Rucete ✏ AP Biology In a Nutshell
16. Biotechnology — Practice Questions 3
This chapter explores the methods and applications of modern biotechnology, including gene cloning, CRISPR editing, bacterial transformation, PCR amplification, and DNA analysis using gel electrophoresis.
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(Multiple Choice — Click to Reveal Answer)
1. A scientist uses a restriction enzyme to cut DNA and then uses DNA ligase to combine it with a plasmid. What is this process called?
(A) RNA editing
(B) Recombinant DNA technology
(C) Polymerase chain reaction
(D) DNA sequencing
Answer
(B) — Recombinant DNA technology involves combining DNA from two sources using restriction enzymes and DNA ligase.
2. In PCR, what determines the specificity of the DNA fragment that is amplified?
(A) The type of polymerase used
(B) The length of the DNA template
(C) The primers used in the reaction
(D) The number of thermal cycles
Answer
(C) — Primers bind to specific sequences flanking the region of interest, dictating which segment of DNA is amplified.
3. Why is it important that a plasmid used for cloning has an origin of replication?
(A) To attach RNA polymerase
(B) To ensure the plasmid replicates in the host cell
(C) To prevent plasmid degradation
(D) To create sticky ends
Answer
(B) — The origin of replication allows the plasmid to be duplicated within the host cell, ensuring the inserted gene is copied.
4. Which of the following would be best for visualizing the results of a successful CRISPR-Cas9 gene edit?
(A) Transformation assay
(B) Gel electrophoresis followed by DNA sequencing
(C) Bacterial conjugation
(D) Reverse transcriptase PCR
Answer
(B) — Gel electrophoresis can show fragment length changes, and sequencing confirms the presence or absence of edits at the nucleotide level.
5. A gene coding for a fluorescent protein is inserted into a plasmid with an antibiotic resistance gene. After transformation, what does fluorescence in the bacteria indicate?
(A) Successful digestion of plasmid
(B) Uptake and expression of the recombinant plasmid
(C) CRISPR mutation was corrected
(D) The plasmid was degraded
Answer
(B) — Fluorescence shows the gene was expressed, and the presence of antibiotic resistance confirms plasmid uptake.
6. What is one reason bacteria are commonly used in genetic engineering experiments?
(A) They lack DNA
(B) They can perform mitosis
(C) They reproduce rapidly and can express foreign genes
(D) They contain organelles for protein sorting
Answer
(C) — Bacteria grow quickly and can be engineered to express foreign genes, making them ideal for gene cloning and protein production.
7. What allows CRISPR-Cas9 to be used for precise gene editing?
(A) Use of radioactive tracers
(B) Antibody labeling
(C) Custom guide RNA sequences
(D) Primer mismatch correction
Answer
(C) — Custom-designed guide RNAs allow Cas9 to target any desired DNA sequence for editing.
8. During gel electrophoresis, why do smaller DNA fragments travel farther than larger ones?
(A) They have more negative charges
(B) They form more hydrogen bonds
(C) They encounter less resistance moving through the gel matrix
(D) They are more dense
Answer
(C) — Smaller DNA fragments pass more easily through the pores of the gel, allowing them to travel farther toward the positive electrode.
9. Which of the following is a major advantage of PCR compared to traditional DNA cloning?
(A) It does not require enzymes
(B) It produces RNA instead of DNA
(C) It rapidly amplifies DNA without the need for living cells
(D) It edits genes directly in organisms
Answer
(C) — PCR can amplify specific DNA regions in vitro without growing bacteria or using plasmids.
10. What is the function of ethidium bromide or SYBR Green in gel electrophoresis?
(A) Cut DNA at restriction sites
(B) Label DNA for visualization under UV light
(C) Slow down DNA migration
(D) Prevent DNA contamination
Answer
(B) — These dyes bind to DNA and fluoresce under UV light, making DNA fragments visible in the gel.
