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CHAPTER 18—DNA TECHNOLOGIES: ANALYZING AND MODIFYING GENES
MULTIPLE CHOICE
1. Which of the following is NOT an example of biotechnology?
a.
genetic engineering
b.
yogurt manufacturing
c.
cheese production
d.
crop fertilization
e.
analysis of genomes
2. When cloning DNA into bacteria,
a.
the DNA sequence is inserted into a plasmid.
b.
the DNA sequence is inserted into the bacterial chromosome.
c.
the linear DNA sequence is circularized, thus generating a plasmid.
d.
the new DNA is directly introduced into the bacterial cell.
e.
either the approach in b or c is used; the choice depends on the type of bacteria.
3. What is the natural function of restriction endonucleases?
a.
research applications
b.
DNA manipulation in vitro
c.
defense against viruses that infect bacteria
d.
regulating bacterial gene expression
e.
breaking phosphodiester bonds in mammalian DNA
4. Some restriction endonucleases cut the DNA in such a way that short, single stranded regions are
created. We call these regions
a.
hydrogen-bonding ends
b.
sticky ends
c.
tacky ends
d.
blunt ends
e.
jagged ends
5. What do restriction endonucleases do?
a.
break phosphodiester bonds
b.
break ester bonds
c.
break glycosidic bonds
d.
break peptide bonds
e.
break hydrogen bonds
6. The DNA sequence recognized by restriction endonucleases is generally how long?
a.
1−2 nucleotides
b.
4−8 nucleotides
c.
10−15 nucleotides
d.
24−32 nucleotides
e.
36−47 nucleotides
7. Cloning plasmids generally contain genes for which two traits?
a.
lacZ and ampR
b.
beta-galactosidase enzyme production and ampicillin resistance
c.
DNA ligase production and sticky ends
d.
DNA circulase and DNA recombinase enzyme expression
e.
DNA replication ability and antibiotic resistance
Enzyme
Recognition sequence and cut site (vertical line). Only the top strand of the double
stranded DNA sequence is shown.
Eco RI
G|AATTC
Hind III
A|AGCTT
Bam HI
G|GATCC
Cla I
AT|CGAT
Pvu I
CGAT|CGC
Use the table above for the following question(s).
8. Which enzyme did I use if I cut a DNA fragment and ended up with a double stranded sequence that
looks like this:
AATTC---------G
G---------CTTAA
a.
Eco RI
b.
Hind III
c.
Bam HI
d.
Cla I
e.
Pvu I
9. Which enzymes did I use if I cut a DNA fragment and ended up with a double stranded sequence that
looks like this:
GATCC---------AT
G---------TAGC
a.
Eco RI and Hind III
b.
Hind III and Bam HI
c.
Bam HI and Cla I
d.
Cla I and Pvu I
e.
Pvu I and Bam HI
10. Which enzymes should I use to generate a double stranded sequence that looks like this:
AGCTT---------G
A---------CCTAG
a.
Eco RI and Hind III
b.
Hind III and Bam HI
c.
Bam HI and Cla I
d.
Cla I and Pvu I
e.
Pvu I and Bam HI
11. Why is it important to have an antibiotic resistance gene in the cloning plasmid?
a.
It makes the bacteria resistant to the antibiotic.
b.
It provides a way for researchers to sort the bacteria that have the cloning plasmid from
the bacteria that don't have the cloning plasmid.
c.
It aids scientists in developing new antibiotic treatments.
d.
It provides a way for sorting bacteria that have the gene of interest from the bacteria that
just have an "empty" cloning plasmid.
e.
It makes the bacteria produce more clones.
12. Why do scientists insert their DNA of interest into the middle of the lacZ coding sequence?
a.
It makes the bacteria resistant to the antibiotic.
b.
It provides a way for researchers to sort the bacteria that have the cloning plasmid from
the bacteria that don't have the cloning plasmid.
c.
It aids scientists in developing new antibiotic treatments.
d.
It provides a way for sorting bacteria that have the gene of interest from the bacteria that
just have an "empty" cloning plasmid.
e.
