MICRO exam 2 chp 17
Rank the following steps in the correct order to describe the process of seamless cloning. 1. DNA to be inserted is amplified by PCR and contains 5' sequences that match the vector's junction site 2. Vector is linearized and mixed with the amplified DNA 3. T5 exonuclease cleaves the 5' ends of all fragments leaving 3' sticky ends 4. The vector and amplified DNA hybridize at their complementary 3' sticky ends 5. DNA polymerase extends the 3' ends of the vector and inserted DNA strands 6. DNA ligase seals the gaps between vector and insert to close the recombinant circular molecule
231546
A microbiologist would like to use a noncompetent genus of streptococcal bacteria, Enterococcus faecalis, as a cloning host to express genes from Streptococcus pneumoniae, which is naturally competent. Is this possible? A) Yes; electroporation or chemical transformation can be used to make Enterococcus competent, and then genes from Streptococcus can be introduced via a cloning vector. B) Yes; electroporation or chemical transformation can be used to make noncompetent Streptococcus mutants, from which genes can be inserted into a cloning vector and introduced into Enterococcus. C) No; competence factor is an essential protein that enables the uptake of foreign DNA, therefore a cloning host such as Enterococcus that lacks competence factor protein is unable to be transformed by electroporation or chemical transformation. D) Yes; since the cloning host and the DNA to be introduced are both from bacteria that are streptococci, natural competence in this case is not necessary as long as the appropriate vector is used.
a
A molecular biologist treats a 600-bp length of linear DNA with HindIII restriction endonuclease. Following gel electrophoresis, the biologist observes that there is only one band on the gel and it corresponds with the migration distance of the 200-bp molecular weight marker. Assuming that no procedural errors were made, what can be concluded from these results? A) Two HindIII recognition sequences were present in the original DNA. B) Three HindIII recognition sequences were present in the original DNA. C) There were no HindIII recognition sequences present in the original DNA. D) One HindIII recognition sequence was present in the original DNA.
a
A(n) ________ vector is a plasmid that can be replicated in several different organisms because it has at least one origin of replication that will function in each host. A) shuttle B) chimeric C) expression D) phage
a
Antibiotics incorporated into the culture medium can ________. A) select against organisms that have not incorporated the plasmid B) select against organisms that have incorporated a plasmid not containing the desired gene C) enhance production of recombinant proteins D) select against organisms that have not incorporated the plasmid and select against organisms that have incorporated a plasmid not containing the desired gene
a
As a newly hired molecular biologist for a company that produces genetically modified seeds, your first project is to ensure that a GM cotton plant carrying a bacterial insecticide gene is expressed in tissues of seedlings as well as mature plants. To better understand regulation of the gene, you decide to use ________ GFP fusion to detect activity of the promoter and also use ________ GFP fusion to determine the location of the protein in plants tissues at various stages of growth. A) transcriptional; translational B) translational; transcriptional C) transcriptional; transcriptional D) translational; translational
a
Cas9 endonuclease differs from a restriction enzyme in that ________. A) restriction enzymes recognize a 4-8bp target sequence in the DNA and directly cleave at that site, whereas Cas9 contains a guide RNA that is complementary to a specific DNA site and a polypeptide that cleaves once hybridization is complete B) Cas9 recognizes a 4-8bp target sequence in the DNA and directly cleave at that site, whereas restriction enzymes contain a guide RNA that is complementary to a specific DNA site and a polypeptide that cleaves once hybridization is complete C) restriction enzymes recognize 4-8bp sequence of the DNA, whereas the amino acids in Cas9 recognizes sequences that are approximately 20bp in length D) restriction enzymes can cleave any loci in the DNA molecule, whereas Cas9 can only cleave DNA fragments that originated as mobile genetic elements such as phages and transposons
a
During the investigation of a convenience store robbery, witnesses report that the perpetrator exited through a main entry door without wearing gloves. The police are holding a prime suspect in custody while samples from the door are collected and analyzed for a DNA match. Since the door was handled by many people before the robbery, how will the forensics department distinguish the suspect's DNA from the DNA of others using PCR? A) Sequence-specific primers to the suspect's DNA will be used for PCR amplification. B) Sequence-specific primers to the suspect's DNA will be added following PCR amplification. C) PCR will be used to make sequence-specific primers that are complementary to the suspect's DNA. D) Since multiple people touched the doorknob without gloves, it is impossible to use PCR to distinguish the suspect's DNA.