11. Which statement best describes a “reporter gene” like GFP (green fluorescent protein)?
(A) A gene that codes for a restriction enzyme
(B) A gene used to signal successful gene expression
(C) A bacterial gene that confers immunity
(D) A sequence that prevents transformation
Answer
(B) — Reporter genes produce a detectable signal (like fluorescence) that indicates the success of transformation or expression.
12. What is the purpose of the denaturation step in PCR?
(A) Primers attach to DNA
(B) DNA polymerase is activated
(C) DNA strands are separated by heat
(D) DNA fragments are visualized
Answer
(C) — Denaturation uses high heat to separate double-stranded DNA into single strands for replication.
13. A plasmid has been successfully cut and foreign DNA inserted. What should be done next to confirm success?
(A) Run the sample through a mass spectrometer
(B) Transform bacteria and select for antibiotic resistance
(C) Freeze the sample immediately
(D) Discard all untransformed plasmids
Answer
(B) — Transformation followed by antibiotic selection ensures only bacteria with the recombinant plasmid survive.
14. What makes Taq polymerase ideal for PCR reactions?
(A) It contains its own primers
(B) It can synthesize RNA
(C) It withstands high temperatures without denaturing
(D) It cuts DNA at palindromic sequences
Answer
(C) — Taq polymerase is thermostable and does not denature during the high heat cycles of PCR.
15. In which of the following situations would gel electrophoresis be least useful?
(A) Verifying plasmid insertion
(B) Comparing DNA fingerprinting profiles
(C) Amplifying DNA sequences
(D) Measuring DNA fragment sizes
Answer
(C) — Amplification is performed by PCR, not by gel electrophoresis.
16. What is a transgenic organism?
(A) An organism with a mutated genome
(B) One that cannot undergo transformation
(C) One that contains foreign DNA from another species
(D) One that has been exposed to PCR
Answer
(C) — Transgenic organisms have genes from other species inserted into their genome using recombinant DNA technology.
17. Which of the following DNA features is most important for designing an effective PCR primer?
(A) Having thymine at the 3′ end
(B) Matching a specific sequence near the target
(C) Being at least 50 nucleotides long
(D) Avoiding any guanine bases
Answer
(B) — Primers must be complementary to sequences flanking the target region to initiate replication.
18. A student uses CRISPR-Cas9 to target a gene but sees no edit in sequencing results. What could explain this?
(A) Cas9 made a perfect repair
(B) The guide RNA failed to match the target sequence
(C) The gel was run too long
(D) Restriction enzymes degraded the target DNA
Answer
(B) — Without proper base pairing, Cas9 cannot recognize or cut the target DNA sequence.
19. In biotechnology, what is a “template strand”?
(A) The coding DNA used in transcription
(B) The strand used as a pattern to synthesize complementary DNA in PCR
(C) The mRNA translated by ribosomes
(D) The RNA that guides CRISPR activity
Answer
(B) — In PCR and replication, the template strand is the original DNA strand used to build new complementary strands.
20. Which term refers to the direct uptake of plasmid DNA by a bacterial cell?
(A) Hybridization
(B) Transformation
(C) Transcription
(D) Conjugation
Answer
(B) — Transformation involves a bacterial cell incorporating foreign DNA, such as a plasmid, from its environment.
21. A researcher uses a DNA probe labeled with a radioactive isotope. What is this probe used for?
(A) To amplify DNA
(B) To sequence DNA
(C) To detect specific sequences by hybridization
(D) To inhibit translation
Answer
(C) — A DNA probe binds to complementary sequences and can be used to locate specific DNA fragments in a mixture.
22. A scientist runs a PCR reaction but sees multiple bands in gel electrophoresis. What is the most likely reason?
(A) Guide RNA mismatch
(B) Off-target primer binding
(C) DNA denaturation was skipped
(D) The DNA ladder was incorrect
Answer
(B) — If primers bind nonspecifically, multiple DNA fragments can be amplified, producing multiple bands.
23. What happens during the extension step of PCR?
(A) The DNA strands separate
(B) Primers bind to single-stranded DNA
(C) DNA polymerase adds nucleotides to synthesize new strands
(D) DNA is cut into smaller fragments
Answer
(C) — In extension, DNA polymerase synthesizes new DNA starting from the primers.