It makes the bacteria produce more clones.
13. What is the substrate for beta-galactosidase in bacterial screening assays that check for colonies having
recombinant DNA?
a.
lactose
b.
X-gal
c.
sucrose
d.
blue azure
e.
bromophenol blue
14. If multiple fragments from a genome have been cloned into bacteria, how can a researcher identify the
bacteria that contains the particular sequence they are interested in?
a.
The bacteria containing the gene of interest are antibiotic resistant and unable to digest X-
gal.
b.
The DNA purified from individual colonies of bacteria is probed with a short, single
stranded DNA fragment that is complementary to the gene of interest.
c.
The scientists have to sequence all of the DNA from all of the white bacterial colonies;
fortunately computers let us automate this process.
d.
The scientists have to sequence all of the DNA from all of the blue bacterial colonies;
fortunately computers let us automate this process.
e.
We have to isolate the DNA of interest before transforming it into the bacteria. That is the
only way to know which bacteria have the DNA of interest.
15. Why would researchers want to have both genomic libraries and cDNA libraries from a particular
organism?
a.
The genomic library is generally too big to be helpful; a cDNA library, being smaller, is
much easier to study.
b.
A cDNA library will be the same in every cell; the genomic library tells researchers which
genes are being expressed in individual cell types.
c.
The cDNA library helps us understand how retroviruses work; the genomic library does
not.
d.
The cDNA library is a lot easier to clone than a genomic library.
e.
A genomic library will be the same in every cell; the cDNA library tells researchers which
genes are being expressed in individual cell types.
16. What is/are true of the polymerase chain reaction?
a.
PCR allows for the mass production of a specific DNA sequence without cloning.
b.
PCR permits scientists to target a single copy of a single gene among millions of other
sequences.
c.
PCR allows scientists to study DNA from samples of finite amounts, such as from fossils,
or small tissue samples left at a crime scene.
d.
PCR cannot be used to clone DNA of unknown sequence
e.
all of these
17. I need to determine if the gene for a particular protein is the same length in frogs, humans, and trees.
Which method or methods will be most helpful to me?
a.
PCR and agarose gel electrophoresis
b.
DNA cloning and DNA hybridization
c.
cDNA library construction and agarose gel electrophoresis
d.
genomic library construction
e.
agarose gel electrophoresis alone
18. Identify the correct steps and the correct order for cloning a gene of interest.
1.
Transform the bacteria.
2.
Mix the postrestriction endonuclease gene of interest with the postrestriction endonuclease
cloning plasmid.
3.
Incubate the combined DNA fragments with the liagase enzyme.
4.
Use restriction endonucleases to cut the gene of interest and the target DNA.
5.
Spread bacteria on medium containing lactose and ampicillin.
6.
Spread bacteria on medium containing X-gal and ampicillin.
a.
4, 2, 3, 1, 6
b.
4, 3, 2, 1, 5
c.
4, 3, 2, 1, 6
d.
4, 2, 3, 1, 5
e.
2, 4, 1, 5, 3
19. I've cloned a genomic library and know that my bacteria have foreign DNA fragments inserted into the
cloning vector. I now need to identify the specific bacteria that have the gene coding for protein X.
How can I locate these bacteria?
a.
DNA hybridization of the different colonies
b.
PCR of the same bacterial mixture that was spread on agarose plates
c.
restriction endonuclease treatment of DNA from some of the bacterial colonies
d.
agarose gel electrophoresis of DNA isolated from the bacterial colonies
e.
transfer the bacterial colonies to filter paper
20. Which of the following do I need to amplify the DNA for gene X and not for gene Y?
a.
Some prior knowledge of the sequence of gene X so appropriate primers can be designed.
b.
a heat-stable DNA polymerase
c.
restriction endonucleases that cut gene X and not gene Y
d.
plasmid vectors that have multiple antibiotic resistance genes.
e.
A cDNA library and access to probes for gene X
21. Why does the polymerase chain reaction require the samples to undergo numerous cycles, each
containing three different temperatures?
a.
