Genetics Chapter 20
5) One of the primary reasons for generating a large number of clones in a eukaryotic genomic library is that ________. A) each cosmid replicates in coordination with the host chromosome B) lysogenic phages continue to integrate their DNA into the host chromosome, thus reducing the number of desired recombinant clones C) each vector can take up only a relatively small fraction of the eukaryotic DNA D) each ligation product is sequence specific E) the host range of the vector is limited
C Section: 20.1
20) What is meant by the designation EcoRI?
one of the first restriction enzymes isolated from E. coli Section: 20.1
22) What term is used to refer to the process in which DNA can be introduced into host bacterial cells?
transformation (or transfection in some cases) Section: 20.1
15) Assume that a given plasmid vector to be used in a cloning experiment contains 4000 base pairs of DNA. Assume also that the restriction endonuclease Cuj cuts this plasmid at the following sites (starting from an arbitrary zero point): 1000, 1500, and 3000. Given complete digestion of the plasmid with the endonuclease so that only linear fragments are produced, what sizes of DNA are expected?
500 bp, 1500 bp, and 2000 bp Section: 20.1
2) Restriction endonucleases are especially useful if they generate "sticky" ends. What makes an end sticky? A) single-stranded complementary tails B) blunt ends C) poly-A sequences D) 5′ cap E) interference
A Section: 20.1
3) List two especially useful characteristics of cloning vectors. A) high copy number and antibiotic resistance gene(s) B) virulence and lysogenicity C) ability to integrate into the host chromosome and then causing a lytic cycle D) nonautonomous replication and transposition E) reverse transcriptase and ligase activities
A Section: 20.1
7) In the context of molecular genetics, reverse transcription PCR (RT-PCR) refers to ________. A) assembling a DNA sequence from an mRNA B) assembling an RNA sequence from a DNA sequence C) translating in the 3′ to 5′ direction D) transcribing first, then translating E) making an amino acid sequence from a DNA sequence
A Section: 20.3
9) Nucleic acid blotting is widely used in recombinant DNA technology. In a Southern blot, one generally ________. A) hybridizes filter-bound DNA with a DNA probe B) hybridizes filter-bound RNA with a DNA probe C) examines amino acid substitutions with radioactive probes D) cleaves RNA with restriction endonucleases E) ligates DNA with DNA ligase
A Section: 20.4
11) A ddNTP, used often in DNA sequencing, lacks a(n) ________ at the ________ and ________ carbons. A) OH; 2′; 3′ B) methyl; 2′; 3′ C) carboxyl; 5′; 3′ D) OH; 2′; 5′ E) None of the above is correct.
A Section: 20.5
29) What is a cDNA molecule?
A cDNA molecule is a DNA copy of an RNA molecule. Section: 20.2
38) What method might be used to study a knockout mouse that, by virtue of the lost gene or genes, generates a lethal condition?
A permissive environment involving a conditional knockout can sometimes be used to allow the organism to survive a critical period of development. Section: 20.6
13) In the context of recombinant DNA technology, what is meant by the term vector?
A vector is a vehicle to carry recombinant DNA molecules into the host cells where independent replication can occur. Most common vectors are plasmids, bacteriophages, and cosmids. Section: 20.1
28) Under ideal conditions, how many copies of all the sequences of the host genome should be represented in a genomic library?
At least one should be represented. Typically, library construction includes a several-fold greater number of clones than necessary for one representative of each fragment in order to increase the likelihood of cloning difficult fragments and stochastic loss. Section: 20.2
10) In which of the following biochemical reactions is it common to use ddNTPs (dideoxyribonucleoside triphosphates)? A) citric acid cycle B) DNA sequencing C) restriction digestion D) electron transport E) plasmolysis
B Section: 20.5
14) Molecular biologists rely on many, often sophisticated, techniques to pursue their discipline. One may list ultracentrifugation, electron microscopy, X-ray diffraction, electrophoresis, and computer interfacing as fundamental tools. Model organisms provide the raw materials for study. List three "organisms" (or organismic groups) often used by recombinant DNA technologists and describe a major advantage of each group.
