Question 1

In classic CaCl2-mediated transformation of E. coli (conventional method), plasmid DNA interacts with competent cells under which environmental conditions before heat shock?

Accepted Answer

Introduction / Context: Conventional chemical transformation uses divalent cations to render E. coli competent to take up plasmid DNA. Understanding the temperature and ion conditions during the binding stage explains why a subsequent heat shock is required. Given Data / Assumptions: Method: CaCl2 chemical competence (not electroporation). Focus: the environment while DNA associates with the cell surface prior to heat shock. Heat shock at ~42 °C occurs after this interaction step. Concept / Approach: At low temperature (ice), Ca2+ neutralizes negative charges on DNA and lipopolysaccharides, allowing plasmid DNA to adhere to the cell envelope. The system is then briefly heat-shocked to create a thermal imbalance that drives DNA uptake through transient pores. Step-by-Step Solution: 1) Competency induction: cells are incubated in cold CaCl2 to stabilize membranes and screen charges.2) DNA addition: plasmid is mixed with cells while kept on ice, maximizing surface binding.3) Heat shock: a short high-temperature pulse promotes DNA entry; this comes after, not during, the binding step.4) Therefore, the interaction environment is low temperature with Ca++ present. Verification / Alternative check: Standard protocols (Mandel–Higa, Hanahan) specify cold CaCl2 (often with additional salts) for binding, followed by heat shock; Mg2+ alone is not the defining ion in the classic version. Why Other Options Are Wrong: High temperature and Ca++: heat is used later for shock, not during initial interaction.High temperature and Mg++ / Low temperature and Mg++: Mg2+ may be present in some recipes, but Ca2+ at low temperature is characteristic of the binding step. Common Pitfalls: Confusing the binding step with heat shock, or assuming Mg2+ is the key ion for the conventional method. Final Answer: Low temperature and Ca++

Question 2

In agarose gel electrophoresis of nucleic acids, negatively charged DNA migrates in which direction relative to the electrodes?

Accepted Answer

Introduction / Context: Agarose gel electrophoresis separates DNA by size based on movement in an electric field. Recognizing electrode polarity and DNA charge is fundamental for interpreting band positions and troubleshooting runs. Given Data / Assumptions: Analyte: duplex DNA with a negatively charged phosphate backbone. Field applied across a submerged agarose gel. Electrode names: cathode (negative), anode (positive). Concept / Approach: Negatively charged molecules move toward the positive electrode. Thus, DNA migrates from the cathode (negative) toward the anode (positive). Orientation on the bench (left/right) is arbitrary; only electrode polarity determines the true direction. Step-by-Step Solution: 1) Assign charges: DNA is net negative due to phosphates.2) Electric field: opposite charges attract; negative species move to the positive pole.3) Therefore, DNA migrates from cathode to anode. Verification / Alternative check: Dye fronts (e.g., bromophenol blue) are often tracked; their movement toward the anode coincides with DNA migration in standard TAE/TBE buffers. Why Other Options Are Wrong: Left to right / A to B: meaningless without electrode labels.Anode to cathode: opposite of the expected direction for negatively charged DNA. Common Pitfalls: Confusing physical left/right with electrode polarity, or swapping anode/cathode definitions. Final Answer: Cathode to anode

Question 3

Which feature is shared by both DNA replication and transcription in cells, considering polarity and chemistry of chain elongation?

Accepted Answer

Introduction / Context: DNA replication and transcription are distinct processes performed by different enzymes, yet they share core polymer chemistry. This question checks understanding of the universal polarity of nucleic acid synthesis. Given Data / Assumptions: Replication uses DNA polymerases and deoxynucleotides. Transcription uses RNA polymerases and ribonucleotides. Both extend nucleic acid chains by phosphodiester bond formation. Concept / Approach: Both polymerases add nucleotides to the 3'-OH of the growing strand, proceeding 5'→3'. However, replication incorporates deoxynucleotides, whereas transcription incorporates ribonucleotides, and the enzymes are unrelated in subunit composition and specificity. Step-by-Step Solution: 1) Identify shared property: chain growth polarity (5'→3').2) Exclude deoxynucleotide incorporation: true for replication, not transcription.3) Exclude shared enzyme use: DNA vs RNA polymerase are different.4) Therefore, the commonality is 5'→3' synthesis. Verification / Alternative check: Mechanistically, both form a bond between the 3'-OH of the primer strand and the alpha phosphate of an incoming NTP/dNTP, releasing pyrophosphate—consistent with 5'→3' growth. Why Other Options Are Wrong: Deoxynucleotide incorporation: specific to replication.Same enzyme: false; different polymerases and co-factors.None of the above: incorrect because polarity is shared. Common Pitfalls: Assuming symmetry between processes or overlooking ribose vs deoxyribose differences. Final Answer: Synthesis in the 5'-3' direction

DNA Structure and Replication Questions