a
I am a ribonucleoprotein; I contain both a polypeptide and an RNA molecule. My RNA binds to unique sequences on a DNA molecule, targeting them for cleavage by my polypeptide. I occur naturally in microbes to protect against viral attack, and I can be engineered in the lab for precision DNA editing. What am I? A) Cas9 nuclease B) EcoR1 restriction enzyme C) RNA polymerase D) 3' oligonucleotide
a
In order to express eukaryotic genes in a bacterium, the ________ must first be removed. A) introns B) exons C) enhancers D) 3' poly A sequence
a
Plasmid DNA having one EcoRI recognition sequence is treated with EcoRI restriction endonuclease. Following gel electrophoresis, how many bands should be visible on the gel? A) One B) Two C) Four D) Zero
a
Restriction endonucleases were discovered by ________. A) Arber and Smith B) Jackson, Symons, and Berg C) Boyer and Cohen D) Temin and Baltimore
a
Sputum from a patient with a history of tuberculosis due to Mycobacterium tuberculosis bacteria is collected and sent to the microbiology lab for analysis. X-ray analysis and an acid-fast smear made from the sputum indicate that the patient has active tuberculosis. The physician has requested that the sputum be analyzed for pathogen load, but because colonies of Mycobacterium typically take between 3 to 8 weeks to appear on agar, results from traditional culture methods are often delayed. If you were the microbiologist in this case, what would you do? A) Using the sputum, apply real-time PCR with primers to Mycobacterium tuberculosis DNA to estimate the pathogen load, then follow up with a traditional culture. B) Using the sputum, apply end-point PCR with primers to Mycobacterium tuberculosis DNA to estimate the pathogen load, then follow up with a traditional culture. C) Set up a traditional culture on the sputum, then use the colonies for real-time PCR with primers to Mycobacterium tuberculosis DNA to quantify the pathogen load. D) Set up a traditional culture on the sputum, then use the colonies for end-point PCR with primers to Mycobacterium tuberculosis DNA to quantify the pathogen load.
a
The Cas9 nuclease is able to cleave target DNA at a single precise site by ________. A) using a guide RNA that hybridizes to a unique sequence on the target DNA molecule B) recognizing a palindromic sequence on the DNA and cleaving at that site leaving sticky ends C) associating with a single-stranded DNA molecule that binds to its complement on the genome to be cleaved D) altering its active site to bind to and cleave any DNA sequence that is targeted
a
The advantage of homologous recombination is that ________. A) following a splice with Cas9, the ends of the chromosome can be seamlessly joined with a piece of donor DNA that has been engineering to contain flanking sequences homologous to the broken chromosome ends B) following a splice with a restriction enzyme, the sticky ends can be combined with a piece of donor DNA that has the same sticky ends C) following a splice with Cas9, the ends are joined back together with the induction of a frameshift mutation resulting in an inactive protein D) genes from two different sources can be joined together when the wild-types contain homology between the two molecules, allowing DNA ligase to join the naturally-occurring sticky ends
a
When separating DNA fragments by gel electrophoresis, what is the purpose of including molecular weight markers? A) Markers are used as a control to determine the relative size of restriction fragments. B) Markers provide an indication as to the total number of restriction fragments on the gel. C) Markers are needed to estimate the relative charges on each of the restriction fragments. D) Markers ensure that fragments having a greater molecular weight migrate at the same rate as those having a lighter molecular weight.
a
Which cloning vector should be used to express a 500-kb DNA fragment in Saccharomyces cerevisiae? A) YAC B) BAC C) pUC19 D) λ1059
a
Which is most analogous to the role of GFP in recombinant DNA technology? A) Using dyes to stain and detect bacteria under light microscopy from the sputum of a patient diagnosed with tuberculosis. B) Comparing the RFLP patterns of DNA from various suspects to DNA isolated from the scene of a crime using gel electrophoresis. C) Creating a metagenomic library representing archaea bacteria that thrive in the hot springs of Yellowstone National Park. D) Monitoring the spread of multi-drug resistant pathogens on a hospital floor by comparing antibiotic susceptibility patterns.
a
Which of the following PCR procedures includes all of the others? A) DNA is amplified for one cycle. B) DNA is denatured at 95oC. C) DNA is reannealed at 50oC D) Primers are extended at 72oC.
