: replacing a defective gene gene silencing: production of subunit vaccines
gene silencing: production of subunit vaccines
Which of the following places the steps in the PCR procedure in the correct order? 1) Incubate at 94°C to denature DNA strands; 2) Incubate at 72°C for DNA synthesis; 3) Incubate at 60°C for primer hybridization.
1 3 2
List the 5 steps of the typical modification procedure.
1) Isolate Bacterial Plasmid 2) Select gene of interest and insert into plasmid 3) Plasmid taken up by bacterium 4) Bacteria replicate 5a) Cloned gene harvested from bacteria 5b)Protein product harvested from bacteria
Which of the following methods would be used to introduce the plasmid shown in the figure into E. coli? gene guns Ti plasmids and Agrobacterium microinjection transformation
transformation
In a laboratory setting, which of the following scenarios would benefit from utilizing a technique such as PCR? -You want to study the effects of a protein on different cell types, but the bacterial species that the protein originates from is considered a pathogen. -You need to amplify an entire bacterial chromosome to characterize the genes present. -You would like to make copies of a bacterial gene of interest for sequencing and characterization. -You have a fastidious organism that is hard to grow in large numbers and you need to isolate an operon for transferring to a bacterium for expression.
-You want to study the effects of a protein on different cell types, but the bacterial species that the protein originates from is considered a pathogen. -You would like to make copies of a bacterial gene of interest for sequencing and characterization. -You have a fastidious organism that is hard to grow in large numbers and you need to isolate an operon for transferring to a bacterium for expression.
Cells will not express genes that are on DNA fragments floating in their cytoplasm. To express the genes on such a fragment, you will need a vector, a DNA construct that will deliver your genes into the new cell and will have the necessary DNA promoter sequences to initiate transcription. The vector that will suit your needs for this project is an expression plasmid. A plasmid is a small circular strand of a DNA double helix that will have a site for insertion of your DNA fragment and the necessary promoters for expression of the genes. The sequence of the circular plasmid will be digested with the same restriction enzyme you used to cut out the fragment from the bacterial chromosome. The fragment will then be fused into the opened plasmid using the enzyme DNA ligase. The ends of the fragment will be paired with the cut ends of the plasmid based on DNA nucleotide sequences. Since the same enzyme was used to cut the fragment and the plasmid, this will result in the ends having identical DNA base sequences, which repair enzymes will recognize and ligate the DNA sequences together. The question now is which plasmid of the hundreds of commercially available plasmids you should use. Which plasmid characteristics would be required for optimal expression of a DNA fragment to yield a peptide? -a strong promoter for expression of the inserted DNA fragment -a DNA sequence that signals for trafficking of the plasmid to the nucleus -a region with multiple sites for restriction digestion with different enzymes -a sequence for insertion of the expressed protein into the cell wall
-a strong promoter for expression of the inserted DNA fragment -a DNA sequence that signals for trafficking of the plasmid to the nucleus -a region with multiple sites for restriction digestion with different enzymes
Arrange the following statements in chronological order
1) The Ti plasmid is isolated and prepared for the insertion of foreign DNA. 2) The gene for Bt toxin is isolated from the bacterium and inserted into the plasmid. 3) The plasmid is taken up by a bacterium. 4) The bacterium is used to introduce the Bt toxin gene into the chromosome of plant cells. 5) A single plant is chosen to produce a plant that now produces Bt toxin.
Scientists like to use fluorescent proteins for various types of recombinant DNA procedures. You have a very small amount of the gene for a fluorescent protein. You'd like to make a fluorescent bacterium. Which of the following represents the correct sequence of procedures that you would use? Amplify the gene using PCR. Transform the vector into the bacteria. Insert the gene into a plasmid vector. Transform the vector into the bacteria. Amplify the gene using PCR. Insert the gene into a plasmid vector. Amplify the gene using PCR. Insert the gene into a plasmid vector. Transform the vector into the bacteria. Insert the gene into a plasmid vector. Amplify the gene using PCR. Transform the vector into the bacteria.
Amplify the gene using PCR. Insert the gene into a plasmid vector. Transform the vector into the bacteria.