The initial cycles don't produce many copies of the original gene since the amplification is
exponential in nature.
b.
The three temperatures are for different processes; the highest temperature separates the
double stranded DNA into single strands, the lower temperatures allow a target primer to
bind and a DNA polymerase to generate new sequences.
c.
The enzyme used is from an organism that thrives in high temperatures, so high
temperatures must be used.
d.
Non-target DNA is made during the first 10 cycles so further cycles are required to
specifically amplify the target DNA.
e.
Once the enzyme starts working, you cannot stop the reaction until there are no more
nucleotides.
Use the figure above for the following question(s).
22. In the figure above, which DNA fragment is the smallest?
a.
A
b.
B
c.
C
d.
D
e.
E
23. In the figure above, which DNA fragment is closest to the positive electrode?
a.
A
b.
B
c.
E
d.
G
e.
H
24. In the figure above, which lane shows a very large, linear DNA fragment that was cut by one enzyme?
a.
1
b.
2
c.
3
d.
4
e.
5
25. In the figure above, which lane shows a very large, linear DNA fragment that was cut by two
enzymes?
a.
1
b.
2
c.
3
d.
4
e.
5
26. Restriction fragment length polymorphisms are used to
a.
test the effectiveness of different restriction enzymes on a sequence of DNA.
b.
compare the DNA sequences between individuals by looking for changes in restriction
enzyme digest patterns.
c.
compare DNA sequences by determining the full DNA sequence.
d.
prepare DNA for further sequence analysis.
e.
treat sickle-cell anemia.
Use the figure above for the following question(s).
27. Using the information presented in the figure above, where nuclear DNA was digested with MstII,
subjected to gel electrophoresis, and probed in a Southern blot with the indicated probe, which lane
shows an individual that is heterozygous for sickle cell?
a.
1
b.
2
c.
3
d.
4
e.
5
28. Using the information presented in the figure above, where nuclear DNA was digested with MstII,
subjected to gel electrophoresis, and probed in a Southern blot with the indicated probe, which lane
had a DNA sample from a palm tree?
a.
1
b.
2
c.
3
d.
4
e.
5
29. Using the information presented in the figure above (where nuclear DNA was digested with MstII,
subjected to gel electrophoresis, and probed in a Southern blot with the indicated probe), which lane
probably had a DNA sample from a nonhuman source, at least partial homology to the beta-globin
gene, and a different arrangement of MstII restriction enzyme sites than found in humans?
a.
1
b.
2
c.
3
d.
4
e.
5
30. DNA fingerprinting is
a.
a way to identify the species a sample came from.
b.
a way to distinguish between two individuals of the same species.
c.
a method that examines the DNA sequences encoding for the tiny ridges and valleys that
provide texture to the pads of human fingers.
d.
a way of extracting DNA samples from human fingerprints.
e.
a way of determining the sequencing of the human genome.
31. In standardized DNA fingerprinting used in the United States,
a.
13 different noncoding loci are examined.
b.
13 different coding loci are examined.
c.
only loci with short tandem repeats are tested; the number varies with individuals.
d.
the loci with long tandem repeats are tested; the number varies with individuals.
e.
the sequence of short tandem repeats is determined.
32. Genetic engineering has been used to
a.
induce bacteria to mass-produce human insulin.
b.
produce a vaccine against hoof-and-mouth disease.
c.
create mice with altered genomes so we can model certain human diseases.
d.
create plants that are more resistant to certain pests or herbicides.
e.
all of these
33. The main difference between germ-line gene therapy and somatic gene therapy is
a.
only somatic gene therapy can result in the modified genes being passed to the next
generation.
b.
only germ-line gene therapy will have results limited to the current generation.
c.
in germ-line gene therapy, the gametes are altered; in somatic gene therapy, that doesn't
happen.
d.
in somatic gene therapy, the gametes are altered; in germ-line gene therapy, only the body
cells are altered.
e.
only germ-line gene therapy is ethical for research purposes in rodents.