Bacteriophage: useful vector, relatively simple genome, short generation time. Bacteria: relatively simple, short generation time, simple growth requirements, well understood genetics, transformable. Viruses: capable of carrying recombinant DNA and infecting eukaryotic cells. Yeast: relatively simple for a eukaryote, short generation time, simple growth requirements, transformable. Section: 20.1
1) Words such as level, civic, and kayak have something in common compared to the fundamental tool of recombinant DNA technology. In the context of recombinant DNA technology, which term would be used to describe such words? A) lysogenic B) prototrophic C) palindromic D) conjugation E) insertion
C Section: 20.1
4) Some vectors such as pUC18 and others of the pUC series contain a large number of restriction enzyme sites clustered in one region. Which term is given to this advantageous arrangement of restriction sites? A) palindrome B) consensus sequence C) multiple cloning site D) β-galactosidase E) complementation
C Section: 20.1
8) The PCR (polymerase chain reaction) protocol that is currently used in laboratories was facilitated by the discovery of a bacterium called Thermus aquaticus in a hot spring inside Yellowstone National Park, in Wyoming. This organism contains a heat-stable form of DNA polymerase known as Taq polymerase, which continues to function even after it has been heated to 95oC. Why would such a heat-stable polymerase be beneficial in PCR? A) Each cycle includes a "hot" saturation phase (95°C), which allows the primers to anneal to the target DNA. B) Each cycle includes a "hot" denaturation phase (95°C), which serves to sterilize the culture. C) Each cycle includes a "hot" denaturation phase (95°C), which activates the Taq polymerase. D) Each cycle includes a "hot" denaturation phase (95°C), which separates the hydrogen bonds that hold the strands of the template DNA together. E) More than one of the above are correct.
D Section: 20.3
6) Assume that a plasmid (circular) is 3200 base pairs in length and has restriction sites at the following locations: 400, 700, 1400, 2600. Give the expected sizes of the restriction fragments following complete digestion. A) 400, 800, 1000 (2 of these) B) 400, 1200, 1600 C) 300, 700, 2200 D) 700, 400, 1400, 2600 E) 300, 700, 1000, 1200
E Section: 20.1
41) In general, the main goal of cloning is to include as many different genes as possible in a single cloning vector.
FALSE Section: 20.1
42) To isolate a bacterium with a plasmid that carries a desired DNA fragment cloned within the ampicillin resistance gene, we should grow bacteria in a medium that contains ampicillin.
FALSE Section: 20.1
44) In recombinant DNA technology, a YAC is an enzyme isolated from a large South American, four-legged mammal.
FALSE Section: 20.1
45) E. coli is a common YAC.
FALSE Section: 20.1
46) In recombinant DNA technology, YAC, RFLP, and λ have identical uses.
FALSE Section: 20.1
48) Reverse transcriptase is often used as the heat-stable enzyme in PCR.
FALSE Section: 20.3
49) In a typical PCR, primers are used to cleave specific regions of the DNA template.
FALSE Section: 20.3
50) During a PCR, heat is provided to inactivate the polymerase enzyme.
FALSE Section: 20.3
52) The main purpose of a probe is its insertion in plasmid DNA.
FALSE Section: 20.4
53) The function of a ddNTP in DNA sequencing is to methylate guanine.
FALSE Section: 20.5
54) A knockout animal, in the context of recombinant DNA technology, is one that has been completely anesthetized.
FALSE Section: 20.6
34) Nucleic acid blotting is commonly used in molecular biology. Two types, Southern blots and northern blots, involve gel electrophoresis and a filter, which holds the nucleic acid. Briefly describe the procedure of "blotting" in this context and differentiate between Southern and northern blots.
In a Southern blot, the DNA to be "probed" is cut with a restriction enzyme(s); then the fragments are separated by gel electrophoresis. Alkali treatment of the gel denatures the DNA, which is then "blotted" onto the filter (nylon or nitrocellulose). A labeled probe (RNA or DNA) is then hybridized to complementary fragments on the filter. In a northern blot, RNA is separated in the gel and "probed" with the labeled DNA. Section: 20.4
19) What might be a reasonable function of restriction endonucleases in a bacterium, distinct from their use by molecular biologists?
Isolated from bacteria, restriction endonucleases restrict or prevent viral infection by degrading the invading nucleic acid of the virus. Section: 20.1
33) In what way are specific DNA sequences of the template amplified in the polymerase chain reaction? In other words, how does one target the target?
Oligonucleotide primers associate by hydrogen bonding to specific sections; primers are then extended. Section: 20.3
12) What is recombinant DNA technology? What would you list as safety issues associated with recombinant DNA technology?
Recombinant DNA technology refers to the creation of new combinations of DNA molecules that are not normally found in nature. Safety issues generally center on the creation and release (accidental or intentional) of genetically engineered organisms that are a threat to human health or to animals and plants in the environment. Many organisms that are "genetically engineered" carry genes for antibiotic resistance. Section: 20.1
26) If one wishes to clone a gene using typical restriction endonucleases, how does the restriction endonuclease recognize genes in the genome?