a
Which of the following bacterial hosts should be used to avoid degradation of DNA that is introduced via a cloning vector? A) Escherichia coli that lack endonucleases B) RecA-expressing Escherchia coli C) Escherchia coli RecA mutants D) Saccharomyces cerevisiae wild type cells
a
Which of the following components of PCR ensures amplification of the target DNA sequence only, despite the presence of many other DNA sequences? A) Primers B) Polymerases C) dNTPs D) High temperatures
a
Which of the following is not part of a yeast artificial chromosome (YAC)? A) The F factor B) A selectable marker C) An ARS D) A CEN sequence
a
Which of the following statements is false regarding the process of seamless cloning? A) The 5' ends of the oligonucleotide primers contain a recognition site for a restriction enzyme that allows sticky ends to form between the inserted DNA and the vector. B) The 5' ends of the oligonucleotide primers are specific to nucleotides at the desired junction site of the vector. C) Following PCR, the generated insert DNA is mixed with the cleaved vector and an exonuclease is added to degrade the 5' ends of all strands. D) 3' sticky ends on both the insert and the vector hybridize, and DNA polymerase and DNA ligase complete the circular recombinant molecule.
a
Which of the following statements is true regarding the use of oligonucleotide primers in PCR? A) Only a portion of the oligonucleotide primers need to hybridize to the 3' template DNA strand; the 5' "overhang" can contain a restriction site that will be copied during the second and all subsequent cycles of PCR. B) Oligonucleotide primers must hybridize completely to the template DNA strand in order for PCR to proceed; nonhybridized DNA will be degraded. C) The use of oligonucleotide primers is obsolete with the Gibson method of seamless cloning; instead, DNA polymerase and DNA ligase are able to copy the DNA independently. D) Oligonucleotide primers must hybridize to the 5' end of the template strand leaving a 3' primer "overhang"; that sequence will be copied and incorporated into the daughter strands allowing seamless cloning to occur.
a
Which of the following techniques exemplifies phenotypic rescue in a host cell? A) Introducing a genomic library from a cell that produces a particular gene product into a mutant host cell auxotroph that cannot synthesize the product. B) Introducing a genomic library from a mutant cell auxotroph for a particular gene product into a host cell that can synthesize that product constitutively. C) Removing genes that encode a particular gene product from a cell that expresses that product constitutively before creating a genomic library. D) Replacing a mutant gene with a functional gene in a DNA library from an auxotrophic mutant cell prior to introducing the library into a related host cell.
a
Which of the following terms is most closely related to genetic complementation? A) Phenotypic rescue B) Naked DNA C) Reverse transcription D) SOS response
a
Which of the following types of cloning vector can carry the largest amount of foreign DNA? A) Bacterial artificial chromosome B) Bacteriophage C) Cosmid D) Plasmid
a
You and a friend are student assistants in a research laboratory that investigates the anticancer properties of proteins isolated from marine organisms. Your friend mentions that she is using a BAC vector to insert shark DNA into Escherichia coli, but after repeated attempts, has found that the bacterial cells fail to synthesize the encoded protein. What is your advice? A) If the shark DNA is unmodified, it contains introns that are not recognized by bacteria, therefore protein synthesis will not occur. B) Since shark DNA is eukaryotic, the cloning vector should be a YAC derived from yeast rather than a BAC derived from bacteria. C) It is impossible to clone eukaryotic DNA into a bacterial host, since eukaryotic DNA has introns and prokaryotic DNA does not. D) Because Escherichia coli is not naturally competent, it cannot serve as the cloning host for foreign DNA.
a
The production of large quantities of a particular DNA sequence is known as gene ________.
amplification
A(n)________ ________ is a piece of DNA with all of the features necessary for chromosomal replication and that can carry large (up to 1000 kb) pieces of foreign DNA into a host organism
artificial chromosome
) Which of the following is not true of cloning vectors? A) They usually contain multicloning sites or polylinkers. B) They contain at least two replication origins. C) They can be replicated within an appropriate host. D) All of these choices are correct.
b
A DNA molecule used to carry a foreign gene into a host organism is called a ________. A) plasmid B) vector C) probe D) blot
b
An enzyme that cleaves internal phosphodiester bonds of a DNA molecule is a(n) ________. A) exonuclease B) endonuclease C) ligase D) methylase
b
If DNA were a positively charged rather than negatively charged, what change to the gel electrophoresis procedure must be made? A) Migration time of restriction fragments must be increased. B) DNA must be loaded at the positive pole rather than at the negative pole. C) Type of buffer used must be basic rather than neutral. D) All of the choices are correct.
