Question 1

Separating charged biomolecules — name the technique  Charged molecules (DNA, RNA, proteins) can be separated based on different migration rates through a hydrated solid matrix when subjected to an electric field. What is this analytical technique c…

Accepted Answer

Introduction / Context: Laboratories routinely separate nucleic acids and proteins to assess size, purity, and identity. A key method exploits charge-based migration through a porous gel under an electric field. Recognizing the correct term helps distinguish it from related but distinct techniques such as blotting and autoradiography, which are downstream detection steps. Given Data / Assumptions: Molecules carry net charge depending on pH and composition. Porous matrices (agarose for nucleic acids, polyacrylamide for proteins) impede larger molecules. An applied electric field drives migration toward the oppositely charged electrode. Concept / Approach: Gel electrophoresis separates by size and charge: DNA/RNA mainly by size (uniform charge density), proteins by size and charge (native PAGE) or by size alone when denatured with SDS (SDS-PAGE). Autoradiography visualizes radio-labeled molecules after separation; blotting transfers separated molecules to membranes for probing; photoreactivation is a DNA repair process, not a separation technique. Step-by-Step Solution: Prepare gel and buffer appropriate to analyte.Load samples with tracking dyes and standards.Apply electric field to induce migration through the matrix.Visualize separated bands by stains or labeled probes. Verification / Alternative check: Resolution and band patterns correlate with known ladders and molecular weights/lengths, confirming proper electrophoretic separation. Why Other Options Are Wrong: Photoreactivation is enzymatic repair; autoradiography detects radioactivity; blotting is transfer, not separation; isoelectric focusing is a form of electrophoresis but typically performed in gels or strips and not the general name requested. Common Pitfalls: Confusing detection or transfer methods with the core separation step. Final Answer: Gel electrophoresis

Question 2

Biolistics — equipment for shooting DNA-coated particles into cells  What is the laboratory device called that physically introduces DNA into cells using high-velocity, DNA-coated microprojectiles (biolistic transformation)?

Accepted Answer

Introduction / Context: Transforming difficult-to-transfect cells (plant cells, some tissues) often requires physical delivery methods. The biolistic approach accelerates microscopic particles coated with DNA into target cells to achieve transient or stable integration. Recognizing the correct device name is essential for interpreting methods sections and designing protocols. Given Data / Assumptions: DNA can be delivered by chemical, electrical, or physical means. Biolistics uses microprojectiles (gold or tungsten) and kinetic energy. The tool is purpose-built for this delivery method. Concept / Approach: The instrument is commonly termed a gene gun or biolistic particle delivery system. It uses pressurized helium or explosive charges to propel particles through cell walls/membranes. Lasers and inoculating needles are not used for ballistic DNA delivery; DNA probes detect sequences, not deliver them; electroporation is a different method relying on transient membrane permeabilization by electric pulses. Step-by-Step Solution: Coat metal particles with plasmid or linear DNA.Load particles into the gene gun apparatus.Fire particles into tissue or cells under vacuum/controlled pressure.Recover transformed cells for selection or analysis. Verification / Alternative check: Successful transformation is confirmed by reporter gene expression (e.g., GFP) or selection marker growth, validating gene gun delivery. Why Other Options Are Wrong: Laser microdissection isolates cells; probes detect nucleic acids; a needle inoculates cultures; electroporation is non-ballistic. Common Pitfalls: Overlooking tissue integrity and particle size, which affect viability and transformation efficiency. Final Answer: Gene gun (biolistic particle delivery system)

Question 3

Restriction enzymes — which type cuts within its recognition site?  Restriction endonucleases are grouped into Types I, II, and III based on structure and cleavage behavior. Which type typically cuts the DNA within its specific recognition sequence,…

Accepted Answer

Introduction / Context: Restriction enzymes revolutionized molecular cloning by enabling precise DNA cutting at defined sequences. The operational differences among Types I, II, and III determine how useful an enzyme is for mapping and cloning applications. The question focuses on the type that cleaves directly at its recognition site. Given Data / Assumptions: Type II enzymes recognize palindromic sequences and cut at or near those sites. Type I and III enzymes are multi-subunit, ATP-dependent, and cut away from the recognition sequence. Cloning requires predictable, reproducible overhangs or blunt ends. Concept / Approach: Type II restriction endonucleases (e.g., EcoRI, HindIII, BamHI) bind defined sequences and cleave at specific positions within them, producing consistent sticky or blunt ends ideal for ligation. Types I and III have more complex activities and cleave at variable distances, which is less practical for routine cloning. Step-by-Step Solution: Identify the need: precise cleavage at known locations.Recall enzyme classes: Type II performs site-specific cleavage within recognition sites.Eliminate types that cut remotely (I and III).Choose Type II as the best cloning enzyme class. Verification / Alternative check: Standard cloning protocols and maps list Type II restriction sites exactly at cut positions, confirming their utility and behavior. Why Other Options Are Wrong: Type I and III cleave at variable distances from the site; “All” is incorrect; Type IV refers to modification-dependent endonucleases and is not relevant here. Common Pitfalls: Confusing recognition with cleavage location; only Type II reliably cuts within the sequence. Final Answer: Type II

Recombinant DNA Questions