34. Which of the following mammals has NOT been successfully cloned?
a.
sheep
b.
pigs
c.
humans
d.
nonhuman primates
e.
dogs
35. Why might we want to introduce new genes into plants?
a.
to improve their nutritional value to primary consumers
b.
to produce a genetically modified plant
c.
to create plants that can produce pharmaceutical drugs.
d.
to produce plants resistant to certain kinds of damage
e.
all of these
36. Which of the following techniques amplifies a specific sequence of DNA?
a.
PCR
b.
DNA sequencing
c.
Southern blotting
d.
cDNA synthesis
e.
DNA hybridization
37. If a restriction endonuclease cuts a circular DNA three times, how many fragments should there be
after the DNA is subjected to electrophoresis through an agarose gel?
a.
1
b.
2
c.
3
d.
4
e.
5
38. If a restriction endonuclease cuts a linear DNA three times, how many fragments should there be after
the DNA is subjected to electrophoresis through an agarose gel?
a.
1
b.
2
c.
3
d.
4
e.
5
39. Which of the following is a method of introducing genes into plants?
a.
using Ti plasmid and Rhizobium radiobacter bacteria to infect cells with the new genes
b.
creating transgenic plant cells in cultures to produce a callus which then grows into a
transgenic plant
c.
crossing plants with desirable traits with other plants with the same trait for multiple
generations
d.
both using Ti plasmid and Rhizobium radiobacter bacteria to infect cells AND creating
transgenic plant cells to produce a callus that grows into a transgenic plant.
e.
both using Ti plasmid and Rhizobium radiobacter bacteria to infect cells AND crossing
plants with desirable traits with other plants with the same trait for multiple generations.
40. Why is Southern blotting used?
a.
to identify samples with specific DNA sequences
b.
to transfer DNA samples to a special membrane or filter paper
c.
to separate DNA based on size
d.
to amplify a genomic region of interest
e.
to prepare DNA for cloning
41. At which stage is the foreign gene introduced into the developing embryo?
a.
blastocyst, via a transgenic cell
b.
egg, via direct microinjection of the new DNA
c.
sperm, via direct microinjection of the new DNA
d.
in cell culture, prior to fertilization
e.
it all depends on the method the particular researcher prefers.
42. How is the Ti plasmid used to create transgenic plants?
a.
The Ti plasmid allows all of the plant cells to host the bacteria Rhizobium radiobacter and
thus express the foreign gene that was inserted into the bacterial chromosome.
b.
The Ti plasmid inserts the foreign gene directly into the plant's nuclear DNA.
c.
Once the Rhizobium radiobacter forms a tumor in the plant, the tumor cells can take up
the foreign DNA and incorporate it into their chromosomes.
d.
The Ti plasmid inserts the foreign DNA into the DNA in the chloroplast.
e.
The Ti plasmid inserts the foreign DNA into the plant's mitochondrial DNA.
43. Why did scientists turn to genetic engineering for rice to create resistance to blight?
a.
The bacteria causing blight cannot be identified.
b.
Efforts to cross-breed crop rice with blight-resistant rice produced rice unsuitable as a food
source.
c.
It is the cheapest and easiest way to produce blight-resistant rice.
d.
Almost of the crop rice planted is lost each year to blight.
e.
It provided the funding necessary to sequence the rice genome.
44. All of the following are true of gene therapy except
a.
gene therapy could be used to treat inherited disorders.
b.
viruses are used to deliver genes to target cells.
c.
cystic fibrosis has been successfully treated by gene therapy.
d.
gene addition incorporates an additional gene into target cells.
e.
gene correction corrects the gene mutation in the target cells.
45. I generate cDNA libraries for the same cell line under different conditions. When I compare the
libraries, I find some cDNAs in one library that are missing from the other. What is the best
explanation for this result?
a.
One of the cDNA libraries is really a genomic library.
b.
Even the same cell type will change gene expression as conditions change. The difference
in cDNAs tells me which genes are expressed in each set of conditions.
c.
There was an error in generating one of the cDNA libraries and some of the sequences
were not cloned properly.
d.
The probe I used in the Southern blot of the libraries must have been poorly constructed.
e.
There is no explanation other than experimental error.