b
Plasmid cloning vector DNA is usually introduced into bacterial hosts by ________. A) ligation B) transformation C) transduction D) plasmolysis
b
Plasmid vectors often contain ________ genes that can be used to screen for recombinants. A) metabolic activation B) antibiotic resistance C) insertion sequence D) promoter/operator
b
Recombinant DNA technology does not rely on which of the following enzymes? A) Restriction endonucleases B) RNA methylase C) DNA ligase D) Reverse transcriptase
b
Restriction endonucleases are produced by ________. A) fungi B) bacteria C) protozoa D) plants E) All of the choices are correct
b
Restriction endonucleases in bacteria may have evolved in order to ________. A) carry out natural genetic engineering B) protect the bacteria from infection by viruses C) use nucleic acids as a food (energy) source D) All of the choices are correct.
b
The three steps that take place in each cycle during PCR occur in which order? A) DNA annealing, denaturation, and synthesis B) DNA denaturation, annealing, and synthesis C) DNA synthesis, denaturation, and annealing D) DNA denaturation, synthesis, and annealing
b
When a eukaryotic gene is cloned into a bacterium, the advantage of a complementary DNA (cDNA) gene being used instead of fragments of genomic DNA is that ________. A) the promoter and terminator are found in the cDNA gene but not in the genomic fragment B) the introns have been removed from the cDNA gene but not from the genomic fragment C) the cDNA is made with the nucleotides found in the prokaryote but not in the eukaryote D) There is no advantage to using a cDNA gene rather than a genomic fragment
b
A PCR procedure that allows a determination of the amount of a particular DNA fragment that is present in a sample is called ________. A) quantitative PCR B) analytical PCR C) real-time PCR D) reverse PCR
c
A ________ is a DNA molecule used in hybridization reactions to detect the presence of a particular gene in separated DNA fragments. A) plasmid B) vector C) probe D) blot
c
A(n) ________ vector contains promoters that result in high-level transcription of the gene cloned within a multicloning site. A) shuttle B) chimeric C) expression D) phage
c
Cloning a gene involves all of the following EXCEPT ________. A) isolating the fragment of DNA containing the desired gene B) insertion of the gene into an appropriate vector C) expression of the vector and the gene in a cell-free environment D) introducing ligated DNA into E. coli cells
c
Movement of charged molecules in an electrical field, which is used to separate nucleic acid fragments for recombinant DNA work, is called ________. A) iontophoresis B) nucleophoresis C) electrophoresis D) plasmaphoresis
c
Which is a true statement regarding the size of PCR products? A) Since the number of DNA products extends beyond each primer with each cycle, when PCR is completed, the majority of products are of various sizes. B) Since the length of primers increases with each cycle, when PCR is completed, the majority of products are of various sizes. C) Since the number of DNA products ending exactly between both primers increases with each cycle, when PCR is completed, the majority of products are of similar size. D) None of the choices are correct.
c
Which of the following is true about restriction endonucleases? A) They make a blunt cut on the two DNA strands so that there are no single-strand regions. B) They make staggered cuts on the DNA so that single-strand ends are formed that can be used to insert foreign DNA cut with the same enzyme. C) Some make a blunt cut on the two DNA strands so that there are no single-strand regions and some make staggered cuts on the DNA so that single-strand ends are formed that can be used to insert foreign DNA cut with the same enzyme. D) Depending on the incubation conditions, the same enzyme can either make a blunt cut on the two DNA strands so that there are no single-strand regions OR make staggered cuts on the DNA so that single-strand ends are formed that can be used to insert foreign DNA cut with the same enzyme
c
Which procedure is most useful in quantifying the active transcription of botulism toxin genes in a can of food that is contaminated? A) Southern blot B) End-point PCR C) Real-time PCR D) DNA hybridization
c
Which situation(s) might warrant the removal of a histidine tag when purifying a recombinant protein? A) Histidine residues bind preferentially to nickel resins on the column during purification, and are not washed away. B) The expression vector used for inserting the gene of interest already contains histidineencoding sequences. C) Histidine residues inhibit protein folding, thereby decreasing the functionality of the protein. D) All of the choices are correct.