Which of the following are used by the Centers for Disease Control and Prevention to track outbreaks of food borne disease? -DNA fingerprints -DNA fingerprints, restriction fragment length polymorphisms, and reverse-transcriptase PCR(rtPCR) -reverse-transcriptase PCR (rtPCR) -DNA fingerprints and restriction fragment length polymorphisms -restriction fragment length polymorphisms
DNA fingerprints, restriction fragment length polymorphisms, and reverse-transcriptase PCR(rtPCR)
With your fragment now in an expression plasmid, you will need to check to ensure that the fragment is in the correct reading frame from the promoter so that a functional enzyme can be made. You will utilize the polymerase chain reaction (PCR) to amplify a section of the plasmid containing your insert so that it can be sequenced. PCR uses a DNA polymerase isolated from a hyperthermophilic bacterial species to synthesize a new DNA strand off a template strand of DNA. You are supplying a template in the form of the plasmid with the inserted fragment. PCR also uses the requirement of DNA polymerase to have a pre-existing primer with an open 3' end for new strand synthesis. You will provide the primers that will bind to specific DNA sequences before and after the inserted fragment and will act as boundaries for what part of the plasmid will be amplified. The resulting short, amplified fragments will be identical in base sequence to the original plasmid templates. Once PCR amplification is complete, you will have enough template fragments to send for DNA sequencing to ensure that you have inserted the genes correctly into the plasmid. Correct insertion will result in the production of an mRNA strand that will have the first gene codon in the correct reading frame for a working enzyme to be produced. Of the following components, which ones are required for the amplification of a specific section of DNA using the polymerase chain reaction? RNA polymerase DNA polymerase that is heat stable a strand of RNA to act as a template DNA primers
DNA polymerase that is heat stable DNA primers
Which of the following best describes why PCR protocols contain numerous cycles of the denaturation/annealing/extension steps? -The denaturation step of each cycle only separates some of the source DNA. By performing numerous cycles, PCR generates copies of all the target sequences. -Each cycle of PCR doubles the amount of DNA synthesized, but the number of copies starts out small. Numerous cycles are required to produce a sufficient number of copies. -Each cycle of PCR allows Taq polymerase to partially synthesize the target sequence. Numerous cycles are necessary for the target sequence to be fully copied. -Each cycle of PCR incorporates some of the included primers into amplicons. Numerous cycles of PCR are required to ensure all primers are incorporated.
Each cycle of PCR doubles the amount of DNA synthesized, but the number of copies starts out small. Numerous cycles are required to produce a sufficient number of copies.
Bt crops are engineered in the lab to produce Bt toxins due to the presence of a bacterial gene from B. thuringiensis. Why is it advantageous for the plants to produce the Bt toxin? The Bt toxin will protect the plant from pathogenic bacteria. The plant will release chemicals that will repel all nearby insects. People who eat the food produced by a Bt crop will be resistant to bacterial infections. Insects that normally destroy non-toxin-producing crops will be killed when they eat plants that do produce the toxin.
Insects that normally destroy non-toxin-producing crops will be killed when they eat plants that do produce the toxin.
Which of the following best explains how scientists are able to introduce the bacterial gene for Bt toxin into the cotton plant genome? The bacterial gene for Bt toxin is isolated, and the DNA is put into tiny bullets (like BB's) that are "shot" into the cotton plant using a gene gun. The Bt toxin gene is isolated and inserted into a Ti plasmid from Agrobacterium tumefaciens. The engineered Ti plasmid is taken up by a bacterium that infects the cotton plant. A virus is engineered to contain the Bt toxin gene. This virus is then used to infect the plant and pass on the gene. The Bt toxin gene is added to water that is sprayed on the cotton plants. The gene is taken up through the roots of the plant.
The Bt toxin gene is isolated and inserted into a Ti plasmid from Agrobacterium tumefaciens. The engineered Ti plasmid is taken up by a bacterium that infects the cotton plant.
Which statement best describes restriction enzymes? They can cut only circular plasmid DNA. They randomly cut DNA molecules to generate numerous fragments. They are necessary for the polymerase chain reaction (PCR) to occur. They are important for cloning applications because they can be used to cut DNA at specific nucleotide sequences.
They are important for cloning applications because they can be used to cut DNA at specific nucleotide sequences.
How do restriction enzymes cut DNA sequences? They have the ability to cut DNA randomly. They cut DNA at sequences that have lots of adenine bases. They cut DNA at sites, called recognition sites, that have specific nucleotide sequences.
They cut DNA at sites, called recognition sites, that have specific nucleotide sequences.
What is the end goal of PCR? To quickly increase the number of copies of a specific DNA sequence To allow cells to make DNA faster, thereby growing faster To increase the pool of different DNA sequences
To quickly increase the number of copies of a specific DNA sequence
You have to this point cut out your genes of interest, inserted them into an expression plasmid, amplified the section of the plasmid carrying the genes, and sequenced them to check for proper insertion. Now you have to get the plasmid into the yeast cell to see whether yeast can express the new bacterial proteins. You could just add the plasmid to a yeast culture and some cells would eventually take up the plasmid on their own. This would be extremely inefficient, though, and would result in very few transformed cells in a population numbering in the millions. You need a technique that will allow for many--if not all--of the cells to take up the plasmid, thus increasing your chances of finding a cell that can successfully express a gene off of the plasmid. One way to do this would be via electroporation. In electroporation, you will place a sample of cells in an electrical field suspended in a salt solution. This combination will trigger all of the ion-gated channels on the cytoplasmic membrane of the cell to be opened at once. The opened channels will allow for an influx of fluid into cells containing the plasmid. In an effort to yield a high percentage of transformed bacteria, you may choose to utilize electroporation to increase the probability of what occurring? -that the cells will be active and have the ability to take up the plasmid -The plasmid requires an electoral field to be activated for use by the bacterial cell. -that a few of the cells will be able to take up the expression plasmid -that nearly all of the cells present in the field will take up at least one copy of the plasmid
that nearly all of the cells present in the field will take up at least one copy of the plasmid