MATCHING
Match each of the following terms with its correct definition.
a.
This uses DNA technologies to alter genes in a cell or organism.
b.
Bacterial enzymes that recognize and cut specific DNA sequences.
c.
A method of hybridizing labeled DNA to DNA fragments that were previously subjected
to gel electrophoresis.
d.
The products of a restriction endonuclease's action.
e.
A collection of clones that together contain every DNA sequence in a genome, including
non-coding sequences.
f.
The use of a short ssDNA sequence, labeled with some sort of tag, to bind to
complementary DNA and thus identify it.
g.
A collection of clones that together contain a DNA version of every mRNA sequence
expressed in a particular cell type.
h.
This procedure can be described as "a photocopy machine for specific DNA sequences."
i.
DNA from two different sources that have been joined together into a single molecule.
j.
This term is used to describe organisms that have been subject to genetic engineering.
k.
This technique is used to separate DNA molecules based on their relative sizes.
l.
A method of identifying individuals based on their individual patterns of short tandem
repeats located on certain loci of their genome.
46. genetic engineering
47. recombinant DNA
48. restriction endonucleases
49. restriction fragments
50. genomic library
51. DNA hybridization
52. cDNA library
53. PCR
54. agarose gel electrophoresis
55. Southern blot
56. DNA fingerprinting
57. transgenic
Classification
Use the processes listed below for the following question(s).
a.
DNA library
b.
Restriction enzyme digest
c.
Gene cloning
d.
Southern blotting
e.
PCR
f.
DNA fingerprinting
g.
Transgenics
58. You'd use this tool if you wanted to check and see if a particular gene was contained in an organism's
genome.
59. This technique will let you move a gene into a vector for expression in different cells.
60. You could use this technique to see if a DNA sequence is present in an individual's genome without
having to use Sanger sequencing.
61. This technique is used to amplify specific sequences of DNA.
62. You'd use this to cut DNA at a selected site.
63. How can you mass-produce a gene in a bacterial cell that doesn't normally have this gene?
64. You use this method determine the presence or absence of particular 20 bp nucleotide pattern in a
noncoding region of a genome.
65. You want to generate mice that express a fluorescent protein in every cell. What methodology would
be used?
66. What technique can be used to determine if a suspect is likely guilty of a crime?
SHORT ANSWER
67. Define the purpose of cloning a gene into a plasmid cloning vector.
68. Explain the ethical concerns with germ-line gene therapy in humans.
69. What is the major difference between a genomic library and a cDNA library?
70. What is DNA hybridization?
71. If the DNA from a crime scene matches a suspect’s DNA at four different loci, what should be
concluded if the DNA does not match the suspect’s DNA at a fifth locus?
MODIFIED TRUE/FALSE
If the statement is true, answer "T". If the statement is false, answer "F" and make it correct by
changing the underlined word(s) and writing the correct word(s) in the answer blank(s).
72. DNA fingerprinting is limited to human studies.
73. There are often different methodologies that can be used to obtain the same information about a DNA
sequence.
74. Gene therapy is an available treatment for numerous genetic disorders.
75. Gene therapy has been successful in curing some individuals of autoimmune disorder and sickle cell
disease.
76. Genetically modified crops have been banned in the United States.
77. Some genetically modified tobacco plants can express the light-producing chemicals present in
fireflies.
78. When using embryonic germ-line cells to create transgenic mice, the offspring of the genetically
modified animal will have all transgenic cells in their bodies.
79. Blight-resistant crop rice has been tested and found to be nutritionally equivalent to the non-
genetically modified crop rice.
80. Shotgun sequencing is the method of choice for generating whole genome sequences.
81. Open Reading Frames include the start and stop codons for a protein.
ESSAY
82. Choose one molecular biology technique discussed in this chapter and argue why it is the single most
important technique for forensic scientists. Justify your answer with the benefits of this method
compared to the others that are available to you.
83. What is a GMO and what are two major concerns about these organisms?
84. What is the difference between a selection and a screen with regard to cloning genes into plasmids and
transforming them into host cells?