24. Why might a plasmid contain both a reporter gene and a selectable marker?
(A) To avoid gene silencing
(B) To ensure efficient mRNA processing
(C) To confirm both uptake and expression of foreign DNA
(D) To reduce DNA replication errors
Answer
(C) — The selectable marker confirms plasmid uptake; the reporter gene confirms that the inserted DNA is being expressed.
25. Which tool allows scientists to introduce specific changes to DNA sequences in living cells?
(A) Restriction mapping
(B) Southern blotting
(C) CRISPR-Cas9
(D) Protein electrophoresis
Answer
(C) — CRISPR-Cas9 enables targeted gene editing by introducing precise DNA changes in vivo.
26. A researcher introduces a mutation in the PAM sequence required for CRISPR-Cas9 activity. What is the expected result?
(A) Increased binding efficiency of guide RNA
(B) Cas9 will cut more accurately
(C) Cas9 will fail to bind and cut the target DNA
(D) Cas9 will permanently inactivate the gene
Answer
(C) — The PAM sequence is essential for Cas9 to recognize and bind the DNA; mutation in PAM prevents cutting.
27. Why are two different restriction enzymes often used when preparing a plasmid and insert for cloning?
(A) To reduce ligation time
(B) To prevent self-ligation of the plasmid and ensure directional insertion
(C) To shorten the DNA insert
(D) To increase PCR yield
Answer
(B) — Using two enzymes creates non-identical ends, which prevents the plasmid from closing without the insert and ensures directionality.
28. A scientist wants to knock out a gene in mice and study its effect. Which combination would be most effective?
(A) PCR and bacterial transformation
(B) Restriction digestion and gel electrophoresis
(C) CRISPR-Cas9 and embryo injection
(D) DNA laddering and Southern blot
Answer
(C) — CRISPR-Cas9 can induce targeted mutations, and injecting it into early embryos can create knockout mice.
29. In a gel electrophoresis experiment, the ladder bands appear but no sample bands are visible. What is the most plausible explanation?
(A) The DNA was too concentrated
(B) The DNA was not stained or degraded
(C) The voltage was too low
(D) The gel lacked buffer
Answer
(B) — If DNA was not stained or degraded, it will not be visible even if present in the gel.
30. A PCR product is not visible after 35 cycles. What is the best next step to troubleshoot this issue?
(A) Reduce annealing temperature slightly to allow better primer binding
(B) Increase primer length to over 100 bases
(C) Use a higher percentage agarose gel
(D) Skip the denaturation step
Answer
(A) — Slightly reducing annealing temperature may improve primer binding and increase product yield.
31. Which of the following accurately compares CRISPR-Cas9 and RNA interference (RNAi)?
(A) Both permanently change the genome
(B) CRISPR edits DNA while RNAi silences gene expression post-transcriptionally
(C) RNAi works only in prokaryotes
(D) CRISPR requires mRNA to function
Answer
(B) — CRISPR-Cas9 modifies DNA; RNAi blocks translation or promotes degradation of mRNA.
32. A scientist designs a guide RNA that has one mismatch at the 3′ end. What is the most likely result in CRISPR activity?
(A) Cas9 will cut normally
(B) Cas9 will cut randomly throughout the genome
(C) Cas9 may fail to bind or cut the target DNA
(D) Cas9 will only bind RNA instead
Answer
(C) — Mismatches in the guide RNA, especially near the 3′ end, reduce binding affinity and editing efficiency.
33. A biotechnology company creates a plasmid with a heat-shock promoter. What is the purpose of this element?
(A) To allow plasmid replication only in warm temperatures
(B) To trigger expression of the inserted gene at high temperatures
(C) To cut DNA under heat stress
(D) To increase bacterial transformation rate
Answer
(B) — Heat-shock promoters activate gene transcription in response to elevated temperatures.
