Introduction and History
What is the technology called that combines DNA sequences from different sources?
Recombinant DNA technology
A researcher wants to detect and quantify a DNA target while amplification is occurring. Which PCR method should they use, and what signal is monitored?
Quantitative PCR (qPCR); fluorescence is monitored during amplification.
Which mechanism of DNA transfer involves the use of a viral vector to deliver foreign DNA into the cell?
Transduction
Which sequencing technology reads DNA by measuring changes in electrical current as it passes through a pore?
Oxford Nanopore
Name another type of cloning other than traditional cloning mentioned in the presentation.
Golden Gate assembly or Gibson assembly
How did the work of Cohen and Boyer help move recombinant DNA technology from a laboratory concept toward a practical tool?
They demonstrated that DNA fragments could be combined with bacterial plasmids and propagated in bacterial cells.
You double the dNTP concentration to "boost yield," and the reaction fails. Why?
dNTPs chelate Mg²⁺, so free Mg²⁺, which Taq needs, drops. Excess dNTPs also lower fidelity.
What effect did the difference in heat-shock duration (45 seconds vs. 90 seconds) have on transformation efficiency as researched by Chan et al.?
They found no meaningful difference between both durations.
You need to confirm the sequence of a short region in one recombinant plasmid. What sequencing method would you suggest?
Sanger Sequencing
Why is antibiotic screening not enough to let us know that the cloning process was successful?
Antibiotic screening only verifies that the plasmid/vector is in the cell but not that the insert is in the vector.
Case study — Bacterial secretion system: The researchers engineered E. coli Nissle 1917 to deliver therapeutic proteins orally. What was the purpose of deleting the nlpI gene, and how could this help protein delivery?
Deleting nlpI increased outer membrane vesicle (OMV) production. These vesicles could package therapeutic proteins and help protect them during gastrointestinal transit, supporting their delivery.
Reagents are still in excess at cycle 35, yet amplification stops being exponential. Give three reasons.
Product strands reanneal with each other instead of with primers, the enzyme loses activity over repeated heating, and pyrophosphate and other byproducts accumulate and inhibit it. Primer depletion can also contribute
Bacteria grow on an antibiotic-containing plate after a transformation experiment. Why does this result not prove that they contain the desired recombinant plasmid?
The bacteria may carry a plasmid with the antibiotic-resistance marker but no target insert. Additional screening, such as colony PCR or blue-white screening, is needed.
You designed primers to amplify a gene for cloning, but PCR produces several bands instead of one. How could primer design explain this, and how could bioinformatics help?
Primers may bind to unintended DNA regions, producing additional PCR products. Researchers could use Primer-BLAST to check potential off-target amplification and redesign the primers to improve specificity.
How did CMC agar and Congo red dye help researchers determine whether or not cloned colonies have cellulase activity?
Colonies that had a cellulase activity enzyme were able to break down CMC which caused a yellow halo colour change when Congo red dye was applied.