c
is a bacterial plasmid vector. A) Lambda B) T4 DNA ligase C) pUC19 D) SV40
c
A molecular biologist is interested in purifying a recombinant protein by His-tagging, but the protein and vector both lack histidine residues. In this case, the molecular biologist could use which of the following technique(s) to achieve purification? A) Add a 6xHis-tag to the C-terminus of the protein B) Add a series of histidine residues to the N-terminus of the protein C) Amplify the histidine-encoding sequence by PCR and add it to the gene of interest D) All of the choices are correct.
d
Complementary DNA (cDNA) probes are produced using ________. A) restriction endonucleases B) RNA polymerase C) DNA ligase D) reverse transcriptase
d
The enzyme reverse transcriptase was discovered by ________. A) Arber and Smith B) Jackson, Symons, and Berg C) Boyer and Cohen D) Temin and Baltimore
d
The polymerase chain reaction (PCR) can be used to produce ________ of copies in a few hours. A) hundreds B) thousands C) millions D) billions
d
Which is the most frequently chosen prokaryotic host for use in cloning techniques? A) Saccharomyces cerevisiae B) Bacillus subtilis C) Staphylococcus aureus D) Escherichia coli
d
Which of the following best describes the basis for separation of DNA fragments during agarose gel electrophoresis? A) The fragments with the highest percentage of G and C will migrate fastest. B) The fragments with the highest percentage of A and T will migrate fastest. C) The largest fragments will migrate fastest. D) The smallest fragments will migrate fastest.
d
Which of the following can be used as vectors for cloning DNA fragments? A) Plasmids B) Cosmids C) Bacteriophages D) All of the choices are correct.
d
Which of the following was first produced commercially using recombinant DNA technology? A) Human growth hormone B) Interleukins C) Hepatitis B vaccine D) Human insulin
d
In ________, cells are mixed with recombinant DNA and exposed to a brief pulse of highvoltage electricity to cause the membrane to become permeable and allow the uptake of DNA from its environment.
electroporation
A researcher wants to cleave the DNA of a target organism at a single, precise location in the genome. The best way to achieve this is to select a restriction enzyme that recognizes a 4-8 bp palindromic sequence on the double-stranded DNA.
false
Cosmids are so named because they can be used to express foreign genes in a variety of different hosts.
false
If all of the PCR products of a DNA sequence are 4000 bp, then the DNA sequence that was amplified must have been 8000 bp.
false
Promoters for genes that code for proteins can be isolated from a cDNA library.
false
The difference between nonhomologous end joining (NHEJ) and homologous recombination is that NHEJ introduces a piece of donor DNA with seamless integration between the chromosome and donor DNA, whereas homologous recombination introduces a frameshift mutation when the repair is made between the broken chromosome ends.
false
The thermostable enzyme most commonly used in PCR is reverse transcriptase.
false
Transposons are frequently used as cloning vectors.
false
When a eukaryotic gene is expressed in a bacterium, the eukaryotic regulatory sequences should be maintained in order to achieve maximum expression of the gene.
false
A genomic ________ is a sufficiently large collection of recombinant DNA molecules in which the inserted sequences together represent the entire genome of an organism.
library
A microbial ecologist who is interested in studying how gene expression is influenced by different species of bacteria in a biofilm would most likely find a metagenomic library more useful than a genomic library
true
Cosmids are plasmids that can be packaged into capsids of the bacteriophage lambda; therefore, they can be transmitted like phages, but they can exist and replicate in a cell like plasmids.
true
Electroporation is commonly used to introduce recombinant DNA molecules into cells.
true
Genetic engineering methods have been used to produce vaccines
true
If all of the restriction fragments in a DNA digest were of very low molecular weight, then all of the fragments on the electrophoretic gel will be closest to the positive pole and furthest from the negative pole.
true
Oligonucleotide primers require only a portion of the sequence to hybridize to the template DNA; the non-hybridized 5' end of the primer can be engineered to contain a recognition sequence for a restriction endonuclease that will be incorporated into the double-stranded daughter DNA after the second cycle.
true
One of the major advantages to using plasmids as cloning vectors is that very high copy numbers can be achieved with many types of plasmid vectors.
true
Regardless of the exact approach taken to recombinant DNA technology, one of the keys to successful cloning is choosing the right vector.
true
Some plasmid vectors have incorporated the regulatory sequences of the lactose operon so that the expression of the recombinant gene can be induced at the appropriate time.
true