Restriction endonucleases do not recognize functional regions in the genome (genes). They can recognize only relatively short DNA sequences, which have no relationship to functionality. Section: 20.1
35) Assume that you have cut λ DNA with the restriction enzyme HindIII. You separate the fragments on an agarose gel and stain the DNA with ethidium bromide. You notice that the intensity of the stain is less in the bands that have migrated closer to the "+" pole. Give an explanation for this finding.
Since the smaller fragments migrate toward the "+" pole, away from the origin, they bind less stain than the larger fragments near the origin. Section: 20.4
39) A common term for a plasmid or other DNA element that serves as a cloning vehicle is vector.
TRUE Section: 20.1
40) Restriction endonucleases typically recognize palindromic DNA sequences and often generate "sticky ends" or single-stranded DNA overhangs at cut sites.
TRUE Section: 20.1
43) Some restriction endonucleases are capable of producing blunt ends; others can generate "sticky" ends.
TRUE Section: 20.1
47) A restriction map provides the location of sites cleaved by restriction enzymes.
TRUE Section: 20.1
51) In a PCR, primers are complementary to stretches of DNA with which they anneal.
TRUE Section: 20.3
16) Some restriction enzymes cleave DNA in such a manner as to produce blunt ends. Most often ligation of blunt end fragments is enhanced by the use of the enzyme terminal deoxynucleotidyl transferase. Speculate on the function of deoxynucleotidyl transferase in terms of using blunt end fragments in cloning.
Terminal deoxynucleotidyl transferase extends single-stranded ends by the addition of nucleotide tails. If complementary tails are added, the fragments can hybridize and the recombinant molecules can be ligated. Section: 20.1
36) What is the fundamental purpose of creating a knockout organism?
The idea is to disrupt or eliminate a specific gene or genes and then assay the physiological and/or anatomical outcome. Section: 20.6
32) In the polymerase chain reaction, what is the purpose of the initial high temperature? What is the purpose of cooling in the second step?
The purpose is to denature the target (template) DNA, annealing the primer to the target. Section: 20.3
37) What is a popular approach that is often used to introduce the targeting vector into cells?
Using electroporation, the vector is often introduced into embryonic stem cells. Section: 20.6
27) Following are four processes common to most cloning experiments: a) transforming bacteria b) plating bacteria on selective medium c) cutting DNA with restriction endonucleases d) ligating DNA fragments Place components of this list in the order in which they would most likely occur during a cloning experiment.
cutting DNA with restriction endonucleases, ligating DNA fragments, transforming bacteria, plating bacteria on selective medium Section: 20.1
18) List, in order, the steps usually followed in producing recombinant DNA molecules in a plasmid vector.
isolation of DNA (foreign and plasmid), digestion of DNAs with an appropriate restriction endonuclease, ligation of fragments, transformation of host cells Section: 20.1
25) When propagating a clone in the lambda phage, would you have more immediate success if the phage entered the lysogenic or the lytic cycle?
lytic cycle Section: 20.1
31) What is the name of the process by which bacterial colonies (cells) are transferred from one agar plate to another, maintaining the same spatial pattern?
replica plating Section: 20.2
17) Over the years, sophisticated plasmid vectors have been developed for use in recombinant DNA technology. List at least two features that have been introduced in particularly useful vectors.
small size to allow large inserts, high copy number, large numbers of unique restriction sites (multiple cloning sites), variety of selection schemes (pigmented colonies, antibiotic resistance) Section: 20.1
21) Name at least two typical characteristics of a DNA cloning plasmid?
small size, high copy number, multiple cloning site in a selectable marker Section: 20.1
30) What is the specific application of reverse transcriptase in the preparation of cDNA?
synthesis of DNA to form an RNA-DNA duplex Section: 20.2
24) Assume that one conducted a typical cloning experiment using a typical plasmid, transformed an appropriate host bacterial strain, and plated the bacteria on an appropriate X-gal medium. Blue and white colonies appeared. Which of the two types of colonies, blue or white, would most likely contain the recombinant plasmid?
the white colonies because of insertional activation of the lacZ component Section: 20.1
23) Of what advantage is it to have a multiple cloning site (multiple unique restriction sites) embedded in the lacZ component of a plasmid?
An insert of DNA in the multiple cloning site inactivates the lacZ component and allows identification of recombinant plasmids under proper genetic and environmental conditions. Section: 20.1
