Chapter 3
A researcher heats a double-stranded DNA sample and observes that regions rich in A/T base pairs separate before regions rich in G/C base pairs. Based on nucleic acid structure, what property best explains this observation?
A/T base pairs form fewer hydrogen bonds than G/C base pairs, making them less thermally stable.
A plasmid is protected from digestion in E. coli despite containing an EcoRI recognition site. What single molecular feature most likely prevents cutting, and why?
Methylation of the EcoRI recognition sequence prevents enzyme binding and cleavage.
A PCR reaction produces a strong low–molecular weight band and a weak target band. Analyze the most likely cause.
Primer-dimer formation dominated the reaction, reducing amplification of the target sequence.
Two DNA samples from different organisms are heated and then slowly cooled together. Hybrid DNA strands form, but only a small fraction of the strands successfully re-anneal. What does this result most directly indicate about the two DNA samples?
They share limited sequence similarity.
BamHI and BglII fragments ligate efficiently, but the resulting DNA can no longer be cut by either enzyme. What structural change explains this outcome?
Ligation destroys the original recognition sequences, creating a hybrid site no longer recognized by either enzyme.
A PCR reaction produces amplification in early cycles but plateaus quickly. Evaluate one likely molecular reason.
Reaction components such as primers or dNTPs became limiting, reducing amplification efficiency.
A mutation changes the primary structure of a protein by substituting a single amino acid near the protein’s active site. Which conclusion is most justified regarding the protein’s function?
The protein’s activity may be altered because changes in primary structure can affect folding and active site formation.
You need to permanently block transcription at a specific DNA locus without degrading the DNA. Which nucleic acid analog is most effective, and what is the key reason?
Peptide nucleic acid (PNA); it forms a stable triple helix that physically blocks transcription.
A researcher performs RT-PCR on two samples. After 35 cycles, Sample A shows a faint band, while Sample B shows a very bright band on an agarose gel.
Using your understanding of RT-PCR data, determine whether the researcher can conclude that Sample B has exactly five times more mRNA than Sample A. Justify your answer based on PCR mechanics.
No, the researcher cannot conclude that there is exactly a five-fold difference in mRNA abundance. Because this is end-point RT-PCR, the reaction has likely reached the plateau phase by cycle 35, where reagents such as primers or dNTPs become limiting and amplification is no longer proportional to starting template amount. While the brighter band suggests Sample B had more initial mRNA (making RT-PCR semi-quantitative), precise numerical differences cannot be determined from band intensity alone. To accurately measure a five-fold difference, real-time qPCR would be required, as it quantifies amplification during the exponential phase when signal is proportional to starting template.
You want to confirm whether a fluorescent FISH signal corresponds to a gene deletion or merely reduced expression. What single additional experiment would you perform to distinguish these possibilities?
Perform Southern blotting; absence of the band indicates deletion, presence indicates reduced expression.
GC-rich DNA templates often fail under standard PCR conditions. Design a modified PCR strategy to successfully amplify a GC-rich target and justify each change.
PCR conditions could be modified by increasing the denaturation temperature or time to improve strand separation, optimizing the annealing temperature to balance specificity and binding, and adjusting reaction chemistry (such as adding GC-friendly additives) to reduce secondary structure formation and improve polymerase access.