Lytic vs Lysogenic
Describe the key difference between the lytic and lysogenic cycles.
Lytic = active replication & lysis; Lysogenic = genome integration & dormancy
What polymerase is commonly used in PCR and why?
Taq polymerase; heat-stable at 95°C
What feature makes ddNTPs terminate synthesis?
They lack a 3’ hydroxyl group.
What is the purpose of using a DNA probe when screening for recombinant DNA?
To detect a specific complementary DNA sequence.
What is the main role of the Cro protein in lambda phage regulation?
Cro promotes the lytic cycle by repressing CI expression.
What host bacterial protein helps induce prophage escape during the SOS response?
RecA
Why do primers determine PCR specificity?
They define the start/end points for DNA synthesis.
Why are fluorescent labels used in automated Sanger sequencing?
Each ddNTP has a unique color allowing size and base identification.
Why does recombinant DNA often appear as a different-sized band on a gel?
Insertion of foreign DNA increases the plasmid size, shifting its migration.
What does the CII protein activate that allows the phage to enter lysogeny?
CII activates transcription of cI and integrase (int) genes.
Predict what happens if integrase is nonfunctional.
Phage cannot enter lysogeny → only lytic pathway
Predict what happens if annealing temperature is too high
Primers fail to bind → little or no amplification
You see a band at the bottom of a gel in the G lane. What does that represent?
A short fragment ending in G near the 5’ end
A colony hybridization experiment shows no probe signal. Give ONE explanation.
The bacteria lack the target insert / the recombinant plasmid is absent.
What two DNA sites recombine during integration of lambda phage into the host genome?
The phage attP site recombines with the bacterial attB site.
Explain how mutations in the CI repressor affect phage lifecycle choice.
Loss of function → cannot maintain lysogeny → forced lytic activation.
Explain exponential amplification in PCR
Each cycle doubles DNA; product = 2ⁿ where n = cycles.
Why must only a small proportion of ddNTPs be added to a Sanger reaction?
Too many ddNTPs = all chains terminate prematurely → unreadable.
Explain why restriction enzymes are essential for creating recombinant DNA.
They generate specific, predictable cuts producing sticky or blunt ends for ligation.
Explain how CI maintains the lysogenic state once established.
CI represses lytic promoters (PR, PL), activates its own expression (PRM), and keeps Cro/CII levels low, stabilizing lysogeny.
A lambda phage infects an E. coli cell that is experiencing severe DNA damage. However, instead of entering the lytic cycle, the phage unexpectedly establishes lysogeny. Provide TWO molecular explanations for how this could occur.
Mutation in RecA → RecA cannot cleave CI, so the SOS response cannot induce lysis.
Overexpression or hyperstability of CII → CII activates CI and integrase despite DNA damage.
Host protease (HflB/FtsH) defect → CII is not degraded → lysogeny favored.
CI repressor mutation making it non-cleavable → phage cannot enter lytic cycle even under SOS conditions.
A PCR reaction produces a single, clean band at the correct size but yields no product when the same primers are used to amplify genomic DNA from a related bacterial strain. Provide TWO molecular reasons why this could happen and ONE troubleshooting step for each reason.
Sequence variation/mutation at the primer binding sites in the related strain
Troubleshooting: Design degenerate primers or redesign primers to target conserved regions.
Genome has a secondary structure or GC-rich region blocking polymerase progression
Troubleshooting: Add DMSO/betaine or increase denaturation temperature.
The related strain contains inhibitors in the DNA prep (e.g., polysaccharides, salts)
Troubleshooting: Re-purify DNA or perform dilution to reduce inhibitor concentration.
Target gene absent due to horizontal gene loss
Troubleshooting: Use a different marker gene or verify gene presence by sequencing.
Explain why capillary electrophoresis replaced slab gels in automated sequencing.
Capillaries provide higher resolution, faster separation, and automation compatibility.
You digest a plasmid with two restriction enzymes and expect two fragments, but your gel shows three. Provide TWO possible reasons.
Unexpected internal cut site
Partial digestion or star activity
Plasmid rearrangement or recombination event
CII is extremely unstable and rapidly degraded by host proteases. Give TWO environmental conditions that increase CII stability and explain why they favor lysogeny.
Answer:
Low nutrient growth / slow host metabolism: protease activity decreases; CII lasts longer → lysogeny favored.
High multiplicity of infection (MOI): more CII produced overall → easier to reach threshold for CI + integrase activation.