Rucete ✏ AP Biology In a Nutshell
16. Biotechnology — Practice Questions 2
This chapter explores tools such as bacterial transformation, PCR, gel electrophoresis, and CRISPR-Cas9, and their use in cloning, gene analysis, and targeted editing for scientific and medical applications.
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(Multiple Choice — Click to Reveal Answer)
1. Which of the following best explains why DNA moves through the gel during electrophoresis?
(A) It is actively pulled by enzymes
(B) DNA has a negative charge due to its phosphate backbone
(C) Its bases are magnetic
(D) It is positively charged and repelled by the positive electrode
Answer
(B) — DNA moves through the gel because of its negative charge from phosphate groups, which is attracted to the positive electrode.
2. A scientist uses CRISPR-Cas9 to delete a segment of DNA in a gene. What is this approach commonly called?
(A) Amplification
(B) Knockout
(C) Cloning
(D) Transformation
Answer
(B) — Removing or disabling a gene's function using CRISPR-Cas9 is called a "knockout" and helps determine gene function.
3. What is the primary reason scientists use heat-stable polymerase in PCR reactions?
(A) It ensures DNA is cut at correct locations
(B) It prevents primer degradation
(C) It withstands the high temperatures needed to denature DNA
(D) It provides energy for replication
Answer
(C) — Heat-stable polymerase, such as Taq polymerase, is necessary because PCR cycles include high-temperature denaturation steps that would denature ordinary enzymes.
4. What would be the most efficient way to identify bacteria that successfully incorporated a recombinant plasmid with antibiotic resistance?
(A) Use a fluorescent dye
(B) Plate the bacteria on nutrient agar without antibiotics
(C) Plate the bacteria on agar containing the relevant antibiotic
(D) Observe the size of their DNA fragments on a gel
Answer
(C) — Only bacteria that incorporated the plasmid will grow in the presence of the antibiotic, making this a selectable marker system.
5. A researcher wants to replace a faulty gene sequence in a mouse with a corrected version. Which approach is most likely to achieve this?
(A) PCR with random primers
(B) Gel electrophoresis
(C) CRISPR-Cas9 with donor DNA
(D) Transformation with mRNA
Answer
(C) — CRISPR-Cas9 can be used with a donor DNA template to induce homology-directed repair and replace a faulty sequence with the correct version.
6. Which of the following is required to initiate a PCR reaction?
(A) DNA polymerase, primers, nucleotides, and a template
(B) Restriction enzymes and gel
(C) mRNA and ribosomes
(D) Cas9 and tRNA
Answer
(A) — PCR requires DNA polymerase, primers, free nucleotides (dNTPs), and a DNA template to initiate amplification.
7. What determines the movement speed of DNA fragments in gel electrophoresis?
(A) Their nucleotide sequence
(B) Their length in base pairs
(C) The type of dye used
(D) The temperature of the gel
Answer
(B) — Shorter DNA fragments move faster and travel farther through the gel compared to longer fragments.
8. What is the function of a restriction site in biotechnology?
(A) It allows ribosomes to bind DNA
(B) It is where primers bind during PCR
(C) It is a DNA sequence recognized and cut by a restriction enzyme
(D) It indicates where RNA polymerase terminates transcription
Answer
(C) — Restriction sites are specific sequences where restriction enzymes cleave DNA.
9. Which of the following best describes a cloning vector?
(A) A protein that signals replication
(B) A DNA molecule used to carry foreign DNA into a host cell
(C) An RNA molecule that binds DNA
(D) A mutated gene inserted into a virus
Answer
(B) — Cloning vectors (often plasmids) are used to transfer and replicate inserted DNA in host cells.
10. What is one advantage of using CRISPR-Cas9 over traditional gene-editing methods?
(A) It produces longer DNA fragments
(B) It only works in bacterial cells
(C) It targets specific sequences using guide RNA
(D) It can be used without primers or templates
Answer
(C) — CRISPR-Cas9’s guide RNA allows highly specific targeting of nearly any DNA sequence for editing.