34. Which of the following is a key difference between blunt-end and sticky-end ligation in DNA cloning?
(A) Sticky ends do not require ligase
(B) Blunt ends are easier to anneal due to base pairing
(C) Sticky ends increase the efficiency and specificity of ligation
(D) Blunt ends are more stable for sequencing
Answer
(C) — Sticky ends form complementary overhangs, increasing alignment and ligation success.
35. A student performs a transformation using a plasmid with a lacZ gene and observes blue colonies on X-gal plates. What does this result indicate?
(A) The bacteria are untransformed
(B) The lacZ gene was disrupted by an insert
(C) The lacZ gene is intact, and the plasmid did not carry the foreign DNA insert
(D) The plasmid was not replicated
Answer
(C) — Blue colonies express intact β-galactosidase, meaning the plasmid did not receive the DNA insert that would have disrupted lacZ.
36. Explain why Cas9 requires both a guide RNA and a PAM sequence to perform a DNA cut.
Answer
The guide RNA directs Cas9 to a complementary DNA sequence, but Cas9 will only cut if a nearby PAM sequence (e.g., NGG) is present, ensuring specificity and preventing accidental cuts.
37. Describe one advantage of using CRISPR over traditional restriction enzymes for gene editing.
Answer
CRISPR can be programmed with guide RNA to target any sequence, allowing for precise and flexible editing, whereas restriction enzymes can only cut at specific, naturally occurring sequences.
38. A student accidentally omits DNA ligase in a ligation reaction. What would be the expected result?
Answer
The DNA fragments and plasmid may align but will not be covalently bonded, preventing stable recombinant plasmid formation.
39. In a transformation experiment, why are some bacteria plated on media without antibiotic?
Answer
To verify that the cells are viable and capable of growth even if they didn’t receive the plasmid with the antibiotic resistance gene.
40. How could you confirm that a plasmid taken up by bacteria contains your gene of interest?
Answer
You could perform colony PCR, restriction digestion followed by gel electrophoresis, or sequencing to verify the presence and orientation of the insert.
41. A researcher wants to silence gene expression without editing DNA. What method should they use?
Answer
RNA interference (RNAi) can silence gene expression by degrading mRNA or blocking translation without altering the DNA sequence.
42. What is the purpose of using ampicillin in bacterial transformation experiments?
Answer
It selects for bacteria that have taken up the plasmid with the ampicillin resistance gene, allowing only transformed cells to grow.
43. A plasmid includes a Lac operon promoter. How can gene expression be turned on?
Answer
By adding an inducer like IPTG, which binds to the repressor and allows transcription from the Lac promoter.
44. Explain how CRISPR-Cas9 can be used to insert a specific gene into a genome.
Answer
CRISPR creates a double-strand break at the target site, and if a donor DNA with homologous ends is provided, the cell can use homology-directed repair (HDR) to insert the new gene.
45. Why must primers be specific to the target sequence in PCR?
Answer
Specific primers ensure that only the desired DNA fragment is amplified, minimizing off-target amplification and background noise.
46. What is the function of a multiple cloning site (MCS) in a plasmid vector?
Answer
The MCS contains multiple restriction sites, allowing for flexible insertion of foreign DNA using various enzymes.
47. A mutation prevents Cas9 from cutting DNA. How might this mutation affect gene editing?
Answer
Cas9 would bind the target sequence via guide RNA but fail to create a double-strand break, preventing any editing or repair from occurring.
48. A sample shows multiple bands after PCR. What steps could help improve specificity?
Answer
Use higher annealing temperatures, redesign primers for better specificity, or reduce cycle numbers to minimize nonspecific amplification.
49. Describe how gel electrophoresis helps verify successful PCR amplification.
Answer
The amplified DNA is run on a gel, and the presence of a band at the expected size indicates successful amplification of the target sequence.
50. A scientist wishes to clone a gene with directional insertion. What must be done during restriction digestion?
Answer
Use two different restriction enzymes to create non-identical ends, ensuring the insert is ligated into the plasmid in the correct orientation.