11. Which step occurs first in a standard PCR cycle?
(A) Extension
(B) Annealing
(C) Denaturation
(D) Ligation
Answer
(C) — Denaturation is the first step, where double-stranded DNA is heated and separated into single strands.
12. What is one reason a scientist would include a DNA ladder in a gel electrophoresis experiment?
(A) To increase contrast of bands
(B) To provide known fragment sizes for comparison
(C) To absorb excess voltage
(D) To neutralize the gel buffer
Answer
(B) — A DNA ladder contains fragments of known sizes, which serve as references to estimate the size of unknown DNA fragments.
13. Which of the following is NOT typically included in a plasmid vector?
(A) Origin of replication
(B) Selectable marker
(C) Stop codon
(D) Multiple cloning site
Answer
(C) — While plasmids include replication origins, markers, and insertion sites, stop codons are part of gene sequences, not vector structure.
14. What is the primary role of DNA ligase in recombinant DNA technology?
(A) To create sticky ends
(B) To amplify DNA
(C) To catalyze the bonding of sugar-phosphate backbones between DNA fragments
(D) To convert mRNA into DNA
Answer
(C) — DNA ligase seals gaps between adjacent DNA fragments, joining them into a single continuous strand.
15. Why is bacterial transformation useful in biotechnology?
(A) It allows rapid RNA sequencing
(B) It generates multiple copies of a protein-coding gene
(C) It is used to synthesize CRISPR guide RNA
(D) It destroys unwanted plasmids
Answer
(B) — Transformation allows bacteria to replicate inserted genes, enabling mass production of proteins or cloning of DNA.
16. A gel electrophoresis image shows DNA fragments with different distances traveled. Which fragment is the smallest?
(A) The one closest to the well
(B) The brightest band
(C) The one that moved farthest
(D) The one with the largest sequence
Answer
(C) — Smaller DNA fragments move farther through the gel, so the farthest-traveled band is the smallest.
17. Why is the DNA in PCR repeatedly heated and cooled?
(A) To activate RNA polymerase
(B) To synthesize proteins
(C) To denature and re-anneal DNA strands during replication cycles
(D) To create point mutations
Answer
(C) — PCR cycles include denaturation, annealing of primers, and extension to replicate DNA through thermal changes.
18. What characteristic makes CRISPR-Cas9 useful for precision medicine?
(A) It randomizes mutations
(B) It only works in bacterial genomes
(C) It allows editing of specific sequences in human genes
(D) It amplifies DNA fragments from RNA templates
Answer
(C) — CRISPR-Cas9 can be programmed with guide RNA to edit disease-causing mutations at specific sites in the genome.
19. Which of the following processes requires primers to begin replication?
(A) CRISPR gene editing
(B) PCR
(C) Transformation
(D) Gel electrophoresis
Answer
(B) — Primers are essential for initiating DNA synthesis by DNA polymerase during PCR.
20. Which of these best describes a “sticky end” produced by restriction enzymes?
(A) A blunt cut with no overhang
(B) A strand of RNA attached to DNA
(C) A single-stranded overhang that can base pair with complementary sequences
(D) A chemically modified nucleotide
Answer
(C) — Sticky ends are overhanging single-stranded regions that enable complementary base pairing with other DNA fragments.
21. What is the purpose of using a loading dye in gel electrophoresis?
(A) To stain DNA permanently
(B) To slow down DNA movement
(C) To track the progress of DNA migration during the run
(D) To cut the DNA at specific points
Answer
(C) — Loading dye helps visualize sample movement through the gel and does not bind DNA directly.
22. A plasmid contains an ampicillin resistance gene. What will happen if transformed and untransformed bacteria are grown on ampicillin-containing media?
(A) Both types will grow equally
(B) Only bacteria with the plasmid will survive
(C) DNA fragments will be visible on a gel
(D) The bacteria will lose the plasmid
Answer
(B) — Only transformed bacteria carrying the resistance gene will survive on ampicillin-containing plates.
23. What is one function of the multiple cloning site (MCS) in a plasmid vector?
(A) Provides a place for CRISPR guide RNA to bind
(B) Marks the transcription start site
(C) Contains many unique restriction sites for DNA insertion
(D) Prevents antibiotic resistance
Answer
(C) — The MCS contains several restriction sites, enabling the insertion of various DNA fragments into the plasmid.
24. Which feature of Taq polymerase makes it essential for PCR?
(A) It binds to restriction sites
(B) It is resistant to high temperatures used in DNA denaturation
(C) It removes introns from eukaryotic DNA
(D) It creates sticky ends in DNA
Answer
(B) — Taq polymerase does not denature during the high temperatures required to separate DNA strands during PCR.
25. A researcher wants to make copies of a short DNA sequence within a few hours. Which technique is best?
(A) DNA ligation
(B) CRISPR-Cas9
(C) PCR
(D) Gel electrophoresis
Answer
(C) — PCR is the most efficient method for rapidly amplifying specific DNA sequences in vitro.
26. A researcher mutates the recognition site of a restriction enzyme in a plasmid. What is the likely outcome when trying to insert foreign DNA?
(A) The plasmid will replicate faster
(B) The restriction enzyme will cut at multiple new locations
(C) The plasmid will no longer be cut properly, preventing insertion
(D) The DNA will be inserted randomly
Answer
(C) — If the recognition site is mutated, the enzyme won’t cut, preventing proper insertion of the DNA fragment.
27. A CRISPR-Cas9 experiment results in a single nucleotide deletion within a coding region. What is the most likely effect on the protein product?
(A) No change at all
(B) Protein with one amino acid change
(C) Frameshift that alters all downstream amino acids
(D) Temporary pause in gene expression
Answer
(C) — A single nucleotide deletion causes a frameshift, changing the reading frame and potentially producing a dysfunctional protein.
28. Why are sticky ends more useful than blunt ends for recombinant DNA technology?
(A) Sticky ends inhibit transcription
(B) Sticky ends allow base pairing with complementary sequences
(C) Sticky ends prevent ligation
(D) Sticky ends do not require enzymes
Answer
(B) — Sticky ends can hydrogen bond with complementary overhangs, making ligation more efficient and specific.
29. In a gene cloning experiment, why is it important that the plasmid and the foreign DNA are cut with the same restriction enzyme?
(A) To create identical protein products
(B) To prevent contamination
(C) To ensure matching sticky ends for ligation
(D) To increase DNA yield
Answer
(C) — Using the same enzyme ensures that both DNA sources have compatible sticky ends that can be joined by DNA ligase.
30. How does CRISPR-Cas9 achieve specificity in gene editing?
(A) Cas9 only binds eukaryotic promoters
(B) The guide RNA base-pairs with the target DNA sequence
(C) DNA ligase directs Cas9 to the mutation
(D) Transposons insert guide RNA into coding regions
Answer
(B) — Guide RNA is complementary to the target DNA sequence and directs Cas9 to the correct editing site.
31. A DNA sample does not appear in a gel after electrophoresis. Which of the following is the most likely cause?
(A) The DNA was too large to enter the gel
(B) The gel was run with the wrong voltage
(C) The DNA was not stained or visualized with dye
(D) The loading dye was too concentrated
Answer
(C) — Without staining (e.g., ethidium bromide or SYBR Green), DNA cannot be visualized under UV light after electrophoresis.
32. What is the primary reason CRISPR-Cas9 is considered more versatile than restriction enzymes?
(A) It cuts RNA instead of DNA
(B) It can target virtually any sequence with programmable RNA guidance
(C) It produces sticky ends exclusively
(D) It only works on linear DNA
Answer
(B) — CRISPR’s guide RNA can be engineered to target any sequence, offering greater flexibility than restriction enzymes, which have fixed recognition sites.
33. A scientist uses PCR to amplify a DNA segment but observes nonspecific products. What could resolve this issue?
(A) Use higher concentration of dNTPs
(B) Lower the temperature of the denaturation step
(C) Increase the annealing temperature for greater primer specificity
(D) Shorten extension time
Answer
(C) — Higher annealing temperatures improve primer binding specificity and reduce off-target amplification.
34. What role does agarose concentration play in gel electrophoresis?
(A) It changes DNA sequence
(B) It determines voltage direction
(C) It affects the resolution of DNA fragments of different sizes
(D) It neutralizes the buffer
Answer
(C) — Higher agarose concentrations resolve smaller fragments better, while lower concentrations allow better separation of larger fragments.
35. Which feature of plasmids allows them to replicate independently within bacterial cells?
(A) Selectable marker gene
(B) Origin of replication (ori site)
(C) Promoter for the host genome
(D) Restriction enzyme site
Answer
(B) — The origin of replication is the DNA sequence that initiates plasmid replication inside the host cell.
36. Explain why using the same restriction enzyme on a plasmid and a DNA insert is critical in recombinant DNA experiments.
Answer
Using the same restriction enzyme ensures compatible sticky or blunt ends between the plasmid and insert, which allows successful ligation and stable incorporation of the foreign DNA.
37. Describe how CRISPR-Cas9 can be used to knock out a gene in a eukaryotic organism.
Answer
CRISPR-Cas9 introduces a double-strand break in the gene, and the cell's repair mechanisms often result in insertions or deletions (indels) that disrupt the reading frame, effectively silencing the gene.
38. A DNA sample runs as a single band in gel electrophoresis, but the researcher expected two bands. Suggest one likely explanation.
Answer
The restriction enzyme digestion may have failed, leaving the DNA uncut and resulting in a single, larger fragment on the gel.
39. What is the role of a selectable marker in a plasmid vector used for transformation?
Answer
A selectable marker, such as an antibiotic resistance gene, allows only cells that have successfully incorporated the plasmid to survive in selective media.
40. Why is the annealing temperature critical in a PCR reaction?
Answer
If too low, primers bind nonspecifically and generate incorrect products; if too high, primers may not bind at all. An optimal temperature ensures specificity and efficiency.
41. How does gel electrophoresis distinguish DNA fragments of different sizes?
Answer
Smaller DNA fragments move faster through the gel matrix under electric current, while larger fragments move more slowly, resulting in separation by size.
42. A researcher uses CRISPR-Cas9 but sees no mutation in the target gene. Suggest one possible technical error.
Answer
The guide RNA may have been incorrectly designed or not delivered effectively, preventing Cas9 from binding and cutting the target DNA.
43. Describe how PCR can be used to detect genetic mutations.
Answer
Primers specific to normal or mutant sequences can amplify only matching DNA. Comparing product size or sequence reveals the presence or absence of mutations.
44. Why is transformation efficiency important in bacterial cloning experiments?
Answer
High transformation efficiency increases the chances of obtaining colonies that carry the desired recombinant plasmid, saving time and resources.
45. What is the function of the origin of replication in a plasmid?
Answer
It is the site where DNA replication begins, enabling the plasmid to be copied independently of the bacterial chromosome.
46. A researcher uses two restriction enzymes to cut both plasmid and insert DNA. What is the advantage of this strategy?
Answer
Using two different enzymes ensures directional cloning, preventing the insert from being inserted backward into the plasmid.
47. How does Cas9 differ from traditional restriction enzymes in terms of DNA targeting?
Answer
Cas9 is guided by RNA to a specific DNA sequence, allowing precise editing, while restriction enzymes recognize fixed DNA sequences and cut at or near those sites.
48. Why is it important to include a negative control (no template DNA) in a PCR reaction?
Answer
To ensure that any observed PCR product is due to the target DNA and not contamination or nonspecific amplification.
49. A student runs gel electrophoresis and finds that all samples have identical banding patterns. What does this suggest?
Answer
It suggests that all DNA samples are likely identical or contain the same fragment sizes, possibly from the same organism or construct.
50. Outline the basic steps of PCR and their purpose.
Answer
1. Denaturation (95°C): Separates DNA strands. 2. Annealing (50–65°C): Primers bind to target DNA. 3. Extension (72°C): DNA polymerase synthesizes new DNA from primers. These steps repeat for amplification.